Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with³H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.     

Cytotoxicity of mouse peritoneal cells after alloimmunization]

CBA Mice were immunized by two intraperitoneal injections of 30 X 10(6) DBA/2 or C57BL/6 spleen cells at days--12 and--2. Peritoneal cell population was obtained at day zero by washing the peritoneal cavity of Mice. Adherent cells were then separated using a 2 hrs. incubation in "Falcon" plates followed by washing. This macrophage-rich peritoneal cell population was found nonspecifically cytotoxic against 51Cr labeled tumoral target cells: P815 X DBA/2 mastocytoma cells, EL4 X C57BL/L lymphoma cells and spontaneous lymphoma AKR cells (same H--2k as CBA). This adherent peritoneal cell cytoxicity was demonstrated after 24 hrs. incubation with the target cells. It was found in nonspecific combination as well as when using target cells syngeneic to the donor. These findings suggest that adherent peritoneal cell cytotoxicity could be at least partly due to macrophages and result from factor (s) released by sensitized lymphocytes in vivo in the same way as has been previously demonstrated in vitro.     

Functional inhibition of isolated pancreatic cells, new technic for the detection of macrophage cytotoxicity]

It is usually accepted that macrophages "activated" by lymphokines may be found cytotoxic against tumoral target cells but show no detectable cytotoxicity in in vitro tests using normal non tumoral cells as target cells. These data have been obtained mainly with the chromium-release test. The present paper describes a new test using normal isolated pancreatic cells as target cells and evaluating the effect of activated or non-activated macrophages on the insulin secretion response to glucose stimulation. The results show a striking decrease in this response following an 18-hr incubation of pancreatic islet cells with activated macrophages, as compared to that of the same cells incubated with control macrophages. This is clear evidence that activated macrophages may alter normal cells and suggests that their cytotoxic properties are not restricted to tumoral target cells.     

Identification of cytotoxic cells responsible for lysis of target cells pretreated with concanavalin A in spleen populations of pregnant mice with suppressive activity]

During an alloimmunization, killer cells which lyse target cells only in the presence of a lectin are generated. That these cells, as well as suppressive cells, share immunocytological properties with specific killer cells, leads to the hypothesis that these cells may be concerned with the mechanism of immunosuppression. Two experimental results presented in this paper are consistent with this hypothesis: 1) Spleens from H-2k mice pregnant by H-2d males which bear a high suppressive activity also contain a relatively large number of killer cells having the ability to lyse Concanavalin A treated target cells and 2) supernatants of suppressive systems generated through an MLC block the cytolysis of specific target cells by the bound killer cells.     

Cytotoxicity of human T-lymphocytes sensitized by Epstein-Barr virus. Role of HLA antigens at the surface of target cells infected by the virus]

Peripheral T-lymphocytes from patients with infectious mononucleosis are cytotoxic towards target cells from Epstein-Barr virus-genome carrying human lumphoblastoid lines. Thus, these T-cells appear to be sensitized to viral coded determinants. Daudi cells, that carry EBV-genome, but lack HLA antigens are resistant to specific cytolysis in this model. These results suggest direct HLA involvement in the target structure recognized by birus-sensitized cytolytic T-lymphocytes in human.     

Differentiation and tumorigenicity of human malignant melanocytes. Comparative study of two "non pigmented" and pigmented lines from the same fibroblastoid cells]

A morphological, caryological and biochemical study of two established cell lines of human malignant melanocytes derived from the same original tumour, when compared with heterotransplantation results in nude Mice, showed that "non-pigmented" cells are more tumorigenic than pigmented cells. Differentiation and tumorigenicity, although concomitant, here seem to be two independent phenomena.     

Transformation, in vitro, of cerebral hamster cells by polyoma virus]

A cell line called HCxPy was obtained in vitro by transformation of dissociated hamster brain cell cultures by polyoma virus. The first foci of transformed cells became evident 90 to 120 days after viral infection. This cell line is now at the 46th passage. The cells appear tumorigenic for hamsters after subcutaneous and intracerebral injection. They carry the polyoma virus T and cell surface antigens. Good evidence for astrocytic differentiation can be found by morphological examination of the tumours and of the cultured cells.     

In vitro stimulation of chicken pituitary growth hormone and prolactin secretion by chicken hypothalamic extract

Summary The effect of an acid extract of chicken hypothalami on the in vitro secretion of prolactin and growth hormone (GH) by dispersed chicken pituitary cells has been investigated. Both prolactin and GH release were stimulated in a dose related manner in the presence of the hypothalamic extract (HE). Somatostatin had no effect on the basal or HE stimulated release of prolactin although it did inhibit the HE induced release of GH.     

Inflammatory factors produced by sensitized guinea-pig peripheral blood lymphocytes

Summary The supernatants obtained from stimulated tuberculin-sensitive guinea-pig peripheral blood lymphocytes contain factors that induce a cutaneous inflammatory response in normal guinea-pigs similar to the tuberculin reaction and inhibit the migration of normal guinea-pigs peritoneal exudate cells. There appears to be a correlation between the presence of in vitro migration inhibitory activity and inflammatory activity in vivo.     

Activation of CD47 receptors causes histamine secretion from mast cells

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcɛRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the βγ dimer of a G_i protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G_i protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G_i protein complex. Received 8 December 2008; received after revision 12 January 2009; accepted 29 January 2009     

In vitro stimulation of chicken pituitary growth hormone and prolactin secretion by chicken hypothalamic extract.

The effect of an acid extract of chicken hypothalami on the in vitro secretion of prolactin and growth hormone (GH) by dispersed chicken pituitary cells has been investigated. Both prolactin and GH release were stimulated in a dose related manner in the presence of the hypothalamic extract (HE). Somatostatin had no effect on the basal or HE stimulated release of prolactin although it did inhibit the HE induced release of GH.     

In vitro morphological characterization of the bursal reticuloepithelial (REp) cells of chicken.

The REp cells of the bursa follicle medulla of chicken were isolated in vitro. Culture of the REp cells was maintained over a period of 10 days and the cells were observed at 3 and 10 days by means of transmission electron microscopy (TEM) and immunofluorescence. The use of an anticytokeratin monoclonal antibody confirmed their epithelial nature. TEM observations showed the presence of desmosomes and tonofilaments, which are characteristic of epithelial cells. Furthermore, to some extent the cells regenerated in vitro the network they form in vivo. Though the growth rate becomes slower with time, the features of the REp cells do not significantly change.     

Lysophosphatidic acid up-regulates vascular endothelial growth factor-C and lymphatic marker expressions in human endothelial cells

Lysophosphatidic acid (LPA) is a low-molecular-weight lipid growth factor, which binds to G-protein-coupled receptors. Previous studies have shown that LPA enhances vascular endothelial growth factor-A (VEGF-A) expression in cancer cells and promotes angiogenesis process. However, the roles of LPA in lymphatic vessel formation and lymphangiogenesis have not been investigated. Here, we demonstrated that LPA up-regulated VEGF-C mRNA and protein expressions in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression levels of lymphatic markers, including Prox-1, LYVE-1 and podoplanin, were enhanced in LPA-stimulated tube forming endothelial cells in vitro and in vivo. Moreover, we showed that pretreatment with MAZ51, a VEGFR-3 kinase inhibitor, and introduction of VEGFR-3 siRNA suppressed LPA-induced HUVEC tube formation and lymphatic marker expressions. These results demonstrated that LPA enhances expression of lymphatic markers through activating VEGF-C receptors in endothelial cells. This study provides basic information that LPA might be a target for therapeutics against lymphangiogenesis and tumor metastasis.     

Reverse signaling using an inducible costimulator to enhance immunogenic function of dendritic cells

A costimulatory signal from an inducible costimulator (ICOS) of T cells plays a critical role in immunological homeostasis. This study shows that the interaction of ICOSIg and its ligand (ICOSL) on mouse bone marrow-derived dendritic cells (DCs) induces a p38-MAPK dependent elevation of interleukin 6 (IL-6). It also enhances phagocytosis and the antigen-presentation function of DCs in vitro, further favoring cell-mediated immunity in vivo. As seen for other types of costimulator molecules expressed in the T cells in the CD28 family, it is shown here for the first time that ICOS can also deliver reverse signals through its ligand to ICOSL-expressing cells. These reverse signals in turn transfer positive immunogenic information to bone marrow-derived DCs. Our work therefore provides new recognition of an ICOSL/ICOS signal pathway in immunity and also supplies more evidence that this ICOSL/ICOS signal pathway is a reasonable target for therapeutic drugs.     

In vitro morphological characterization of the bursal reticuloepithelial (REp) cells of chicken

Summary The REp cells of the bursa follicle medulla of chicken were isolated in vitro. Culture of the REp cells was maintained over a period of 10 days and the cells were observed at 3 and 10 days by means of transmission electron microscopy (TEM) and immunofluorescence. The use of an anticytokeratin monoclonal antibody confirmed their epithelial nature. TEM observations showed the presence of desmonsomes and tonofilaments, which are characteristic of epithelial cells. Furthermore, to some extent the cells regenerated in vitro the network they form in vivo. Though the growth rate becomes slower with time, the features of the REp cells do not significantly change.     

Influenza infection induces host DNA damage and dynamic DNA damage responses during tissue regeneration

Influenza viruses account for significant morbidity worldwide. Inflammatory responses, including excessive generation of reactive oxygen and nitrogen species (RONS), mediate lung injury in severe influenza infections. However, the molecular basis of inflammation-induced lung damage is not fully understood. Here, we studied influenza H1N1 infected cells in vitro, as well as H1N1 infected mice, and we monitored molecular and cellular responses over the course of 2 weeks in vivo. We show that influenza induces DNA damage to both, when cells are directly exposed to virus in vitro (measured using the comet assay) and also when cells are exposed to virus in vivo (estimated via γH2AX foci). We show that DNA damage, as well as responses to DNA damage persist in vivo until long after virus has been cleared, at times when there are inflammation associated RONS (measured by xanthine oxidase activity and oxidative products). The frequency of lung epithelial and immune cells with increased γH2AX foci is elevated in vivo, especially for dividing cells (Ki-67-positive) exposed to oxidative stress during tissue regeneration. Additionally, we observed a significant increase in apoptotic cells as well as increased levels of DNA double strand break (DSB) repair proteins Ku70, Ku86 and Rad51 during the regenerative phase. In conclusion, results show that influenza induces DNA damage both in vitro and in vivo, and that DNA damage responses are activated, raising the possibility that DNA repair capacity may be a determining factor for tissue recovery and disease outcome.     

In vitro malignant transformation of fetal hamster brain cells by methylnitrosourea]

Using primary cultures originating from 14 day old fetal Hamster brain, we have obtained a cell line with glial morphology. These cell remain non transplantable during the first year of in vitro culture, but undergo spontaneous transformation during the second year. Following prolonged contact of the glial-like cells with 25 to 50 microgram/ml of methylnitrosourea (MNU), a malignant transformation is observed 5 months after the treatment. The lowest concentrations of (MNU) do not cause malignant transformation, but seem to inhibit (or postpone) the spontaneous transformation. After MNU treatment, cells retain their glial nature.     

Human peripheral blood monuclear cells transfected with messenger RNA stimulate antigen-specific cytotoxic T-lymphocytes in vitro

The efficiency of test vaccines needs to be evaluated by quantification of the triggered cellular immune response. Usually, for these assays, autologous target cells expressing the vaccine antigen are required. In the context of messenger RNA (mRNA)-based vaccinations, the target cells used for the read-out are mRNA-transfected monocyte-derived dendritic cells (Mo-DCs). Their production typically requires samples of 100 ml blood from the patients, and limits the number of assays that can be performed. We show here that fresh peripheral blood mononuclear cells (PBMCs) can be transfected with mRNA by electroporation. Such cells are as efficient as mRNA-transfected Mo-DCs for their ability to activate memory T cells in vitro. Thus, mRNA-transfected PBMCs are a convenient replacement of mRNA-transfected Mo-DCs for the in vitro monitoring of natural or vaccine-induced immune responses.Received 17 February 2005; received after revision 1 May 2005; accepted 7 Juni 2005     

Agglutination of leukemic cells and daunomycin entrapped erythrocytes with lectin in vitro and in vivo

Wheat germ agglutinin (WGA) agglutinated the L1210 leukemic cells and daunomycin entrapped erythrocytes in vitro. Comparing with control preparations, the greatest increase in survival was obtained in vivo when the daunomycin entrapped erythrocytes and WGA were given to BDF1 mice bearing L1210 cells.