Gender influences tumour growth in a model of human oral squamous cell carcinoma.

The pattern of tumour growth was investigated using our unique syngeneic animal model of human oral squamous cell carcinoma of the head and neck (SCCHN). Gender-conditioned tumour fragments of near equal size were transplanted into recipient animals and the pattern of tumour growth was assessed by monitoring tumour size in vivo and/or the weight of tumour al termination of the experiment. Our results confirmed the epithelial nature of tumour cells on fresh tumour tissue sections using morphological and immunocytochemical examination. The mean weights of tumour per animal (n=10) after transplant from female (F) to F recipients, and across and between same gender showed differences if the animals were sexually mature. The results indicated an endocrine sensitive (sex hormone) component of tumour growth. In separate experiments, the recipient animals were immunised with a mechanically dissociated single cell suspension from fresh tumour after irradiation (500 cGys). The test,group (n=5) and control group (n=5, saline injected) animals received tumour fragments by grafting and following a Eur ther three weeks post immunisation, the tumours were removed and weighed. The test and control groups were different with a p-value of 0.001, suggesting an immune participation in the growth of this tumour. The results, taken together, thus suggest the participation of both the endocrine and the immune systems on the pattern of tumour growth in this animal model. The possible clinical relevance of these results is discussed.

The possible relevance of the expression of MHC antigens and of EGF receptor in aggressive oral tumours.

In this study the intensity of expression of major histocompatibility complex (MHC) class I and II antigens, adhesion molecule i.e. ICAM-1, epidermal growth factor receptor i.e. EGFr, T cell marker and cytokeratin were compared in oral squamous cell carcinoma (OSCC) and in the benign ameloblastoma of the jaws. The results showed that: a) There was strong expression of both monomorphic and of polymorphic class I MHC antigens (90% of cases) in both basal and suprabasal cells of controls from normal mucose. b) Whereas up to 4% of OSCCs and 27% of ameloblastomas showed complete loss of monomorphic class I antigens, the frequency of polymorphic class I abnormalities was even more marked in both tumour types. c) Strong expression of class II MHC antigens and of ECFr was observed in the basal cells of most normal controls. d) Both class II (50% of cases) and ICAM-1 (30% of cases) showed strong expression in OSCC but not in ameloblastoma. The statistical values between OSCC and normal basal cells for class II and ICAM-1 were not significant whilst the corresponding values for OSCC compared with ameloblastoma were p<0.001 and p<0.001. In the case of OSCC, there were a large number of infiltrating T cells expressing activation marker i.e. class II antigen. e) Strong expression of EGFr was seen in more than 90% of the OSCC cases compared with only 16% of ameloblastomas (0.01

0.001). Our working hypothesis to explain these abnormalities is that although both tumour types (more so in the case of ameloblastoma) have in place an escape mechanism from the immune system, the overexpression of EGFr in OSCC may in part be responsible for the more aggressive behaviour of the malignancy compared with the locally invasive but benign ameloblastoma.

Local anergy rather than systemic anti-tumour immunity to explain tumour growth in an animal model of oral squamous cell carcinoma.

A chemically induced syngeneic hamster tumour model of human oral squamous cell carcinoma (OSCC) was used to investigate the possible influence of locally transplanted growing rumours on the immune system of the recipient. Cell activation and cell cytotoxicity assays were performed in vitro using the colorimetric MTT assay to measure any possible changes. The fast growing nature of the tumour model if grafted locally as a fragment was confirmed but not if injected as a single cell suspension (SCS). Stimulation (Concanavalin A) of spleen cells from normal and from tumour bearing animals showed that there was a minor though statistically significant decrease in the mitogenic response of the latter. Thus, the respective stimulatory indices (SI) were 4.06+/-1.61 and 2.06+/-0.87 (0.02

0.01). No significant difference was observed when spleen cells were stimulated with interleukin-2 (IL-2), although there was a similar trend. Pre-immunisation of animals with irradiated autologous SCS three weeks prior to grafting, resulted in a significant decrease in the tumour growth rate of subsequently grafted tumour. Thus, the mean If: SD (weight of takes in mg) for the successful takes of untreated (n=10) and treated (n=9) groups were 52.0+/-52.2 and 25.7+/-19.4 (0.02

0.05) respectively. The number of cases with no tumour takes were 2 of 10 (20%) and 6 of 9 (66%) respectively. In a separate experiment groups of 5 animals were immunized with an increasing number of cells as irradiated SCS, the results of which demonstrated an inverse correlation between the rate of tumour growth and the number of injected tumour cells. The addition of irradiated SCS to IL-2 activated normal spleen cells (LAK cells) in vitro led to a dose-related decrease in the efficiency of cytotoxicity of latter when tested against an xenogeneic super-sensitive surrogate tumour target cell line (Fen cells). Thus, the percent killing by IL-2-activated normal spleen cells was 56.4%. The corresponding mean values for IL-2 activated normal spleen cells in the presence of tumour SCS at 25/1 and 50/1 ratios were 35.9% (p<0.05) and 11.9% (p<0.001) respectively. Ln an attempt to establish the presence of T suppressor cells, spleen cells from tumour bearing animals were injected concomitantly with SCS into 5 recipients. After four weeks no tumour growth had occurred. In conclusion we demonstrated that the presence of injected or grafted tumour had only a minor effect on systemic immune function but induced a strong local anergic effect. This local anergic effect was demonstrable as blocking of LAK activity and thus perhaps allowed suppression of the functional activities of incoming immunocompetent cells.

Comparison of immunological changes between subcutaneous and intravenous route of administration of IL-2 in cancer patients.

The effects of the route of administration of interleukin 2 (IL-2) on the immunological parameters of patients with various malignancies were investigated. The percent mean values for IL-2 receptor (Tac) positive cells among mononuclear cells in patients receiving IL-2 subcutaneously (5 cases) or intravenously (6 cases) were 7 and 7%, respectively. The corresponding values of post-IL-2 treatment peak were 18 and 16%. Similarly, the values for class II antigen expressing cells were 12 and 16% and 25 and 26%. In no case the difference between the values for the subcutaneous or the intravenous route reached significance. Using the 51Cr release assay, the mean percent killing activity of IL-2-activated mononuclear cells against Daudi tumour target cells for the subcutaneously and the intravenously treated groups were 27 and 14% and the corresponding values for the post-IL-2 treatment peak were 59% (p > 0.05) and 69% (p > 0.05), respectively. The similar values using K562 tumour as target were 17 and 8%, and the corresponding values for post-treatment peak were 55% (p > 0.05) and 23% (p > 0.05). In all cases the cytotoxic values for the post-IL-2 treatment were significantly greater than the pre-IL-2 values. The mean (+/- SD) values for serum beta2-m levels for 6 subcutaneously and 5 intravenously IL-2-treated patients were 6.5 +/- 4.6 and 5.7 +/- 3.4 mg/l (p > 0.05). The corresponding values for post-IL-2 (15 days) were 9.1 +/- 3.4 and 9.9 +/- 5.1 mg/1, respectively. These results demonstrate that there is no significant difference in the immunological parameters in cancer patients receiving IL-2 via subcutaneous or intravenous routes and provide further support for the current trend for clinical trials to concentrate on outpatients subcutaneous administration.

Immunotoxicity of polyunsaturated fatty acids in serum-free medium.

To test the effect of purified polyunsaturated fatty acids on immune cells in vitro, human peripheral blood mononuclear cells and murine spleen cells were incubated in Opti-MEM medium without serum or even albumin and with 2-mercapto-ethanol, insulin, transferrin and selenium as supplements. The human cells were stimulated with phytohemagglutinin and the murine cells were stimulated with Concanavalin A or lipopolysaccharide. Both human and murine cells were stimulated with recombinant human interleukin-2 to generate lymphokine activated killer cells. Linoleic and linolenic acids inhibited all of the immune responses tested, whereas docosahexaenoic and eicosapentaenoic acids did not. Similar effects were observed with cultured B16 F10 murine melanoma cells. Mixtures of linoleic and docosahexaenoic or eicosapentaenoic acids also inhibited the mitogenic response to phytohemagglutinin. Inhibition of lipid mediator production by indomethacin, quercetin, rutin, or nordihydroguariaretic acid, and addition of vitamins C and E with anti-oxidant activity failed to reverse the effects of linoleic acid. Thus, linoleic and linolenic acids appear to directly inhibit immune and tumor cells, at least under these conditions.

Melanoma lung metastases and cytolytic effector cells in mice fed antioxidant-balanced corn oil or fish oil diets.

(C57BL/6 x DBA/2)F1 mice were fed antioxidant-matched fish oil (FO) or corn oil (CO) diets for weeks, were challenged with B16.F10 melanoma cells, and lung metastases were enumerated 17 days later. Mice fed FO had fewer lung tumors than mice fed CO. This dietary effect persisted in mice injected with monoclonal antibodies which depleted natural killer cells, CD8+ T cells or CD4+ T cells. Generation of specific anti-B16.F10 cytotoxic cells in vitro by splenocytes from immunized mice was greater in mice fed FO. Even though host resistance to lung metastases by B16.F10 cells is mediated in large part by natural killer cells, CO and/or FO may have affected cells not tested here or tumor cells themselves.

Induction of murine hepatic glutathione S-transferase by dietary dehydroepiandrosterone.

The naturally occurring steroid dehydroepiandrosterone (DHEA), when administered as a supplement to the diet of mice and rats, produces alterations in the relative concentrations of specific liver proteins; among these, a protein of Mr approximately 28 K is markedly induced by DHEA action. In the present study we identified the murine hepatic approximately 28 kDa protein as glutathione S-transferase subtype GT-8.7. Glutathione S-transferases belong to a gene superfamily that encode closely related proteins which are induced in liver and other tissues by various chemicals, including carcinogens and chemoprotective agents such as dietary antioxidants. Based on the above finding, we evaluated glutathione S-transferase activity in cytosols and microsomes prepared from liver tissue of mice fed either a control diet or a DHEA-containing diet (0.45%, by weight). The specific activity of hepatic cytosolic glutathione S-transferase in mice treated with DHEA up to 7 days was either unchanged or slightly decreased when compared to controls; however, treatment for 14 days or longer resulted in significant increases in activity. The specific activity of microsomal glutathione S-transferase also was increased by long-term DHEA treatment; however, its activity was approximately one-tenth of that in corresponding cytosols.

Inhibition of pregnancy viability in mice following IL-2 administration.

The influence of administration of interleukin-2 (IL-2) on syngeneic and allogeneic murine pregnancy has been investigated. Human or mouse recombinant IL-2 (rhIL-2 and rmIL-2), or partially purified rat IL-2, was inoculated i.p. into C57B1 mice following syngeneic mating but before embryo implantation. This inhibited subsequent fetal development in up to 100% of cases, compared with mice inoculated with control material, including recombinant human interleukin-1 alpha (IL-1 alpha), where no inhibition of pregnancy viability was observed. Similar data were obtained in both syngeneic and allogeneic matings when rhIL-2 was administered on Day 1 of pregnancy. Administration of rhIL-2 during the second pregnancy, rather than a first pregnancy, was less effective. Administration of rhIL-2 during the first pregnancy does not induce a permanent sterility. Histological examination of the endometrium further demonstrated that mice injected with rhIL-2 on Day 1 of their first pregnancy showed a complete absence of embryonic tissue.

Studies on cell-mediated immunity in women using different fertility regulating methods.

Cell-mediated immunity (CMI) was assessed in women using fertility regulating methods for 1-5 months, 6-11 months or 12-18 months. The CMI as assessed by phytohaemagglutin in (PHA)-induced lymphocyte transformation of treated groups were compared with that of normal subjects who were not using any contraceptive methods and women on conventional methods of contraception. The data obtained indicates that there is no significant alteration of CMI in women fitted with IUCD or women on estrogen progestogen combination. However, a significant suppression of CMI is observed in women in progestogen pills for 12-18 months. The short term therapy did not affect the CMI. In a prospective study it was found that the CMI in women before and after the use of combination therapy for 1-5 and 6-11 months revealed no change. Estradiol and progesterone at concentration on 1 microgram/ml in culture medium suppressed PHA-induced lymphocyte transformation.