Effect of Fc-Receptor Modulation on Mumps-Virus-dependent Lymphocyte-mediated Cytotoxicity in Vitro

The natural cytotoxicity of peripheral blood lymphocytes (PBL) from normal human donors to a variety of tissue culture target cells increases upon brief exposure of lymphocytes to mumps virus. The effector cells operative in this system have Fc receptors for IgG (FcR), since cytotoxicity was abolished when FcR ⁺ cells were removed by passage of the lymphocytes over immune-complex columns. When PBL were treated with immune complexes for 16 h at 37°C, their FcR activity was sharply decreased (modulation), as indicated by a significantly reduced capacity of the treated cells to display antibody-dependent cytotoxicity (ADCC). Modulation had variable effects on natural cytotoxicity. In contrast, the virus-dependent cytotoxicity above the natural cytotoxicity remained essentially unchanged, indicating that a functionally intact FcR is not required in this system for carrying out cytolysis.     

Antibody-dependent Lymphocyte-mediated Cytotoxicity in Man: No Requirement for Lymphocytes with Membrane-bound Immunoglobulin

Normal human lymphocytes, depleted of B-lymphocytes by passage through nylon-wool columns, manifested intact antibody-dependent cell-mediated cytotoxicity. Similar findings were made with lymphocytes from four hypo-gammaglobulinaemic patients lacking B-lymphocytes. It is concluded that antibody-induced cell-mediated cytotoxicity in man is independent of B-lymphocytes, as identifiable by membrane-bound immunoglobulin.     

The Role of Viral Glycoproteins in Mumps-Virus-dependent Lymphocyte-mediated Cytotoxicity in Vitro

Human peripheral blood lymphocytes (PBL) from healthy donors express enhanced natural cytotoxicity to target cells after a brief exposure 10 mumps virus in vitro, We describe here experiments aiming at elucidating the mechanism of this virus-dependant cytotoxicity. Treatment with proteolytic enzymes resulted in virus particles depleted of one or both kinds of their glycoprotein spikes. Removal of both of these components from the virion abrogated their ability to enhance cytotoxicity. This virus-dependent cytotoxicity was significantly but not completed reduced when one of the spike glycoproteins (gp 75, HANA) was removed selectively. Similarly, nucleic-acid-free preparations of the spikes, obtained by detergent treatment of mumps virions, also dialed enhanced cytotoxicity. However, the activity of these preparations was lower than that of untreated virions Further evidence for the importance of II AN A was provided by the use of F(ab')₂ fragments of anti-HANA-specific rabbit antibodies. When these fragments were allowed to react with virus before addition of the virus to PBL. no augmentation of cytolysis was observed. Antibody fragments specific for the other spike protein (gp 61, F) failed to inhibit the virus-dependent enhancement of PBL-mediated cytotoxicity. However, anti-HANA and anti-F blocked this reaction when added directly to the mixture of virus-treated PBL and target cells. The results are compatible with the hypothesis that virus-dependent cytotoxicity requires HANA for anchoring the virus to PBL receptors (and perhaps to bring effector and target cells into closer contact), whereas F may be involved in subsequent events increasing effector cell function.     

Characterization and Possible Mechanisms of Mitogen-induced Cell-mediated Cytotoxicity

Mitogen-induced cell-mediated cytotoxicity (MICC) of human peripheral blood granulocytes (PMN) and lymphocytes (PBL) was studied by using chicken erythrocytes as targets. The possible mechanisms were elicited bidirectionally by means of the MICC inhibition test and by functional analysis of lectin. In our preliminary data it was noted that PMN-mediated cytotoxicity was more potent than that of PBL. In addition, erythrophagocytosis or lysosomal enzymes released from PMN offered little contribution to cytotoxic activity of PMN. From the inhibition study it was realized that intact cytoskeletal structures, functionally preserved membrane lectin receptors, active DNA synthesis, and environmental divalent cations were mandatory for the full expression of MICC activity by both effectors. We also identified the cytotoxic activity of PHA-P as having a unique character that is thermostable and independent of the other biological activities.     

Cytotoxicity ofEchinococcus granulosus cyst fluid in vitro

In a cytotoxicity assay, using rat spleen cells as target cells,Echinococcus granulosus cyst fluid from cattle exerted a marked degree of cytotoxicity in vitro. When trypan blue exclusion or [³H]thymidine incorporation by concanavalin A stimulated spleen cells was used as a measure of cell viability, dialyzed cyst fluid showed maximum cell destruction up to a 1:8 dilution. The effect was dose and time dependent, cells being affected by 24 h after exposure to cyst fluid. The components responsible for cytotoxicity of cyst fluid were heat stable and could be recovered using gel chromatography on Sephadex G 50 and G 15 as a single low molecular weight fraction. It is assumed that the toxic products released by the living parasite can readily penetrate through the cyst wall into the surrounding host tissue. The interference of such substances with immunocompetent cells might account for the long-term survival of the parasite in the intermediate host.Dedicated to Prof. Dr. G. Piekarski on the occasion of his 70th birthday     

Lymphocyte-mediated cytotoxicity in isoniazid-associated hepatitis.

The release of lymphotoxin (LT) from peripheral blood lymphocytes of patients with isoniazid (INH)-induced hepatitis was studied, using L929 fibroblast target cells, as was the cytotoxic effect of these lymphocytes on murine hepatoma cells (L1469) and L929 fibroblasts, using a 3H-proline cytotoxicity assay. Evidence for LT release was found in five out of six patients, following stimulation of the peripheral blood lymphocytes with INH or isonicotinic acid (INA) conjugated to human serum albumin. In the direct cytotoxicity assay, cytotoxic effects on the hepatoma cells were enhanced by preincubation of the target cells with INH in five out of six patients tested. Although specificity with regard to the drug was demonstrable, tissue specificity was less certain in that enhanced killing of the fibroblast cell line was also found to occur following preincubation of the L929 cells with INH.     

磁性复合骨水泥的体外细胞毒性

背景：磁性复合骨水泥作为一种新型骨转移治疗材料,不仅能对骨缺损进行修复,还能在交变磁场下升温杀死骨肿瘤。 目的：检测磁性复合骨水泥对小鼠L929成纤维细胞增殖活性的影响及细胞毒性反应。 方法：采用CCK-8法检测含0%(即磷酸钙骨水泥组),10%,20% Fe3O4的磁性磷酸钙骨水泥浸提液及含0%(即聚甲基丙烯酸甲酯骨水泥组),10%,20%Fe3O4的聚甲基丙烯酸甲酯骨水泥浸提液对对数生长期小鼠L929成纤维细胞的毒性作用,每种浸提液又分为50%与100%两个浓度梯度;以正常培养基培养的小鼠L929成纤维细胞作为阴性对照组。 结果与结论：①在100%浓度梯度下：除含20%Fe3O4的磁性聚甲基丙烯酸甲酯骨水泥组细胞数量较少、细胞形态异常外,其余磁性聚甲基丙烯酸甲酯骨水泥组细胞生长状态良好;含10%Fe3O4的磁性磷酸钙骨水泥组培养24 h时表现出轻度细胞毒性,毒性2级;含20%Fe3O4的聚甲基丙烯酸甲酯骨水泥在24-  72 h为表现出轻、中度细胞毒性,毒性为2,3级。各磁性磷酸钙骨水泥组细胞数量及形态正常。②在50%浓度梯度下：各组细胞形态均正常,细胞毒性为0级或1级。表明磁性聚甲基丙烯酸甲酯骨水泥具有轻、中度细胞毒性,磁性磷酸钙骨水泥细胞相容性良好,基本无细胞毒性。     

Mechanisms of Cell-mediated Cytotoxicity in Mice Rejecting Xenogeneic Human Lymphoblastoid Cells

The in vivo immunization of mice with human lymphoblastoid cell line LHN13 generates direct cell-mediated cytotoxicity by spleen cells. The lytic activity appears as early as day 3 after the intraperitoneal inoculation of 7.5 × 10⁵ cells and persists at least until day 11. The killer cells do not adhere to plastic and are not retained on nylon wool columns or on Degalan heads coaled with mouse Ig plus rabbit-anti-mouse Ig The effector cells are partly inhibited by treatment with anti-Thy-1.2 serum plus complement, but this inhibition appears to be non-specific since antiserum alone or normal mouse serum plus complement have the same effects. Heat-aggregated IgG strongly inhibit cytotoxicity, indicating that the effector cells are Fc-positive and that such receptors are implicated in lysis. Altogether, these features strongly argue for an ADCC phenomenon. The involvement of antibodies is demonstrated by the fact that eluates (56°C, 30 min) from immune cells alone induce lysis in the presence of normal spleen cells as effectors The lytic activity of these eluates can be removed by specific adsorption on protein A coupled to Sepharose heads and on the human lymphoid target cells. Positive complementation between immune and non-immune spleen cells suggests that the arming process may occur in vitro during the assay, when antibodies are released by plasmacytes     

Heparinized polyurethanes: in vitro and in vivo studies

Heparin immobilization chemistry using alkyl spacer arms was adapted to optimize yield on polyurethane (PU) surfaces. The resultant biological activity of immobilized heparin (HI) was examined in vitro and in vivo, and compared with a heparin releasing (HR) system. Immobilized heparin retained its ability to bind and inactivate thrombin and Factor Xa; nonspecific coagulation factor binding was insignificant. Such activity cannot be attributed to the leakage of improperly bound heparin. Immobilized heparin-polyurethane catheters implanted in canine femoral and jugular veins for 1 h periods exhibited significant reduction in thrombus formation compared with untreated PU contralateral controls. Polyurethane catheters coated with a 9% heparin dispersion in PU (HR) system provided even greater improvement in antithrombogenicity.     

Lymphocyte-mediated modification of blood-derived macrophage function in vitro; inhibition of growth of intracellular mycobacteria with lymphokines

Evidence is presented, based on in vitro model-systems, that lymphokine-like agents and lymphocytes can elicit in vitro changes in macrophages which replicate those observed in vivo associated with cell-mediated immunity.

(1) Supernatants (containing lymphokines; filtered or unfiltered) from mixed leucocyte cultures (MLC) from two genetically different rabbits activated rabbit blood-derived macrophages cultured in vitro. Activation comprised proliferation, presence of intracellular phase-lucent vacuoles, elongation and formation of intercellular cytoplasmic bridges and giant cells. No such activation was obtained with supernatants from unmixed leucocyte cultures.

(2) Macrophages cultivated in MLC supernatants, but not unmixed leucocyte supernatants, inhibited the intracellular multiplication of mycobacteria, including the vole bacillus and Mycobacterium lepraemurium.

(3) Similar macrophage activation was obtained in cultures of blood-derived macrophages exposed to M. leprae in vitro from patients with tuberculoid leprosy (high resistant form) in the presence of lymphocytes. No such activation was obtained in absence of lymphocytes. Under similar conditions no activation was observed in cultures of macrophages from patients with lepromatous leprosy (low resistant form of the disease).

     

Immunologic Effector Mechanisms of a Standardized Mistletoe Extract on the Function of Human Monocytes and Lymphocytes in vitro, ex vivo, and in vivo

Even though mistletoe extracts have been in clinical use for centuries their exact mode of action is still unknown. Currently, the application scheme for registered preparations is a dose-escalating scheme to thus reduce side effects. In this study, healthy controls and patients were evaluated for their immunologic response to treatment with a standardized mistletoe extract (Iscador). It shows a strong effect as adjuvant that induces TNF-α and IL-12, which was partly mediated via CD14. Desensitization of the TNF-α response could be shown after repeated application in vitro and in vivo. Furthermore, Iscador induces a specific lymphocyte sensitization upon multiple injections and production of IgG₁- and IgG₃-mistletoe antibodies. Remarkably, a systemic bystander effect (heterologous immunity against other recall antigens) was observed after long-term treatment. In conclusion, dose-escalation reduces the monocyte-related clinical side effects. A T-lymphocyte sensitization stimulates mainly a specific Th1 response. The most interesting clinical long-term effect is the bystander stimulation of various memory T cells that might mediate in vivo antitumor and antiinfectious T-cell response under mistletoe-extract immunization.     

Sex-limited protein: in vitro and in vivo functions.

Mouse complement component C4 exists in two isoforms, C4 and a protein with expression restricted to male animals called sex-limited protein (Slp). Although Slp is about 95% homologous to C4, it is generally believed to be non-functional, at least in conventional haemolytic complement assays. In a previous study, however, we showed that Slp is haemolytically active in a C1-inhibitor (C1INH)-regulated, EDTA-resistant mouse complement activation pathway. To study other possible implications of this finding, we generated constitutively expressing Slp-transgenic mice. The transgene was crossed into otherwise Slp-deficient C57Bl/6J and NZB mice. Members of the third backcross generation of C57Bl/6J mice were tested for functional Slp and classical and alternative complement pathway activities (CH50 and AP50 levels, respectively). Slp-transgenic C57Bl/6J mice showed enhanced CH50, but normal AP50 levels when compared with non-transgenic littermates. To discover a possible protective role for Slp in spontaneous systemic lupus erythematosus (SLE) in NZBxNZW (NZBxW) mice, the third backcross generation of Slp-transgenic NZB mice was mated with NZW mice and the development of SLE in the female offspring was followed. In these introductory experiments, Slp-transgenic NZBxW animals presented with a significantly extended life span. Our results imply that Slp is a mouse complement component with functions which partially resemble some of those of human C4A.