Immune function in mice lacking the perforin gene.

Mice lacking the perforin gene were generated by using targeted gene disruption in embryonal stem cells. When infected with lymphocytic choriomeningitis virus (LCMV), perforin-less (-/-) mice showed clear signs of having mounted an immune response based on activation of CD8 T cells but were unable to clear the LCMV infection. This failure to eliminate virus was accompanied by a failure to generate spleen cells capable of lysing LCMV-infected fibroblasts in vitro. Spleen cells from LCMV-infected -/- mice were able to lyse hematopoietic target cells after exposure to phorbol 12-myristate 13-acetate and ionomycin, provided the target cells expressed the Fas antigen. Spleen cells from -/- mice also responded to alloantigen in mixed leukocyte culture by blastogenesis and proliferation. The resulting cells were able to lyse hematopoietic target cells, although not as well as spleen cells from +/+ littermates sensitized in the same manner. However, lysis by -/- cells was again seen only if the target cells expressed Fas antigen. We conclude that perforin-less -/- mice retain and express the Fas lytic pathway as expressed in vitro but that this pathway is insufficient to clear an LCMV infection in vivo.

Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome.

We have developed a method for the introduction of yeast artificial chromosomes (YACs) into transgenic mice. An 85 kilobase (kb) fragment of the human heavy chain immunoglobulin gene was cloned as a YAC, and embryonic stem cell lines carrying intact, integrated YACs were derived by co-lipofection of the YAC with an unlinked selectable marker. Chimaeric founder animals were produced by blastocyst injection, and offspring transgenic for the YAC were obtained. Analysis of serum from these offspring for human heavy chain antibody subunits demonstrated expression of the YAC-borne immunoglobulin gene fragment. Co-lipofection may prove to be a highly-successful means of producing transgenic mice containing large gene fragments in YACs.

Prevention of doxorubicin-induced hematotoxicity in mice by interleukin 1.

Interleukin 1 alpha and interleukin 1 beta induce peripheral neutrophilia with stimulation of granulopoiesis in bone marrow. The continuous administration of interleukin 1 (100 ng/day) to mice for 7 days by s.c.-implanted Alzet osmotic minipumps induced marked stimulation of granulopoiesis in marrow and spleen in normal mice, and protected against the marked depletion of myeloid and erythroid cells in bone marrow of mice treated with single injections of either 20 or 30 mg/kg doxorubicin (DXN). Interleukin 1 beta infusion also protected against DXN-induced atrophy of thymus and secondary lymphoid organs. Single i.p. injection of either interleukin 1 alpha or interleukin 1 beta at doses up to 1000 ng 24 h prior to treatment with DXN did not protect against the hematopoietic and lymphoid toxicities of DXN.

Enhanced antitumor efficacy of adriamycin in combination with interferon-gamma: effects on macrophages.

The combination of the immunomodulator interferon-gamma (IFN-gamma) with the chemotherapeutic drug adriamycin (ADM) was assessed in vitro and in vivo in murine tumor models. When tested in vivo against the murine Lewis lung carcinoma, significantly greater reduction of spontaneous pulmonary metastases was obtained by combination treatment with IFN-gamma, followed 1 day later by ADM. Intraperitoneal ADM treatment also resulted in an increased recruitment of peritoneal mononuclear cells. It is noteworthy that, although the antitumor efficacy was significantly increased by the IFN-gamma/ADM combination treatment, gross toxicity of ADM was not increased. Thus, a net increase in the therapeutic index of ADM was achieved. In vitro, the effects of ADM on the ability of murine peritoneal macrophages, with or without the addition of immunological macrophage activators, to kill tumor cells was studied. Resident macrophages were able to sequester ADM (when present at 10 micrograms/ml) from the medium, and could subsequently mediate killing of target tumor cells. However, incubation of macrophages with low (ineffective by themselves) doses of ADM (1 microgram/ml) prevented their simultaneous or subsequent activation to the tumoricidal state after incubation with the normal macrophage-activating mixture of IFN-gamma plus a muramyl dipeptide (MDP) analog. When the order of addition of reagents was reversed such that the macrophages were preincubated for 24 hr with IFN-gamma (100 U/ml) plus the MDP analog (0.1-10 micrograms/ml), no antagonism of tumoricidal activity was obtained upon subsequent incubation with ADM. There were no interactions between IFN-gamma and ADM on the direct proliferation of tumor cells. Taken together, these results suggest that the enhanced antitumor efficacy of IFN-gamma/ADM combinations in vivo was not due to direct antiproliferative effects on the tumor cells, but rather may be mediated by direct cytotoxicity of ADM on tumor cells enhanced by phagocytic mononuclear cells.

Prophylaxis and treatment of experimental lung metastases in mice after treatment with liposome-encapsulated 6-O-stearoyl-N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine.

A new lipophilic muramyl dipeptide analog, 6-O-stearoyl-N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine, when incorporated in liposomes, was effective in both the prevention and eradication of experimental pulmonary metastases in mice. Multilamellar vesicles composed of synthetic phospholipids (phosphatidylglycerol and phosphatidylcholine) containing saturated myristoyl or unsaturated dioleoyl acyl chains were found to potentiate the antimetastatic activity of this glycopeptide. Prophylactic and therapeutic efficacy was observed against the three murine tumors tested: FSa, an immunogenic fibrosarcoma; NFSa, a nonimmunogenic fibrosarcoma; and B16 melanoma. Neither the administration of empty liposomes or free glycopeptide, nor their coadministration, had a significant antimetastatic effect. This approach is promising for the therapy of cancer metastases in humans, particularly in the prevention of metastatic seeding and in the treatment of micrometastases.

The effect of variations in vitamin C intake on the cellular immune response of guinea pigs.

The uptake of tritiated thymidine by isolated peripheral blood lymphocytes obtained from male guinea pigs immunized with bovine serum albumin was studied in animals maintained on various amounts of Vitamin C for 28 days. Animals were pair-fed on ascorbate-free diet and were supplemented intraperitoneally with 0, 25, or 250 mg Na-ascorbate per day. Scorbutic animals lost weight rapidly during the final 2 experimental weeks. Their daily food intake averaged only 4 g/day during the last week; thus, pair-fed ascorbate-supplemented groups were also subjected to acute nutritional stress. Lymphocytes from guinea pigs receiving 250 mg Na-ascorbate per day incorporated in vitro the highest amounts of tritiated thymidine both in the absence of nonspecific mitogen and in the presence of concanavalin A or phytohemagglutinin. Responses to lipopolysaccharide were not conclusive. Total circulating white cells counts and relative numbers of T and B lymphocytes were assessed in a second study made under identical constraints. In scorbutic animals the percentage of B lymphocytes increased and that of T lymphocytes decreased continuously over the 4-week period. The opposite effect was observed in vitamin C-supplemented animals. These studies suggest that very high doses of ascorbate support elevated mitotic activity after 4 weeks of much reduced food intake.

Cell-mediated cytotoxicity and humoral immune response in ascorbic acid-deficient guinea pigs.

Guinea pigs were divided into three dietary groups: ascorbic-acid deficient, pair-fed, and ad libitum control. Two weeks later guinea pigs were immunized intradermally with 5 x 10(8) chicken erythrocytes in Freund's complete adjuvant. Hemagglutinating antibody titers to chicken erythrocytes 2 weeks after immunization were comparable in all three dietary groups. In vitro 51Cr release from labeled chicken erythrocyte target cells incubated with lymphoid cells from spleens of ascorbic acid-deficient guinea pigs was significantly less than with spleen cells from pair-fed and ad libitum control guinea pigs. The percentage of splenic lymphoid cells that formed rosettes with rabbit erythrocytes, a T cell marker, was the same in all three dietary groups. The defect of ascorbic acid deficiency may reflect an impairment of T lymphocytes function in cell-mediated cytotoxicity or a change in number or function of another cell type.