Neural progenitor and hemopoietic stem cells inhibit the growth of low-differentiated glioma.

The effects of neural progenitor and hemopoietic stem cells on C6 glioma cells were studied in in vivo and in vitro experiments. Considerable inhibition of proliferation during co-culturing of glioma cells with neural progenitor cells was revealed by quantitative MTT test and bromodeoxyuridine incorporation test. Labeled neural progenitor and hemopoietic stem cells implanted into the focus of experimental cerebral glioma C6 survive in the brain of experimental animals for at least 7 days, migrate with glioma cells, and accumulate in the peritumoral space. Under these conditions, neural progenitor cells differentiate with the formation of long processes. Morphometric analysis of glioma cells showed that implantation of neural progenitor and hemopoietic stem cells is accompanied by considerable inhibition of the growth of experimental glioma C6 in comparison with the control. The mechanisms of tumor-suppressive effects of neural and hemopoietic stem cells require further investigation.

Discrepancy between direct and antibody-dependent cytotoxic activities of human LAK cells.

Human lymphokine-activated killer (LAK) cells display cytotoxic activity against natural killer (NK)-resistant tumor cells in an antibody-independent and -dependent manner. We compared LAK cell-mediated antibody-independent cytotoxicity (LAK activity) and antibody-dependent cellular cytotoxicity (ADCC) against untreated and antibody-coated Raji cells, respectively. Human lymphocytes showed drastically increased LAK activity after stimulation with interleukin-2 (IL-2) for 3 or 7 days when compared to non-activated cells. The level of ADCC was reduced for 3-day-generated LAK cells and augmented for 7-day-generated LAK cells as compared to non-activated cultured lymphocytes. Phenotypical analysis revealed IL-2-induced up-regulation of the proportion of CD11b+ (but not CD16+) lymphocyte subpopulation in 7-day-generated LAK cells. The data imply that human LAK cells exhibit antibody-dependent and -independent cytotoxic activities via distinct effector pathways at different stages of generation. These stages may be associated with changes in adhesion molecule (CD11b/CD18) expression on the surface of IL-2-activated lymphocytes.

Glucosaminylmuramyldipeptide-induced changes in phenotype of melanoma cells result in their increased lysis by peripheral blood cells.

Flow cytometry was used to show that biologically active N-acetylglucosamine-containing muramylpeptides (GMPs) induced in vitro dose-dependent increase in the expression of tumor-associated antigens (TAAs) characteristic for colon and mammary gland carcinomas, melanoma and lung adenocarcinoma. Forty to two hundred percent enhancement in TAA-expressing cells was observed after 18-48 h incubation with GMPs. In contrast, MHC class I antigen expression was not altered. Using MTT and chromium-release assays, melanoma cells treated in vitro with GMDP were shown to be more susceptible to killing by peripheral blood cells of healthy donors than non-treated cells. Fractionation of blood cells revealed that platelets were responsible for this effect.

Down-regulation of LAK cell-mediated cytotoxicity: cancer and ageing.

A comparative study was made of the generation of lymphokine-activated killer (LAK) cells in patients with melanoma and healthy donors of different age groups. Significant reduction of effector cell cytotoxicity in patients following 72 h culture with 1,000 U/ml or recombinant IL-2 (rIL-2) as well as a decreased ability to generate LAK cells in elderly individuals were shown to be correlated with suppressor cell activation in rIL-2 stimulated cell population. Suppressor effect depends on monocytes and T-lymphocytes: partial abolition of suppression in LAK cells was observed following removal of adherent cells or treatment with OKT8 monoclonal antibodies and complement.

Activation of secretion and surface alteration of cytolytic T-lymphocytes interacting with target cells.

Cells obtained in mixed lymphocyte culture (MLC) and memory cells adsorbed on the surface of target cells (TC) were examined using scanning and transmission electron microscopy depending on the time of interaction with TC. Three types of lymphocytes were revealed: type I - cells of spherical shape with a smooth surface or an insignificant amount of microvilli; predominantly small and medium-sized lymphocytes contacting TC with non significant involvement of their surface or by several microvilli; type II - oval or round-shaped lymphocytes evenly covered with microvilli with considerably enlarged region of contact; type III cells - predominantly large lymphocytes and lymphoblasts flattened (spread) on TC, with multiple microvilli, ridge-like projections, and ruffles on their surface. TEM revealed activation of the secretory apparatus in the cytoplasm of such lymphocytes. With increased time of interaction, type III cells increase in number (from 8.6% after 10 min to 90.2% after 60 min of incubation). Memory cells show no morphologic signs of secretion in correlation with the absence of lysis of TC on which they are adsorbed. The surface of the lymphocytes adsorbed on the substrate with poly-L-lysin is not noticeably altered. It is suggested that 3 morphological types of lymphocytes correspond to 3 stages of secretion activation. Lymphocyte contact with TC surface is evidently a specific stimulus for activating secretory apparatus of CTL. SEM can be used for quantitation of activated lymphocytes.

Platelet-mediated cytotoxicity and its enhancement by platelet activating factor.

Platelet cytotoxicity was assessed in 70 cancer patients with various tumor localizations and in 30 normal donors. The data presented reveal that the ACL cell line displays the highest sensitivity to platelet cytotoxicity. Using the ACL cells, we discovered that platelets from oncological patients and normal donors display comparable cytotoxicity. The level of platelet lytic activity is irrelevant to tumor localisation; however, it appears to be dependent on the stage of tumor growth. Incubation of platelets, both from donors and patients, with PAF (concentration range 10 pM to 10 nM) results in a significant rise of the killing activity of platelets. PAF induces greater cytotoxicity enhancement for platelets with lower initial activity, this pattern appearing to be the specific feature of the PAF mediated effect. Hence, platelets can be considered as effector cells relevant to antitumor immunity; PAF-mediated enhancement of platelet cytotoxicity can appear to be useful in the search for new immunotherapeutic drugs.

Proliferative activity, lectin-dependent and natural cytotoxicity in blood, lymph node and spleen from patients with Hodgkin's disease.

Mononuclear cells and T-lymphocytes of the blood, spleen and lymph nodes from 48 patients with Hodgkin disease (HD) and blood donors were tested in assays for lectin-dependent (LD) and natural killer (NK) cytotoxic activity. On average, peripheral blood T cell lectin-dependent cytotoxicity differs from that of the donors. However, cytotoxic activity appears to be dependent on the stage of disease; in the IY stage LD cytotoxicity was decreased 2-fold. The lectin-dependent cytotoxicity was also dependent on the histological type of disease and the lowest level (50% of the control level) was associated with the lymphoid depletion type. The cytotoxic activity of T-lymphocytes from the affected areas of the patients' spleen was more marked than that of the unaffected areas. Spleen cell cytotoxicity showed no other correlations. Cytotoxicity of lymphocytes from the affected lymph nodes was drastically lower than activity of blood and spleen lymphocytes. NK activity of the patients' blood and spleen lymphocytes was twice as low as the control level (healthy donors) and did not correlate with stage and/or histological type of disease. The proliferative activity of lymphocytes from 33 HD patients was tested in vitro using allogeneic mononuclear cells from healthy donors or HD patients and/or PHA as stimulators. The response of patients' lymphocytes to alloantigens appeared to be much less affected than response to polyclonal mitogen. Thus, the results obtained by us demonstrate signs of stimulation of the lymphoid system against a background of general immunosuppression in HD.

Treatment with interleukin-2 in an immunodepressed state.

Cytotoxic T-lymphocyte (CTL) and lymphokine activated killer (LAK) cell (fraction II) precursors with a density of 1.077-1.067 g/ml and suppressor cells (fraction I) with a density of 1.067-1.056 g/ml were isolated by the separation of cancer patient peripheral blood mononuclear cells (MNC) on a Percoll gradient. Cells from fraction I inhibited the generation of CTL in mixed lymphocyte-tumor culture (MLTC) and LAK cells when added to fraction II lymphocytes at a ratio of 1:1 at the beginning of the culture. The effect was dependent on the dose of added suppressor cells and resistant to mitomycin-C treatment. Treatment of cell fractions prior to culture with monoclonal antibodies and complement showed that CTL precursors and suppressor cells were OKT3+/OKT8+. Cells from fraction I possessed suppressor activity in all patients examined but only in 4 of 10 healthy donors. Studies of monocytes and T-lymphocytes isolated from fraction I demonstrated that in cancer patients both monocytes and T-lymphocytes functioned as suppressors whereas in healthy donors, the monocytes mediated suppression. The data obtained provide evidence for an increased suppressor cell activity in cancer patients which can inhibit the generation of cytotoxic antitumor response with interleukin-2 (IL-2) in vitro.

The influence of tumor immunity suppressors on the effector stage of human and animal lymphokine-activated killer cells.

Spleen cells of tumor-bearing mice suppressed the cytolytic activity of syngeneic LAK cells when added to the mixture of LAK cells and target cells at the beginning of the cytotoxicity test. Spleen cells of MC 14 tumor-bearing mice acquired the suppressor potential as early as 10 days after tumor transplantation; the suppressor activity in the EL 4 and X63-Ag8.653 tumor-bearing animals was first revealed at the 30th day and manifested itself up to the 120th day. The suppressor activity was expressed in a dose-dependent manner, both by unfractionated spleen cells and nylon wool-passed and plastic-adherent sub-populations. Similar results were obtained during the analysis of anti-tumor immunity suppressors in bladder cancer patients. MNC, nylon wool-passed and plastic-adherent cells of patients with stages I-II disease suppressed the cytotoxicity of autologous LAK cells in 2/6 cases; all patients [4] with III-IV stage possessed such suppressor activity. Presumably, the tumor growth induces the activity of suppressor T cells and monocytes/macrophages. The suppressor activity can interfere with the antitumor effect of autologous (syngeneic) LAK cells at the effector stage.

Artificial aging of mice.

Clinical signs of aging verified by morphometrical analysis of brain tissue were observed in young mice 4 months after administration of brain extract from old mice (5 intraperitoneal injections).

Recombinant interleukin-2 significantly increases the survival of mice with peritonitis, but not acute Staphylococcus aureus peritoneal infection.

The effect of recombinant interleukin 2 (rIL-2) on survival of mice with peritonitis and acute Staphylococcus aureus strain 5/2 infection was studied. rIL-2 was ineffective in the case of acute infection when administered simultaneously with LD95 dose of bacteria. The antibiotics (gentamycin or a combination of penicillin and streptomycin) administered in the same fashion cured 100% of animals. rIL-2 proved to be a potent healing agent in the two of three models of S aureus peritonitis. In this case animals received bacteria at days 0 and 2, 4, or 6. rIL-2 was injected at day 0 (group 1), days 0 and 2 (group 2), and days 0, 2, and 4 (group 3). Treatment with rIL-2 was ineffective in group 1; however, in groups 2 and 3 rIL-2 increased the survival up to 90% (in comparison with 30% in the untreated animals of group 2 and 64% in group 3). On the contrary, administration of antibiotics instead of rIL-2 in the group 3 decreased survival to 25%. The perspectives of rIL-2 use in the treatment of bacterial peritonitis, including purous ones, and the cases complicated by immunodepression, are discussed.

Block copolymers of ethylene oxide and propylene oxide (pluronics) as immunomodulators and antitumour agents.

Block copolymers of ethylene oxide and propylene oxide (pluronics) are nontoxic water-soluble membranotropic surfactants available as polymers with various compositions, molecular masses, number, and arrangement of blocks. In vivo experiments are reported which demonstrate that these polymers and their functional derivatives stimulate the production of anti-sheep-erythrocyte antibodies in mice. The introduction of reactive (hydroperoxide) groups into the polymers by chemical modification or by solubilization of low-molecular-mass hydroperoxides alters the properties of these immunostimulators. In vitro experiments revealed that these modified polymers enhance the activity of natural killer cells without reducing their viability. It is proposed that the immunomodulatory properties of pluronics and their derivatives play an important role in the antitumour activity of these substances in vivo.