Biomedical subjects

W Holter

Publications and source records attributed to W Holter.

66 records · Page 4Linked to original sources

PubMed1987

Expression of functional IL 2 receptors by lipopolysaccharide and interferon-gamma stimulated human monocytes.

Human peripheral blood monocytes were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) alone or in combination. Stimulated but not resting monocytes displayed the Tac peptide of the interleukin 2 (IL 2) receptor within 24 hr as measured by immunofluorescence staining and [3H] Tac binding. The total number of anti-Tac binding sites on co-stimulated monocytes was 13,700. By using scatchard analysis with radiolabeled IL 2, the activated cells were shown to express low numbers (below 100 sites/cell) of high affinity binding sites with a KD of approximately 15 pM. LPS and IFN-gamma were additive in augmenting the number of IL 2 and anti-Tac binding sites. By using an ELISA assay specific for the soluble released form of the Tac peptide we identified 112 U/ml of IL 2 receptors in the supernatant of monocytes stimulated for 24 hr with IFN-gamma, 233 U/ml after stimulation with LPS, and 519 U/ml after the addition of both stimulating agents. Both the membrane form (55,000 daltons), as well as the soluble form (45,000 to 50,000 daltons) of the Tac, IL 2 receptor, peptide from monocytes were shown by immunoprecipitation and gel electrophoresis to be similar size to the comparable forms of these receptors derived from activated T cells. In addition, monocytes stimulated for 8 hr contained mRNA specifically hybridizing to a cDNA probe coding for the Tac peptide. Finally, activated monocytes responded to the addition of recombinant IL 2 by an increase in H2O2 production that was measured by using fluorescent indicator 2,7-dichlorofluorescein. This response as well as the observed induction of monocytic IL 2 receptors by LPS may point to a functional role for this receptor during monocyte/macrophage responses to microbial infections.

Cell Cycle↗

PubMed1987

Induction of human complement activation without cytolysis by mouse monoclonal antibodies to human leukocyte antigens.

Ten monoclonal antibodies to human leukocyte subsets that had previously been shown to lyse their respective target cells in the presence of rabbit serum as complement source were evaluated for their cytolytic capacity with human complement. Four of the ten were lytic with human complement. All were of IgM type. Antibodies were also evaluated for their capacity to induce C3 binding to target cells. With this method we could demonstrate that, indeed, 3 of the 6 noncytolytic antibodies had the capacity to initiate the human complement activation process and to induce C3 binding. Two of these 3 antibodies were of IgM class (VIT3 and VIM13), one of IgG3 (562). From the practical point of view the most interesting of these 3 antibodies is the nonmitogenic anti-CD3 pan-T cell antibody VIT3. Therefore, this antibody was analyzed in more detail. VIT3 antibody concentrations needed to induce detectable C3 binding to human T cells are very low (down to 1 ng VIT3/ml). Human serum as complement source can also be considerably (100X) diluted before C3 binding becomes undetectable. Activation of C3 is a prerequesite for VIT3-induced C3 binding, and bound C3 seems to lack the C3a fragment. Bound C3, in contrast to the quickly occuring antigenic modulation of the CD3 complex and the simultaneous disappearance of the antibody coat, remains expressed also after prolonged incubation at 37 degrees C. C3 fragments bound to T cells after activation with VIT3 are also recognized by cells bearing C3 receptors of types CR1 and CR2.

Antibodies, Monoclonal↗

PubMed1986

Interleukin 2 receptors on cultured murine epidermal Langerhans cells.

Rat monoclonal antibodies 3C7 and 7D4 detect two distinct functional regions of the murine interleukin 2 (IL 2) receptor. When studying the emergence kinetics of IL 2 receptors in mixed epidermal cell (EC)-lymphocyte cultures by using 3C7 and 7D4 in an indirect immunofluorescence assay, we regularly encountered a distinctive membrane fluorescence not only on lymphocytes, but also on a subpopulation of cells exhibiting a dendritic morphology. Reasoning that these 3C7/7D4-reactive dendritic cells might represent a subpopulation of epidermal dendritic cells, we studied mouse EC for the presence of 3C7/7D4- reactive cells. Although 3C7/7D4 reactivity was never detected on freshly isolated EC or on epidermal sheets, a small number of 3C7/7D4+ cells was encountered after 24 to 48 hr of culture. These cells exhibited a dendritic shape, expressed Ia antigens, lacked Thy-1 antigens, and displayed the ultrastructural features of Langerhans cells (LC) with the notable exception of Birbeck granules. Although after 24 hr, only 20% of Ia+ EC were 3C7/7D4+, the vast majority of LC displayed 3C7/7D4 binding sites after 4 to 5 days of culture. Preincubation of cultured LC-enriched EC with recombinant human IL 2 prevented subsequent 3C7-but not 7D4-binding to these cells. Western blot analysis of 7D4-reactive material of detergent extracts from LC-enriched EC revealed three bands in the same m.w. range as reported for CTLL cells. These results demonstrate that cultured LC express IL 2 receptors and may bear important implications for a better understanding of growth regulation, differentiation, and immunologic functions of LC.

Animals↗

PubMed1986

Use of a cocktail of monoclonal antibodies and human complement in selective killing of acute lymphocytic leukemia cells.

Autologous remission bone marrow is a potential source of repopulative stem cells after ablative chemoradiotherapy of tumor patients. Even with remission bone marrow, one major obstacle to use of autologous bone-marrow support is the danger of reinfusing viable tumor cells. This report describes a purging protocol with human complement, which seems suitable for eliminating acute lymphatic leukemia (ALL) cells of the common ALL type. Lysis of ALL blasts is induced with a cocktail of 3 monoclonal antibodies of IgM type (termed VIB-pool). These are directed against the CALLA (CD 10) antigen (VIL-AI antibody) and against 2 different epitopes of the CD24 surface structure (VIB-C5 and VIB-E3 antibodies). The purging efficiency was evaluated with leukemic cell lines of the common ALL type (Reh-6 and Nalm-6) and with blast cells from common ALL patients. Optimal lysis was obtained with antibody and human serum concentrations as low as 1 microgram/ml and 7% respectively. As a standard purging protocol we propose one 20-min incubation at room temperature with antibody followed by two 30-min incubations at 37 degrees C with 25% human complement. In dye exclusion test 99% purging efficiency and in clonogenic assays detecting elimination of up to 5 logs of clonogenic tumor cells 99.99% (= 4 logs) purging efficiency were achieved. Treatment with VIB-pool and human complement had no negative effect on the growth of the normal hemopoietic progenitor cells CFU-GM, CFU-E and BFU-E.

Animals↗

PubMed1986

Recombinant interferon-gamma induces interleukin 2 receptors on human peripheral blood monocytes.

The present report describes the inducibility of IL 2 receptors on human peripheral blood monocytes. Although freshly isolated monocytes are IL 2 receptor negative, approximately one-third of the cells react with the anti-Tac antibody within 18 hr of culture. IFN-gamma is found to double both the number of positive cells and the number of binding sites, whereas IL 2 has no influence on the IL 2 receptor expression on monocytes. Anti-Tac precipitates from monocyte lysates several protein bands of similar m.w. to those previously found with activated T and B cells. Finally, IFN-gamma-induced, but not resting, monocytes are found to bind recombinant IL 2. We conclude that IFN-gamma induces peripheral blood monocytes to express IL 2 receptors similar in structure to those found on activated T and B lymphocytes.

Antibodies, Monoclonal↗

PubMed1986

T cell stimulation via the erythrocyte receptor. Synergism between monoclonal antibodies and phorbol myristate acetate without changes of free cytoplasmic Ca++ levels.

We observed that certain E-receptor antibodies (CD2 antibodies) can induce proliferation of resting human T cells in the presence of PMA, while other CD2 antibodies fail to have such an effect. The same CD2 antibodies that were mitogenic in the presence of PMA (9.6, X11, VIT13), but not the nonreactive ones, were also able to induce T cell proliferation via the so-called alternative pathway of T cell activation, i.e., when added pairwise in certain combinations to T cells in the absence of PMA. While the simultaneous addition of two comitogenic CD2 antibodies (9.6 or X11 plus VIT13) or the addition of a single nonmitogenic CD3 antibody (VIT3) led to a clearcut elevation of intracellular Ca++ levels, no such effect could be observed after the addition of one CD2 antibody alone. Even in the presence of PMA, one comitogenic CD2 antibody alone was unable to trigger a significant Ca++ response, although this combination induced a proliferative response. These data indicate that, distinguishable by their influence on free cytoplasmic Ca++, there are two different mechanisms of T cell activation via CD2. While simultaneous triggering with two antibodies leads to cell proliferation preceded by an increase of Ca++ levels, stimulation with one antibody plus PMA results in proliferation without a measurable early Ca++ response. We conclude that T cells treated by certain CD2 antibodies alone already recognize an activation signal probably unrelated to Ca++ homeostasis, a signal that can further be developed by PMA to result in a completely developed proliferative response.

Antibodies, Monoclonal↗

PubMed1986

Analysis of CD3-antibody-mediated inhibition of T-cell activation.

In this study the influence of a non-mitogenic anti-CD3 antibody on accessory cell-dependent antigen and mitogen-induced T-lymphocyte proliferation has been investigated. The antibody was found to completely inhibit PHA, Con A, PWM, and tetanus toxoid stimulation, with no effect on the proliferation induced by the calcium ionophore A23187. VIT3 completely abrogated the production of IL-2 by lectin-stimulated T cells. It had no effect, however, on the IL-2-dependent proliferation of preactivated T-cell blasts. In addition, the antibody was able to elevate free cytoplasmic Ca2+ levels within minutes after the addition to T cells. Detailed time kinetic analyses revealed that the time interval critical for inhibition was significantly dependent on the interaction between T cells and accessory cells. Under standard conditions, in the presence of 10% non-T cells as accessory cells 50% inhibition was still achieved when VIT3 was added to PHA-stimulated T cells as late as 8 hr after the onset of culture. Delayed addition or a decrease in the number of added accessory cells significantly prolonged this time period. Lectin-stimulated T cells can thus obviously be inhibited via CD3 as long as they have not received all signals including those delivered by accessory cells. Although the underlying mechanisms are not clear so far, the observation that VIT3 at the same time triggers an early cytoplasmic Ca2+ response might indicate that it thereby actively interferes with antigen and lectin-initiated activation processes.

Antibodies, Monoclonal↗

PubMed1985

[Considerations on the therapeutic use of monoclonal antibodies].

The availability of monoclonal antibodies (MoAb) decisively improved and extended the analytical and diagnostic possibilities both in science and in clinical medicine. Monoclonal antibodies are also important tools for the isolation and purification of biologically active components. However, the therapeutic applications of monoclonal antibodies require manifold clarification. In this manuscript an attempt is made to present a short survey of the current stage of development in the therapeutic applications of MoAb and to discuss the foreseeable future in this research area. Possible fields of application, such as selective cell manipulation, drug targeting, detoxification by neutralization and/or immune clearance, specific antibody substitution are summarized and the negative and positive results so far achieved are discussed. Ex vivo therapy with monoclonal antibodies, which has already been used in a number of hospitals, is dealt with in detail. Problems already encountered, as well as anticipated in the course of the therapeutic implementation of monoclonal antibodies are discussed.

Antibodies, Monoclonal↗

PubMed1985

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes.

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.

Antibodies, Monoclonal↗

PubMed1985

Analysis of T cell activation with a non-mitogenic anti CD3 antibody and the phorbol ester TPA.

We used a non mitogenic anti CD3 antibody, termed VIT3, to study the signals required for the activation of normal resting T lymphocytes. Besides being not mitogenic, this antibody completely inhibits mitogen induced proliferative responses. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), however, VIT3 induces DNA replication and cell proliferation comparable to PHA responses. In addition, T cells cultured with VIT3 plus TPA but not with VIT3 or TPA alone express high levels of interleukin 2 (IL-2) receptors and transferrin receptors. This co-stimulation appears to be accessory cell independent. Purified T cells respond equally well to VIT3 plus TPA as do unseparated mononuclear cells and addition of non-T cells has no enhancing effect. We conclude that the IgM antibody VIT3, although non-mitogenic by itself, still delivers a first and for the activation essential signal. In resting T cells this signal does not induce demonstrable anti-Tac antibody binding nor does it lead to a fully developed proliferative response in the presence of recombinant IL-2. Together with a second signal provided by TPA it serves, however, as a potent inducer of T cell growth.

Animals↗

PubMed1983

[Lymphocyte stimulation in the primed lymphocyte typing test by as yet unknown HLA-linked antigens (WP-alleles)].

Lymphocytes from HLA-identical unrelated donors often show positive stimulation in the mixed lymphocyte culture, possibly caused by alleles of HLA-linked, but not yet defined gene loci. In order to investigate such determinants lymphocytes from HLA-DR identical individuals were primed and tested in the PLT system. Family and population data indicate that such antigens exist and can be detected in vitro.

Alleles↗

PubMed1983

Stimulation in primary MLR caused by a PLT defined non-HLA-D/DR determinant, EP1.

The influence in primary mixed lymphocyte culture reaction (MLR) of a primed lymphocyte typing (PLT) defined non-HLA-D/DR determinant, EP1, was investigated. In primary MLR between HLA-D/DR compatible lymphocytes, the response of the lymphocytes from 14 EP1-negative HLA-D/DR heterozygous individuals towards two EP1-positive homozygous typing cells (HTCs) was on an average approximately 35% higher than the response towards two EP1-negative HTCs (P less than 0.01). The strength of the MLR between lymphocytes from 25 EP1-negative and 10 EP1-positive individuals matched for two HLA-D/DR antigens was investigated. The average responses of EP1-negative lymphocytes against EP1-positive lymphocytes were approximately 40% higher than the average responses against EP1-negative lymphocytes (P less than 0.01). These data indicate that the PLT defined determinant EP1 causes stimulation in primary MLR.

Epitopes↗