Modulation in vitro of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids.

Essential fatty acids (EFA) have been shown in animal studies to have a differential effect on various aspects of immune reactivity. However, there have been few studies in humans. Therefore, we elected to investigate the effects of a variety of EFA [gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in vitro on human blood lymphocyte reactivity, cytokine secretion and natural cytotoxicity. The proliferative response to polyclonal mitogens (phytohaemagglutinin, pokeweed mitogen, concanavalin A), as measured by [3H]thymidine incorporation into newly synthesized lymphocytes, was inhibited (P < 0.05) by all EFAs tested, in a dose-dependent manner (3-15 micrograms/ml). The greatest inhibition of proliferation was caused by EPA and DHA. Similarly, EPA, DHA and GLA significantly reduced cytotoxic activity [expressed as lytic units, using 51 chromium-release assays natural killer (NK) (K562 cells) and lymphokine-activated (LAK) (Daudi cells) cells] (P < 0.05) in a concentration-dependent manner (5-50 micrograms/ml), without affecting cell viability. EPA and DHA exhibited greater suppression than GLA. Furthermore, the inhibition of cell proliferation and suppression of natural cytotoxicity was associated with marked decrease in cytokine [interleukin-1 (IL-1), IL-2, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma)] production in vitro. Our findings demonstrate that EFAs (GLA, EPA, DHA) have the potential to inhibit significantly various aspects of human lymphocyte cell-mediated and humoral immune reactivities.

A novel diagnostic index for use in the breast clinic.

The diagnostic accuracy of clinical examination, mammography, ultrasonography and fine needle aspiration (FNA) cytology in discriminating between benign and malignant breast lesions has been retrospectively assessed in 603 patients who attended the Professorial Breast Clinic and then underwent surgical biopsy, in Aberdeen Royal Infirmary, between January 1989 and September 1992. In this series of patients, FNA cytology gave the highest overall prediction of malignancy (92.6%) with a sensitivity of 87% and a specificity of 98%, whilst ultrasonography gave the lowest correct overall prediction (75.6%), with a sensitivity of 70% and a specificity of 79%. Using all the available findings (clinical examination, mammography, ultrasonography, FNA) as well as the patients' age, a stepwise logistic discriminant analysis was used to derive a mathematical equation for use as a novel diagnostic index. Weighted scores, derived from each variable, were used in the discriminant equation to predict the likelihood of malignancy in patients of different ages (>90%). For young patients (<35 years) the discriminant analysis predicted the diagnosis in 98% of women correctly. Quadruple assessment provides an opportunity for establishing the likely diagnosis in most patients the same day they visit the Breast Clinic. The diagnostic index, currently being evaluated prospectively in our breast clinic, may be a further aid to establishing a correct diagnosis.

Effect of essential fatty acids on natural cytotoxicity in patients with colorectal cancer.

Essential fatty acids (EFAs), have been shown to modulate lymphocyte reactivity and destroy various tumour cells in vitro. Natural cytotoxicity, mediated by NK and LAK cells, is believed to play an important anti-cancer role in vivo. The effect of EFAs, given orally as dietary supplementation, on NK and LAK cell cytotoxic activity in patients with localized (n = 10) and advanced (n = 20) colorectal cancer has been studied, using in-vitro 51Cr release cytotoxicity assays with K562 (NK) and DAUDI (LAK) cells. The activity of effector cells was expressed as lytic units. NK cell activity showed no significant change following 15 days ingestion of EFAs in the group with localized cancer, but was significantly reduced in the group with advanced disease and continued to decline, reaching minimal levels following 6 months supplementation (P = 0.017). LAK cell activity showed no overall alteration after 15 days ingestion of EFAs in patients with localized cancer, but in the group with advanced disease, the reduction in the activity occurred at day 15 and steadily declined on prolonged intake, reaching significant minimal levels after 6 months of supplementation (P = 0.05). Cell surface marker analysis (FACS-CD MABs) revealed reduced absolute numbers of CD16+, CD56+ and CD57+ lymphocytes (P < 0.05) in the patients with advanced colorectal cancer. More importantly, the cytotoxicity of NK and LAK cells returned to the pre-supplementation values, 3 months after cessation of EFA intake. Furthermore, there was no alteration in the cytotoxic activity of NK and LAK cells in the control group (advanced colorectal cancer without EFA supplementation) during the 6 months period of evaluation. These results suggest that prolonged EFA supplementation, in the doses used in this study, may have detrimental effects on natural anti-cancer cytotoxic mechanisms in patients with malignant disease.

Differential responses of human tumor-cells to polyunsaturated Fatty-acids - stimulation of proliferation of a colon-tumor cell-line by docosahexaenoic Acid.

The potential differential effects of polyunsaturated fatty acids (5-100 mu g/ml) on four human tumour cell lines of different origin and a human fibroblast cell line were investigated. Following 6 days exposure to the fatty acids, gamma linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid, culture growth was almost completely abolished at the highest concentration used. At lower concentrations, the tumour cell lines exhibited a differential sensitivity to the inhibitory effects of the fatty acids on cell number (IC50, breast=lung>melanoma>colon). MRC-5 fibroblast cell numbers were significantly increased at low concentrations of gamma linolenate and eicosapentaenoate, but significantly reduced by docosahexaenoate. These effects on cell numbers were rapid in onset. Following only 2 days exposure to low concentrations of the fatty acids, cell numbers in the breast tumour cell line, MCF-7, were significantly reduced relative to controls. In contrast, the colon cell line, WiDR, was largely unaffected at this time, and in some cases, cell numbers were significantly increased. In the normal fibroblast cell line, cell numbers were significantly reduced by docosahexaenoate at concentrations greater than or equal to 20 mu g/ml. Following only 2 days exposure to PUFA, cell death in the breast cell cultures was maximally increased above controls by 20 mu g/ml of docosahexaenoate, whereas cell proliferation was unaffected at this concentration. In contrast, under these circumstances, cell proliferation in the colon cell cultures was significantly increased by this PUFA while there were only small increases in cell death. Our observations have highlighted the differential responses of human tumour cell lines to PUFAs and documented the stimulation of a colon cell line by certain PUFAs.

Modulation of cytokine production in vivo by dietary essential fatty acids in patients with colorectal cancer.

1. The effects of essential fatty acids (gamma-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid), at a dose of 4.8 g/day, given in combination as dietary supplements, on cytokine production were investigated in patients with colorectal cancer. 2. Total serum cytokines--interleukin (interleukin-1 beta, 2, 4 and 6), tumour necrosis factor-alpha and interferon-gamma--were analysed using the enzyme-linked immunosorbent assay technique at different time intervals during the course of essential fatty acid supplementation. 3. Fatty acid uptake and patient compliance were confirmed by a significant increase in serum levels of gamma-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid in all three fractions: tricylglycerol, cholesterol and phospholipid. 4. There was no significant alteration in total serum cytokine concentration/levels in the first 2 months of essential fatty acid ingestion, but the levels of serum cytokines steadily declined thereafter, reaching minimum levels after 6 months of essential fatty acid supplementation. 5. Essential fatty acids, at the dose and duration (6 months) used in this study, reduced total serum interleukin-1 beta levels by 61% (P = 0.044), interleukin-2 by 63% (P = 0.05), interleukin-4 by 69% (P = 0.025), interleukin-6 by 83% (P = 0.030), tumour necrosis factor-alpha by 73% (P = 0.040) and interferon-gamma by 67% (P = 0.050). 6. Three months after cessation of essential fatty acid intake, however, these cytokine levels returned to presupplementation values. 7. This present study has shown that long-term n-3 and n-6 EFA ingestion results in a significant reduction in circulating key cytokines. The precise mechanism of this reduction is unclear.

Essential fatty acids and malignant disease.

EFAs have shown promising anti-tumour effects in cell culture studies and in animal models. However, it is also clear that in man EFAs can have marked immunomodulatory effects, in particular, inhibiting certain aspects of host anti-tumour defence mechanisms (natural cytotoxicity and cytokine production). Further careful studies are required to evaluate more precisely the role of EFAs in the treatment of malignant disease in man.

Effect of essential fatty acids on circulating T cell subsets in patients with colorectal cancer.

The effect of essential fatty acids (EFA), given orally as dietary supplements, on the responsiveness in vitro of peripheral blood lymphocytes (PBL), to the mitogen concanavalin A have been studied in 10 patients with localized and 14 patients with advanced colorectal cancer. The degree of lymphocyte activation was assessed by measuring the amount of tritiated [3H]thymidine incorporated into newly synthesised lymphocyte DNA. The results were expressed as stimulation indices. T cell responses to concanavalin A stimulation showed a significant reduction of stimulation indices following EFA supplementation, in both the localized (P = 0.026) and advanced (P = 0.016) tumour groups, when compared with pretreatment activity in vitro. Mixing experiments, using EFA-supplemented and non-EFA-supplemented lymphocytes with concanavalin A, suggest no enhancement of T suppressor cell activity. Cell surface marker analysis (fluorescence-activated cell sorting for CD phenotyping) revealed a reduction of absolute numbers of CD4+ and CD8+ lymphocytes following EFA supplementation. The stimulation indices returned to pre-supplementation values 3 months following cessation of EFA intake. There was no significant change of these indices in the control (no EFA supplementation) advanced tumour group tested. This study suggests that EFA supplementation in patients with colorectal cancer selectively reduces circulating PBL, and T cell subset (including suppressor cells) numbers and/or activity. Such effects may have an important outcome in patients with malignant disease.