Mechanisms of Listeria monocytogenes-mediated modulation of tumor growth.

Studies using Listeria monocytogenes as an antitumor agent were initiated to determine the requirements for Listeria-mediated tumor inhibition to occur. When Strain 13 guinea pigs were injected with an admixture of viable Listeria and a methylcholanthrene-induced fibrosarcoma in a ratio of 1 bacterium to 100 tumor cells, Listeria had a marked capacity to inhibit tumor growth. This confirms an earlier study in our laboratory (M. M. Dustoor, A. Fulton, W. Croft, and A. A. Blazkovec, Infect. Immun. 23:54-60, 1979). At no time did animals exhibit overt symptoms of disease as a result of Listeria infection. Animals treated with antilymphocyte serum, which had previously been shown to abrogate T-cell functions, were no longer able to suppress Listeria-tumor cell mixtures. Treatment in vivo with carrageenan, a macrophage-inhibitory agent, also abrogated Listeria-mediated tumor inhibition. These results suggest that Listeria-mediated inhibition requires intact T-lymphocyte and macrophage function. Experiments in which Listeria was given in admixture with the tumor cells or in the opposite flank demonstrated that the antitumor effects require intimate association of the Listeria and tumor cells. Histopathological studies, showing that macrophages and lymphocytes are the predominant inflammatory cells present at sites of tumor destruction, further suggest a role for these cells in Listeria-mediated inhibition. Animals which had rejected prior Listeria-tumor cell inocula were resistant to rechallenge with the homologous tumor for more than 1 year. This work thus confirms in vitro studies demonstrating that both lymphocytes and macrophages are required for Listeria-mediated tumor inhibition to occur. This study demonstrates that viable Listeria can have potent antitumor effects without causing overt disease as a result of Listeria infection.

Antitumor activity of Listeria monocytogenes on a guinea pig fibrosarcoma.

Listeria monocytogenes-mediated tumor inhibition was studied in strain 13 guinea pigs by using a methylcholanthrene-induced fibrosarcoma (MCA-1). Mixtures of Listeria and tumor cells in ratios of 1:100, 1:200, or 1:400 (Listeria:MCA-1 cells) led to significant suppression of tumor growth. Intralesional injection of tumors on day 6 posttransplantation led to the regression of a highly significant number of tumors. Animals receiving injections of Listeria, either in a mixture with tumor cells or intralesionally, displayed enhanced skin test reactivity to a tumor extract. Tumor regressors were resistant for at least 2 to 3 months after the initial transplant to rechallenge with MCA-1 cells. Thus, with this particular tumor-host system, Listeria was successfully employed as an antitumor agent with no visibly detrimental side effects to the host.

Cytolytic activity in vitro of lymph node cells after exposure in vivo to tumor cells suspended in blocking sera.

The in vivo effect of blocking sera (bs) on both tumor growth and subsequent in vitro cytolytic activity of regional lymph node cells was determined following injection of hepatoma cells suspended in normal sera (ns) or bs into the hind footpads of guinea pigs. Tumor growth was unaffected by bs but the primary response to tumor by LNC draining tumor/bs sites was significantly lower in 4/6 experiments as compared to cells from lymph nodes draining tumor/ns sites.

Guinea pig cell-mediated tumor immunity: the chromium release assay detects both cytolysis and serum blocking for syngeneic chemically-induced tumors.

The chromium release test (CRT) was used to assess cell-mediated immunity to syngeneic, chemically-induced tumors in guinea pigs. The animal models were Sewall Wright strain 13 guinea pigs with 3-methyl-cholanthrene (MCA)-induced fibrosarcomas and strain 2 guinea pigs with diethylnitrosamine (DEN)-induced hepatocarcinomas. Regional lymph node cells were significantly cytolytic for the immunizing tumors, specifically so for three of the four tumors, and tumor-bearer sera could significantly block cytolysis. The two DEN-induced strain 2 hepatomas, line 1 and line 10, are antigenically distinct by the CRT but the two MCA-induced tumors have tumor specific antigens as well as a common, cross-reactive antigen.

Acquired cellular resistance, delayed hypersensitivity, and altered macrophage migration in Listeria monocytogenes-infected guinea pigs.

A Listeria monocytogenes infection in guinea pigs was used to study the interrelationship between antigen-induced macrophage migration inhibition, delayed-type hypersensitivity, and acquired cellular resistance. Early after infection (at 2 and 7 days), very significant enhancement of macrophage migration was observed. Migration inhibition was detected beginning on day 14 and was uniformly observed only on day 21 of the infection, after which a shift again to enhancement was seen. The early detection (by day 2) of migration enhancement suggested that this assay may be more sensitive than assessment of delayed type hypersensitivity in vivo, which in this system was first detectable only on day 4. Acquired cellular resistance, as measured by enhanced survival following a high dose challenge with Listeria, was present from day 7 after infection until at least day 60. By splenic clearance studies, however, acquired cellular resistance was present only until day 14 after infection, suggesting that in this system splenic clearance was not a very reliable criterion for measuring acquired cellular resistance.

The importance of the ratio between effector and target cells for detection of serum blocking of tumor lymphocytolysis.

Sera from tumor-bearong guinea pigs were tested for their capacity to influence the magnitude of lymphocytolysis of tumor cells in vitro as measured by 51Cr release. Thirty-three sera were tested in 124 experiments in which the effector to target cell ratio (E:T) was 200:1. Significant blocking of cell-mediated lysis occurred in 62% of the experiments, whereas significant potentiation occurred in 7%. Six sera were found which showed significant blocking in some experiments and significant potentiation in other experiments. One of these sera was further evaluated by varying E:T, using lymphocytes from three immune donors. For each of the three populations of immune lymphocytes, significant blocking was demostrable only at one of three E:T tested. Blocking was observed only with an E:T of 200:1 in two cases. In a third case, blocking was observed at an E:T of 50:1, whereas an E:T of 200:1 gave rise to significant potentiation. The effect of these sera on lymphocytolysis in vitro is clearly dependent upon the E:T ratio.

Ontogenetic aspects of sexual dimorphism and the primary immune response to sheep erythrocytes in hamsters from prepuberty through senescence.

Ontogeny of the primary response to sheep erythrocytes of age-matched groups of male and female hamsters was studied at various chronological ages ranging from prepuberty to senescence. Selected organs were likewise weighed upon sacrifice to obtain developmental patterns. Adrenal weights were higher in the male, and pituitary weights were higher in the female; for both organs typical dimorphism was demonstrable by 36 days. Spleen weight and index favored the female by 46 days. Immunological sex dimorphism first appeared in groups injected at 53 days and autopsied at 58 days and persisted through senescence. Sexual dimorphism of antibody-mediated immunity, previously shown to favor the female in both the primary and secondary immune responses, thus follows the dimorphism of total amount of splenic lymphoid tissue and occurs shortly after realization of sexual maturity in the male. These findings support our previous suggestion of the suppressive effect of androgens on the antibody-mediated immune responsiveness of the male hamster.

Blastogenesis as an in vitro correlate of delayed hypersensitivity in guinea pigs infected with Listeria monocytogenes.

Randomly bred guinea pigs of both sexes were injected intracardially with one-half a 50% lethal dose of Listeria monocytogenes. When these animals were skin tested with 30 mug of a water-soluble extract of sonically disrupted Listeria, animals had uniformly detectable levels of delayed-type hypersensitivity (DTH) 6 days after infection. Histological examination of skin test reaction sites, after fixation in Helly fixative and Giemsa staining, revealed a classical tuberculin-type infiltrate consisting primarily of mononuclear cells with few polymorphonuclear cells. Many of the small vessels showed perivascular cuffing. When purified peritoneal exudate lymphocytes from these animals were cultured in vitro in the presence of various concentrations of Listeria antigen, it was fount that the optimal antigenic dose for specific antigen-induced incorporation of [3H]thymidine varied for individual animals. In contrast to the early onset of uniformly detectable levels of in vivo DTH, in vitro lymphocyte blastogenesis was not uniformly demonstrable until 14 days postinfection and remained highly significant on days 21, 28, and 84 postinfection. At 7 days postinfection, lymphocytes from 7 of 17 animals were capable of undergoing sifnificant blastogenesis. The Listeria antigen preparation was not mitogenic for peritoneal exudate lymphocytes from normal animals. It was found that no direct correlation exists between the in vivo levels of DTH and in vitro blastogenesis. Cell donors showing significant in vitro blastogenesis nevertheless were also skin test positive for most animals tested. Humoral antibody was found to play no significant role in the immune response of guinea pigs to a primary infection with Listeria monocytogenes.

Delayed hypersensitivity and acquired cellular resistance in guinea pigs infected with Listeria monocytogenes.

Randomly bred pigs of both sexes were injected intracardially with one-half of a 50% lethal dose of Listeria monocytogenes. When infected animals were skin tested with 30 mug of a water-soluble extract of sonically disrupted Listeria, both males and females had uniformly detectable levels of delayed hypersensitivity (DH) 4 days after infection. In males, cutaneous hypersensitivity to Listeria antigens reached a peak on day 5 or 6 of infection, and high levels of DH persisted through the 7th week. In females, DH reached a peak on day 6 or 7, remained at this level through the 4th week, and then dropped sharply. Cutaneous reactivity was usually higher for males than for females, and differences between the sexes were statistically significant 5, 6, and 7 weeks after infection. Low levels of DH were still present 41 weeks (females) or 46 weeks (males) after infection. Assays to determine the number of viable Listeria present in spleen homogenates indicated that bacterial multiplication occurred only during the first 24 hours of infection. The number of Listeria declined steadily thereafter, and by day 13 no bacteria could be recovered from the spleens of infected animals. Spleen assays indicated that Listeria-infected animals of both sexes were resistant to a small challenge dose of Listeria given 48 hours, 7 days, or 2 weeks after the primary infection. Resistance to re-infection was absent in females challenged at 41 weeks and in males challenged at 46 weeks.

In Vivo and In Vitro Studies of Delayed-Type Hypersensitivity to Toxoplasma gondii in Guinea Pigs.

Delayed-type hypersensitivity develops late in the course of human toxoplasmosis, and a positive skin test is of some value for implicating chronic or eliminating acute forms of toxoplasmosis as a cause of disease. Toxoplasma-infected guinea pigs were studied to determine the onset and development of delayed-type hypersensitivity. Both the toxoplasmin skin test and the in vitro macrophage migration inhibition technique indicated that delayed hypersensitivity to toxoplasma antigen existed as early as 1 week after infection. The mechanism responsible for the observed inhibition of macrophage migration in vitro appeared to be an inhibitory factor(s) released from sensitized lymphoid cells in the presence of antigen.