Effects of dietary fat and protein on DMH-induced tumor development and immune responses.

Although in three different mouse tumor systems with corn oil as dietary fat we previously found that milk protein decreased tumor development compared with beef, the results were reversed in 1,2-dimethylhydrazine (DMH)-injected mice. The purpose of this study was to determine if the latter result was due to the protein source. BALB/c mice (n = 280) were divided into five diet groups and injected 10 times at weekly intervals with DMH (20 mg/kg wt) or saline. Four diets contained 11% protein (casein, milk, or beef) and 5% fat (corn oil or beef tallow), and the AIN-76A diet was used as a control diet. The source of fat was a significant modulator of tumor development. Corn oil markedly increased total tumor volume and the number of tumors per mouse compared with beef tallow. Its tumor-enhancing effects were evident when it was combined with milk but not with casein. In addition, significantly lower lymphoproliferation and T-cell cytotoxicity against colon tumor cell targets was associated with corn oil consumption, whereas nonfat milk as the protein source was related to normal oxidative burst capacity of phagocytes. These results demonstrate that the source of dietary fat, in addition to the protein source, has a profound effect on both tumor development and immune responsiveness in this animal tumor system.

A comparison of schedules combining fractionated hyperthermia and irradiation in mice.

A comparison was made of three study arms delivering localized fractionated hyperthermia followed by irradiation for two weeks. The treatment results demonstrated 18-week survival and NED survival to be 35 per cent (7/20) and 30 per cent (6/20) respectively for heat and irradiation 5 days per week, 57.9 per cent (11/19) and 52.6 per cent (10/19) for combined treatment 3 days per week and 27.8 per cent (5/18) for heat 3 days per week and irradiation 5 days per week. It is felt that thermotolerance will account for the lack of difference between 24 h and 48 h irradiation schedules when irradiation is given daily. Irradiation fraction size, however, is suggested as a moderating variable as well.

Suppression of tumor growth and enhancement of immune status with high levels of dietary vitamin B6 in BALB/c mice.

Effects of dietary vitamin B6 at levels ranging from deficiency to megadoses on the development of herpes simplex virus type 2-transformed (H238) cell-induced tumors and on in vitro responses relating to cell-mediated immunity were examined. Male BALB/cByJ mice (n = 260), 5 weeks of age, were fed 20% casein diets containing pyridoxine (PN) at 0.2, 1.2 for the control diet, 7.7, or 74.3 mg/kg diet for 4-11 weeks. After 4 weeks of dietary treatment, 120 of the mice received an injection of H238 cells; mice without H238 injection served as controls. At 4, 8, and 11 weeks, animals from each group were euthanized and blood and spleen samples obtained. Mice fed 0.2 mg PN developed mild deficiency symptoms and gained significantly less weight than those fed 1.2-, 7.7-, and 74.3-mg PN diets. Thirteen to 16 days after tumor cell injection, primary tumor incidence was lowest in mice fed 74.3 mg PN; later, incidence among groups was similar. Mice fed 1.2 mg PN had the largest primary tumor volume, the highest incidence of lung metastases, and the greatest number of metastatic nodules per animal at 7 weeks post injection. Overall, lower tumor volumes were found in animals fed 7.7 and 74.3 mg PN (14 and 32% less than the tumor volume for those fed 1.2 mg PN, respectively); mice fed 0.2 mg PN had the lowest tumor volume. Blood and spleen lymphoproliferative response to stimulation by phytohemagglutinin or concanavalin A generally tended to be higher in mice fed 7.7 and 74.3 mg PN as compared to that in animals fed either 0.2 or 1.2 mg PN. However, decreased mitogen-stimulated responsiveness was observed in all animals with progressive tumor growth. Tumor growth also resulted in splenomegaly and increased thymic atrophy. Significant negative relationships between tumor volume and tumor pyridoxal 5-phosphate (PLP) concentrations were observed for 1.2-, 7.7-, and 74.3-mg PN diet groups. These data suggest that high dietary intake of vitamin B6 may have suppressed tumor development by either immune enhancement or PLP growth regulation of this tumor.

Hyperthermia and radiation in vivo: effect of 2-deoxy-D-glucose.

Since hypoxic cells rely heavily on glucose metabolism for energy, 2-deoxy-D-glucose (2-DG), an inhibitor of anaerobic glycolysis, would be expected to increase tumor cell killing by heat and thus enhance the effect of concurrent radiation. In order to test this hypothesis two types of BALB/c mouse tumors, one induced by subcutaneous injection of 10(6) herpes virus Type 2-transformed (H238) cells and the other by injection of 1.6 X 10(5) 1,2-dimethylhydrazine-transformed (#51) cells in the right thigh, were subjected to radiation, 2-DG, and heat used singly and in various combinations. Control mice were injected with saline. Three to four weeks after inoculation the mice were assigned to one of eight treatment groups (28 mice/group) so that average tumor volume/group before treatment would be equivalent. A single 2000 rad dose of radiation 3 hr prior to heat and 2-DG injected intraperitoneally at 1 g/kg 30 min before heating were given to some of the groups. Localized heat at 43.5 +/- 0.1 degrees C for 30 min, when used, was administered by means of a water bath. Rectal temperatures were kept below 39 degrees C, whereas intratumor temperatures reached a maximum of 42 degrees C. After treatment, tumor volume, mouse weight, and mortality were noted twice a week for four weeks. In both tumor models, mice receiving radiation plus heat, and radiation plus heat plus 2-DG, had significantly smaller tumors over the entire 4 to 28 day range after treatment than saline-injected control mice. In addition, in the H238 tumor model, addition of 2-DG to treatment with radiation and heat resulted in significantly smaller tumors at 25 days. 2-DG alone or in combination with heat (without radiation) resulted in significantly smaller H238 cell-induced tumors at day 28 post-treatment when compared to the saline controls. The H238 tumor-bearing mice experienced a significant (4.7%) loss in total body weight after heating. It could be that heating trauma produced dehydration and possibly also decreased caloric intake to an extent which could be measured in weight loss. This observation, however, was not made in the heated mice in the #51 tumor model.

Effects of the removal of adherent and phagocytic cells on the spleen cell lymphoproliferative response of tumor-bearing mice.

Cell-mediated immunity was investigated in two BALB/c mouse tumor systems using the lymphoblastogenesis test with phytohemagglutinin as the mitogen. This lymphoproliferative response was quantitated using the Stimulation Index (SI). There was little evidence for suppressor cell activity in cell mixing experiments in which spleen cells from #51 cell-injected mice were mixed with spleen cells from normal mice. Following macrophage removal by Sephadex G-10 columns and carbonyl iron ingestion, there were no significant changes in the SI values for spleen cells from the #51 cell-injected mice. In contrast, spleen cells from mice injected with H238 cells, a herpes virus-transformed cell line, had a significantly lower SI value than that of normal mice. Suppressor cell activity was demonstrated in cell mixing experiments in which spleen cells from H238 cell-injected mice were mixed with normal spleen cells. Removal of adherent cells from spleen cells from H238 cell-injected mice by Sephadex G-10 columns restored the SI value to that of normal mice. An increased SI value was also seen after removal of phagocytic cells by carbonyl iron. These results suggested that cells with the functional properties of macrophages played an important part in the immunosuppression observed in the H238 tumor system. Comparison of the two macrophage depletion methods suggested that another cell population was also involved in the suppressive effect. Results of immunofluorescent techniques with anti-Lyt-1 and anti-Lyt-2 monoclonal antibodies show these cells to be Ly 1-, Ly 2,3+ phenotypes of T-lymphocytes.

Mouse neoplasia and immunity: effects of radiation, hyperthermia, 2-deoxy-D-glucose, and Corynebacterium parvum.

Radiation (XRT), hyperthermia, 2-deoxy-D-glucose (2DG), and Corynebacterium parvum were given in various combinations to BALB/c mice injected with herpes virus type 2-transformed (H238) cells. Addition of heat significantly increased the antitumor effects of XRT, and the combination of XRT + 2DG + heat resulted in the highest incidence of complete tumor regression. Enhanced activity of phytohemagglutinin-responsive T lymphocytes and natural killer cells capable of killing YAC-1 tumor cells was noted in some of the treatment groups while tumor volume was similar for all of the groups. This enhancement was most likely to be achieved when heat was included as part of the treatment protocol.

BALB/c mice fed milk or beef protein: differences in response to 1,2-dimethylhydrazine carcinogenesis.

The development of 1,2-dimethylhydrazine (DMH)-induced colon tumors and immune responses were investigated in male BALB/c mice fed six different equicaloric diets. Milk or beef at a low (11%) or high (33%) level supplied the dietary protein, and corn oil (primarily) at a low (5%) or high (30%) level supplied the fat. Eleven weekly injections of DMH (at 20 mg/kg mouse) or saline were administered. At 59 weeks of age, the milk-fed mice had a significantly higher (P less than or equal to .05) colon tumor incidence than the beef-fed mice, 67 and 16%, respectively. Tumor volume and colon weight in the milk-fed mice were also significantly greater. Low natural killer cell activity against [125I]5-iodo-2'-deoxyuridine-labeled colon tumor cells and high serum blocking of antitumor cell activity were observed in the milk-fed-mice. These mice also exhibited higher T-lymphocyte cytotoxicity against colon tumor cells. These results differ from those of our previous studies and those of numerous epidemiologic investigations.

Modification of spontaneous mammary tumors in mice fed different sources of protein, fat and carbohydrate.

The effects of different sources of dietary protein (milk, soy, wheat, fish and beef), fat (corn oil and butter), and carbohydrate (dextrin and sucrose) on the development of spontaneous mammary tumors in virgin female C3H/HeJ mice were investigated. Weanling mice were randomly divided (28 mice/group) and fed ad libitum one of 14 equicaloric diets containing either 11% or 33% protein and 5% or 30% fat or a standard mouse feed for approximately 2 years. Beginning at 6 months of age, tumor incidence, non-specific deaths, individual weights and amount of food consumed were monitored. Variations in tumor incidence were most pronounced when the mice fed different sources of protein (at a high level) were compared. The mice fed the low fat diets containing either low milk protein (high carbohydrate) or high fish protein generally exhibited the lowest tumor incidence and highest percent survival. High weight gain was correlated with early tumor appearance, but not with tumor incidence later in the experiment. The mice fed a low fat diet containing low milk protein were tumor-free significantly longer than mice fed the diets containing fish or beef. The only groups with 100% tumor incidence by 120 weeks of age were those fed diets containing sucrose (table sugar) or a high fat level.

Modification of a transplantable colon tumor and immune responses in mice fed different sources of protein, fat and carbohydrate.

The effects of different sources of dietary protein, fat and carbohydrate on tumor development and on tests relating to cell-mediated immunity were investigated in male BALB/c mice after subcutaneous injection of 8 X 10(4) 1,2-dimethylhydrazine (DMH)-induced colon tumor (no. 51) cells. Results indicated that mice fed the milk protein source (especially at the low protein level) had smaller tumors, a higher spleen cell proliferative response to stimulation by phytohemagglutinin (PHA), and greater cytotoxic T-cell activity against the tumor cells than those fed the comparable diets containing protein from the other sources. Peripheral blood lymphocytes only from the milk-fed mice, regardless of tumor presence, exhibited a relatively low response to PHA stimulation, thereby suggesting a dietary effect on the migration pattern of PHA-responsive lymphocytes. The level of protein significantly affected both T-cell and natural killer cell cytotoxicity. The tumor-bearing mice fed the diet containing sucrose (table sugar) had a significantly lower spleen cell response to PHA stimulation than those fed the comparable diet containing dextrin. The level or source of fat did not significantly affect any of the parameters tested in this system.

Modification of herpes 2-transformed cell-induced tumors in mice fed different sources of protein, fat and carbohydrate.

The effects of different sources of protein (milk, soy, wheat, fish and beef), fat (corn oil and butter), and carbohydrate (dextrin and sucrose) on tumor development and on spleen characteristics were investigated in BALB/c mice injected subcutaneously with 5 X 10(5) herpes simplex virus Type 2-transformed cells (H238 cells). Low or high levels of protein and fat were used. Several weeks post-injection results indicated that a high level of fat significantly enhanced tumor incidence. A high fat level was also associated with a lower spleen weight and a smaller proportion of mature granulocytes in the spleen. Butter, compared to corn oil, significantly restricted tumor volume. Among the most highly significant findings was the low tumor incidence in mice fed protein from either a milk or a fish source.

Different weekly changes in immune response in virus-transformed cell-injected mice fed two different diets.

Changes in tumor development and in certain immune responses were investigated at 7-weekly intervals after subcutaneous injection of 5 X 10(5) herpes simplex virus Type 2-transformed cells (H238 cells) into male BALB/c mice fed 2 different diets. One diet contained 11% casein and 5% fat while the other had 11% supplemented wheat gluten and 30% fat. Weanling mice (140/group) were fed one or the other of the diets for 12 weeks before injection and subsequent testing of 15 injected and 5 non-injected mice from each diet group each week. In mice fed the low-fat diet containing casein both tumor incidence and tumor volume were significantly lower (P less than or equal to 0.05) than in the group fed the 30% fat diet containing supplemented wheat protein. The casein-fed mice also had less splenomegaly and a higher proportion of mature lymphocytes in the spleen during tumor growth. The proliferative capability of the spleen cells after phytohemagglutinin stimulation was enhanced 2 weeks after H238 cell injection only in the casein-fed mice.

Responses of mouse spleen morphology to the growth of subcutaneously injected virally transformed cells.

The changes in the fractional volume of six structural components in the spleens of Balb/C mice injected with Herpes simplex virus Type 2-transformed cells (H238 tumor cells) were quantitated during progressive tumor growth. Spleen stereology was performed at three time intervals during the early stages of tumor development. The results revealed that the volume of the compact myeloid tissue and reaction center of lymphoid nodules increased about four- to five-fold from 10 to 33 days after H238 tumor cell injection. A progressive increase was also seen in the red pulp volume. Although an increase in volume of the marginal zones around the lymphoid nodules was evident early during the test period, by day 33 the mean value was similar to the control value. These results indicate that the spleen undergoes significant morphological changes in three splenic components during progressive growth of a tumor produced by subcutaneous injection of a virally-transformed cell line.

Effect of L1210 leukemia on the susceptibility of mice to Candida albicans infections.

This study was carried out to determine whether animals bearing L1210 leukemia were more susceptible to candida infection in the absence of immunosuppression and to determine also if the L1210 cells suppressed the inflammatory response of the animal host. Systemic infection was studied by intravenous injection of Candida albicans and checking for the number of candida organisms cultured from the blood and the kidneys. Localized infection was studied by intramuscular injection of C. albicans into the thighs and measuring the changes in the thigh size. Compared with tumor-free controls, the intravenous injection resulted in higher counts of C. albicans from the blood and the kidneys of tumor-bearing animals. No significant difference in the localized swelling was noted between tumor- and nontumor-bearing mice with respect to intramuscular injection of C. albicans. The results thus indicate that L1210 leukemia increases susceptibility of tumor-bearing animals to systemic candida infection. L1210 cells were shown to reduce the accumulation of neutrophils and to suppress the inflammatory reaction elicited by C. albicans.

Change in methylation of Salmonella bacteriophage P3 deoxyribonucleic acid with host-controlled modification by Escherichia coli.

Modification of bacteriophage P3 by passage through Escherichia coli K was correlated with a 54% decrease in the content of 6-methylaminopurine in the phage deoxyribonucleic acid.

Serological relationships of Salmonella A bacteriophages isolated from salmonellae in different Kauffmann-White groups.

Six different Salmonella group A phages from salmonellae in Kauffmann- White groups B, C(1), C(2), and D were examined serologically. Those phages which were specific for a particular somatic antigen were found to be serologically very similar. Antiserum against a phage with one specificity was able to neutralize a different phage with the same specificity but unable to neutralize, in the normal way, a phage with a different specificity. Phages mixed with heterologous phage antiserum responded with an "inhibition response" in which there appeared to be a neutralization of the phage infectivity for the first 10 min, followed by a reversal of the neutralization until, by 20 or 25 min, there was no apparent neutralization. This response was interpreted to indicate that the adsorption antigens, probably situated on the tail fibers, were different for phages with different specificities but sufficiently similar so that heterologous antibodies could react with the antigens; but the antigen-antibody complex was quickly disassociated, resulting in a modification of the antibody molecules but no change in the specificity sites of the antigen. A subgrouping of the Salmonella A phages based on their antigenic specificity is suggested.

Some properties of five new Salmonella bacteriophages.

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C(1). Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.