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N Lempert

Publications and source records attributed to N Lempert.

At least 37 records · Page 2Linked to original sources
PubMed1989

The inhibitory effects of N-ethylmaleimide, colchicine and cytochalasins on human T-cell functions.

The thiol-alkylating agent, N-ethylmaleimide (NEM), was found to inhibit the response of human peripheral blood mononuclear cells to the T-cell mitogen, concanavalin A (Con A). NEM (10 microM) blocked Con A-induced agglutination, production of interleukin-2 (IL-2) and expression of the IL-2 receptors (Tac) without toxicity. In order to determine whether the effects of NEM on lymphokine production were related to inhibition of agglutination, we compared the immunosuppressive effects of NEM with those of cytochalasin A, cytochalasin B and colchicine. NEM did not inhibit E-rosette (ER) formation, suggesting that it does not interfere with actin filaments. Low concentrations of NEM (4 microM) inhibited IL-2 production and Tac expression without inhibiting agglutination, while 6-10 microM NEM blocked agglutination and DNA synthesis as well. In contrast, 5-10 microM cytochalasin B (CB) inhibited ER formation, agglutination, Tac expression and DNA synthesis, but augmented IL-2 production by three- to ten-fold. Colchicine (0.1-10 microM) had no effect on ER formation or agglutination and augmented IL-2 production by as much as 18-fold. However, colchicine blocked Tac expression by greater than 40% and DNA synthesis by greater than 80%. Cytochalasin A (CA), which has the thiol-reactive properties of NEM, the actin filament-disrupting properties of CB, and the microtubule-disrupting properties of colchicine, exhibited the immunosuppressive effects of all three compounds. These studies suggest that the inhibitory effects of NEM on IL-2 production do not appear to be due to reactivity with the cytoskeleton, but are probably due to effects on signal transduction pathways leading to IL-2 production and expression of IL-2 receptors.

Agglutination↗
PubMed1988

Hydrogen peroxide-mediated inhibition of T-cell response to mitogens is a result of direct action on T cells.

Hydrogen peroxide, a reactive oxygen intermediate produced by activated neutrophils, has been shown to inhibit the response of human T lymphocytes to mitogens and alloantigens. Since hydrogen peroxide is known to react with iron and to induce lipid peroxidation, we compared the effects of hydrogen peroxide and a lipid peroxidation product, malondialdehyde, on the response of human peripheral blood mononuclear cells to T-cell mitogens. Peripheral blood mononuclear cells pretreated with 1 mmol/L of malondialdehyde, washed, and resuspended in fresh medium exhibited no inhibition of phytohemagglutinin responsiveness. Peripheral blood mononuclear cells treated in the same manner but with 200 mumol/L of hydrogen peroxide were inhibited by more than 95%. The addition of ferric edetate did not alter the inhibitory effects of 50 to 100 mumol/L of hydrogen peroxide, nor did the addition of deferoxamine, an iron chelator. These studies suggest that exogenous lipid peroxidation does not affect lymphocyte activation but that hydrogen peroxide has a direct inhibitory effect. Although monocytes are necessary for T-cell mitogenic responses, the effect of hydrogen peroxide was found to be directed at T lymphocytes. Exposure of T cells to a single dose of 200 mumol/L of hydrogen peroxide resulted in more than 71% suppression of the proliferative response measured 48 hours later, but the effect was spontaneously reversed by 72 to 96 hours. Repeated exposure of the cells to hydrogen peroxide resulted in continued inhibition of the proliferative response. These findings suggest that hydrogen peroxide produced by inflammatory phagocytic cells might be capable of suppressing the immune response of nearby T lymphocytes.

Deferoxamine↗
PubMed1987

Inhibition of early events in the human T-lymphocyte response to mitogens and alloantigens by hydrogen peroxide.

Hydrogen peroxide (100 to 200 microM) inhibited the response of human peripheral-blood mononuclear cells (PBMCs) in phytohemagglutinin, concanavalin A, and mixed lymphocyte culture assays by more than 90% without affecting cell viability. The response of PBMCs to pokeweed mitogen was stimulated twofold by 50 microM H2O2, but 200 microM H2O2 inhibited the pokeweed mitogen response by more than 95%. A 50 microM concentration of H2O2 completely blocked the generation of cytotoxic T cells in the mixed lymphocyte culture but did not inhibit the production of interleukin 2. Concentrations of 100 microM to 200 microM H2O2 inhibited interleukin 2 production by 45% to 57%. The H2O2 appeared to block early events in T-cell activation, since 200 microM H2O2 was not inhibitory when added one hour after stimulating the cells with phytohemagglutinin. Treatment of PBMCs with 200 microM H2O2 did not decrease the total cellular thiol pool, suggesting that H2O2-mediated inhibition of the proliferative response was not due to thiol oxidation. However, pretreatment of PBMCs with the lipid antioxidants butylated hydroxyanisole, butylated hydroxytoluene, and n-propyl gallate blocked more than 75% of the inhibitory effect of H2O2, suggesting that H2O2 inhibits T-cell activation by inducing lipid peroxidation.

B-Lymphocytes↗
PubMed1987

In vitro immunosuppressive properties of cyclosporine metabolites.

The in vitro biological activity of cyclosporine (CsA) and four of its metabolites (M1, M8, M17, and M21) was determined. M1, M17, and M21 are primary metabolites, while M8 is a secondary metabolite derived from either M1 or M17. The order of inhibitory activity in production assays was phytohemagglutinin (PHA), concanavalin A (ConA), mixed lymphocyte culture (MLC), and interleukin-2 (IL-2) CsA greater than M17 greater than M1 greater than M21 much greater than M8. In the PHA assay, CsA was significantly more inhibitory than M17, but in Con A and MLC assays, the inhibitory activity of M17 approached that of CsA. More importantly, M17 and M1 inhibited the production of IL-2 in the MLC to the same extent as CsA. M21 was significantly less inhibitory than either M17 or M1, and M8 appeared to be largely devoid of biological activity. These experiments demonstrate that single hydroxylations of amino acids 1 (M17) and 9 (M1) do not significantly affect the ability of the molecule to block IL-2 production, but hydroxylation of both amino acids renders the molecule virtually inactive. In addition, the presence of the N-methyl group on amino acid 4 appears to be very important, since removal of this group (M21) greatly diminishes the immunosuppressive activity.

Concanavalin A↗
PubMed1986

Differential inhibition of human T-lymphocyte activation by maleimide probes.

Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processes in the activation sequence by inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 microM) was found to be fully inhibitory in PHA, Con A, and MLC assays only when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15 sec after stimulating the cells with PHA resulted in a loss of greater than 50% of the inhibitory activity. The addition of 50 microM 2-ME 10 min after treating the cells with NEM failed to block the inhibitory effect. NEM (10-20 microM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in response to mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effects of NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative response was associated with the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreased the total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreased the total cellular thiols by 38% but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation.

Cytotoxicity, Immunologic↗
PubMed1986

Immunosuppressive metabolites of cyclosporine in the blood of renal allograft recipients.

Cyclosporine levels by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) were monitored in serial blood samples (n = 177) from 11 renal allograft recipients. HPLC analysis revealed three primary metabolites of CsA (M17, M1, and M21) in peak and trough blood samples; M17 was the preponderant metabolite. In 4 patients on whom serial metabolite assays were performed, M17 was found in the blood at 86-2004 ng/ml; M1 and M21 were found at up to 100 ng/ml. The immunosuppressive properties of purified metabolites M1, M17, M21, and M8 (which was not detected in the blood) were compared with CsA. M17--and, to a lesser extent, M1 and M21--were found to inhibit the in vitro response of human mononuclear cells in the mixed leukocyte culture and in mitogen (phytohemagglutinin [PHA], concanavalin A [Con A], and pokeweed mitogen [PWM]) assays at 1000 ng/ml. M8 exhibited no in vitro inhibitory activity. M17 was further tested at 10-1000 ng/ml in PHA and mixed lymphocyte culture (MLC) assays. M17 had considerably less inhibitory activity (12-43%) than CsA (18-70%) in the PHA assay. However, in MLC experiments M17 blocked the proliferative response by 39-72% at 100-800 ng/ml, which approached the degree of inhibition exhibited by CsA (63-87%). In 34 of 37 (92%) patient blood samples, the level of metabolite M17 was found to exceed the parent drug level and could not be measured accurately by RIA. The observed in vitro immunosuppressive activity of metabolites (particularly M17) and their presence in the blood of renal allograft recipients suggest a possible role for these metabolites in the immunopharmacology of CsA.

Cells, Cultured↗
PubMed1985

The vascular endothelial cell antigen system.

Vascular endothelial cells (VEC) are clearly immunogenic and express antigens unique to vascular endothelial cells. The observation that peripheral blood monocytes also express this VEC antigen system made prospective testing feasible. Preformed antibody to the VEC/monocyte antigen system of the donor usually leads to graft rejection in HLA-identical combinations, but antibody directed against donor monocytes exclusively (monocyte-specific antigens) appears to be benign. Clearly the VEC antigen system is an important immunogen in HLA-identical living-related donor combinations--and, in addition, this antigen system may be equally important in the non-HLA-identical combinations. The identification of antibody directed against the VEC/monocyte antigens of the donor is frequently masked by the concurrent development of anti-HLA antibody. Preliminary family segregation studies support the concept of genetic linkage between the VEC/monocyte antigen system and the major histocompatibility complex. A group of 153 consecutive, prospective monocyte crossmatches performed have yielded approximately a 7% incidence of positive monocyte crossmatches, with traditional crossmatches being negative. This frequency of patients sensitized to the VEC/monocyte antigens of the donor could conceivably account for up to 70% of the observed early graft loss in living-related donors. We think a positive monocyte crossmatch to the donor in the presence of positive reactivity to concordant VEC and monocytes remains a contraindication to transplantation.

Endothelium↗