Neil E. Hubbard

Probiotic Immunomodulation in Health and Disease

Probiotics, microorganisms that have a favorable influence on physiologic and pathological processes of the host by their effect on the intestinal flora, may play a role in improving human health. One of the putative effects is the modulation of immune function. Thus, the mucosal immune system and methods to assess its function are reviewed briefly. Probiotic modulation of humoral, cellular and nonspecific immunity is reviewed, with emphasis placed on immune response in disease models. There are very few reports of human intervention studies with probiotics. However, some of the possible future directions for research with respect to probiotics, immunity, and human health are discussed. Although the application of probiotics has demonstrated trends with respect to altered aspects of immune response, the underlying mechanisms by which that occurs are unclear.

Micronutrients and Innate Immunity

Micronutrients such as zinc, selenium, iron, copper, beta-carotene, vitamins A, C, and E, and folic acid can influence several components of innate immunity. Select micronutrients play an important role in alteration of oxidant-mediated tissue injury, and phagocytic cells produce reactive oxidants as part of the defense against infectious agents. Thus, adequate micronutrients are required to prevent damage of cells participating in innate immunity. Deficiencies in zinc and vitamins A and D may reduce natural killer cell function, whereas supplemental zinc or vitamin C may enhance their activity. The specific effects of micronutrients on neutrophil functions are not clear. Select micronutrients may play a role in innate immunity associated with some disease processes. Future studies should focus on issues such as age-related micronutrient status and innate immunity, alterations of micronutrients in disease states and their effect on innate immunity, and the mechanisms by which micronutrients alter innate immunity.

Reduction of murine mammary tumor metastasis by conjugated linoleic acid

Recent studies have shown that conjugated linoleic acid (CLA) can inhibit the initiation and thus, incidence of mammary tumors in rodents. The concentration of CLA required for these effects was as low as 0.1% of the diet, with no increased effects above 1%. To date, there is little evidence that CLA has any effect on growth or metastasis of mammary tumors. In this report, we demonstrate that CLA, at the concentrations used in previous studies, had a significant effect on the latency, metastasis, and pulmonary tumor burden of transplantable murine mammary tumors grown in mice fed 20% fat diets. The latency of tumors from mice fed CLA was significantly increased when compared with the 0% CLA control diet. The volume of pulmonary tumor burden, as a result of spontaneous metastasis, decreased proportionately with increasing concentrations of dietary CLA. With 0.5 and 1% CLA, pulmonary tumor burden was significantly decreased compared to mice treated with the eicosanoid inhibitor, indomethacin and fed diets containing no CLA. Tumors of mice fed as little as 0.1% CLA and as much as 1% had significantly decreased numbers of pulmonary nodules when compared with diets containing no CLA. The decrease in the number of pulmonary nodules by CLA was nearly as effective as indomethacin, a known suppressor of tumor growth and metastasis in this malignant model. These data suggest that effects of CLA on mammary tumorigenesis may go beyond the reported alterations in tumor incidence and effect later stages, especially metastasis.

Inhibition of growth and linoleate-enhanced metastasis of a transplantable mouse mammary tumor by indomethacin.

The influence of the cyclo-oxygenase inhibitor, indomethacin (IM), on the metastasis, development and prostaglandin E (PGE) levels of line 4526 mammary tumors grown in mice fed high fat (HF, 20%, w/w) diets containing various levels of linoleic acid (18:2) was investigated. Control mice that grew primary tumors and were fed HF diets containing 12% 18:2 (w/w) had 2-3 times the number of lung metastases than mice fed 1%, 4%, or 8% 18:2. Chronic treatment of mice with 10 micrograms/ml IM in drinking water reduced metastasis in 1% and 4% 18:2-fed mice compared to controls and completely inhibited the increased metastasis of mice fed the 12% 18:2 diet. Treatment with IM also increased the latency and decreased the growth rates of primary 4526 tumors of all dietary groups. Treatment of mice with a higher dosage of IM (20 micrograms/ml), decreased tumor metastasis even further compared to controls, but did not decrease tumor growth rate compared to the lower dosage of IM (10 micrograms/ml). Tumor PGE levels, measured by radioimmunoassay (RIA), were decreased by IM treatment. These data provide evidence that arachidonic acid metabolites such as PGE may be involved in the metastasis of 4526 mammary tumors.

Effect of separate conjugated linoleic acid isomers on murine mammary tumorigenesis

Recent studies have linked conjugated linoleic acid (CLA) to altered tumorigenesis of several sites. We showed recently that a mixture of CLA isomers was able to significantly decrease mammary tumor metastasis in mice. That effect was seen with as little as 0.1% CLA in the diet. Other studies with dietary CLA have shown that various isomers may have differential effects. The purpose of this work was to assess which individual CLA isomers had similar effects in alteration of mouse mammary tumor metastasis. For that, we fed six 20% (w/w) total fat diets which contained either no CLA, low (0.1%, w/w) or high (0.25%, w/w) levels of cis9,trans11-CLA (c9,t11), trans10,cis12-CLA (t10,c12) or a mixture of the 2 isomers (0.125% of each, w/w) as free fatty acids. Neither the separate isomers nor the mixture had an effect on the latency or growth of primary line 4526 tumors when compared to the group without CLA. However, all diets containing CLA significantly decreased the total tumor burden (volume of tumor, mm(3)) in lungs of mice from both spontaneous metastasis (reduced by 42-73%) as well as implantation and survival of the metastatic cell (reduced by 46-61%) when compared with diets containing no CLA. Diets containing a greater concentration of either c9,t11 or t10,c12 had a significantly greater effect compared to the lower concentrations of the respective isomers when metastatic nodule size and total tumor burden were assessed. The diet containing both isomers decreased total tumor burden similarly to the diets containing the lower concentration of each of the isomers. Thus, the effects of c9,t11 and t10,c12 may not be additive and possibly share similar mechanisms for decreasing metastatic tumor burden in mice transplanted with mammary tumor cells.

Alteration of murine mammary tumorigenesis by dietary enrichment with n-3 fatty acids in fish oil

We and others have previously shown that dietary fat can alter the growth and metastasis of rodent mammary tumors. Few transplantable tumor models have been used to study the effects of dietary n-6 versus n-3 fatty acids on mammary tumorigenesis. Here we study the effects of fish oil and safflower oil on the growth and metastasis of an animal model that in several ways parallels the human disease. Tumor latency, growth and metastasis were studied in mice fed diets that contained either 10 or 20% total fat which was varied in the type of fat with either menhaden fish oil (FO), safflower oil (SO) or a 50/50 mixture of the two. Tumor latency was significantly longer and tumor growth was significantly slower in mice fed the 20% FO diet. When spontaneous metastasis was assessed, mice fed diets containing FO had significantly decreased numbers of pulmonary nodules and total metastatic load. Likewise, mice fed FO diets had a lower level of implantation and survival of pulmonary metastases. Thus, in our animal model, diets containing n-3 fatty acids in fish oil significantly decrease primary breast tumor growth and its metastasis.

Modulation of murine mammary tumor vasculature by dietary n-3 fatty acids in fish oil.

We have previously shown that mice fed a high (n-3) fatty acid-containing diet with 20% (w/w) total fat had significantly slower mammary tumor growth, decreased numbers of metastatic pulmonary nodules, and decreased total metastatic load. In this study we sought to determine whether tumor vascularization was altered in mice fed diets varying in concentrations of (n-3) and (n-6) fatty acids. Several direct or indirect parameters of vascularization were tested. With 20% dietary fat, fish oil (FO) or a mixture of FO and safflower oil (FS) significantly reduced blood vascular area, mast cell number and macrophage infiltration in solid mammary tumors compared to tumors grown in mice fed safflower oil (SO). A decreasing trend was seen in the percent area of vessels positive for CD31 and vascular endothelial growth factor (VEGF) in the 20% FO and 20% FS compared to the 20% SO dietary groups. VEGF concentrations were twice as high in smaller tumors (100 mm3) from all dietary groups as compared to larger tumors (500 mm3). A two-fold increase in VEGF levels was found in the 20% SO dietary group compared to the 20% FO group in 100-mm3 but not larger tumors. We conclude that at 20% total fat, the n-3 fatty acids found in fish oil may inhibit primary mammary tumor growth through modulation of select determinants of vascularization.

Effects of tumor necrosis factor alpha on leptin secretion and gene expression: Relationship to changes of glucose metabolism in isolated rat adipocytes

OBJECTIVE: Our objective was to determine the effects of prolonged exposure to tumor necrosis factor-alpha (TNF-α) on leptin secretion from and leptin (OB) gene expression in isolated adipocytes. Because glucose uptake and the metabolism of glucose beyond lactate are important determinants of leptin production in adipocytes, we examined the effects of TNF-α on glucose uptake and lactate production and their relationship to leptin secretion.DESIGN AND METHODS: Isolated rat adipocytes were anchored in a defined matrix of basement membrane components and cultured with media containing 5 mM glucose, 0.16 nM insulin and several concentrations of TNF-α. Leptin secretion, steady-state levels of leptin mRNA levels, glucose uptake, and lactate production were assessed over 96 h.RESULTS: TNF-α at concentrations of 0.024, 0.24, 2.4 and 24 ng/ml did not affect leptin secretion over 24 h. TNF-α at concentrations of 0.24 to 24 ng/ml significantly inhibited leptin secretion over 96 h by 19–60%. TNF-α at concentrations of 0.024 to 24 ng/ml significantly decreased steady-state levels of leptin mRNA after 96 h by 32–95%. In addition, TNF-α at concentrations of 2.4 and 24 ng/ml significantly increased glucose uptake and lactate production over 96 h by 30–57%. TNF-α at a concentration of 0.024 ng/ml did not affect leptin secretion, glucose uptake or lactate production. Overall, for the TNF-α concentrations tested, leptin secretion was inversely related to the percent of glucose carbon released as lactate; however, TNF-α did not induce a proportional increase of lactate production from glucose.CONCLUSION: Short-term (24 h) exposure of isolated adipocytes to TNF-α does not affect leptin secretion. Prolonged exposure to TNF-α produces a concentration-dependent inhibition of leptin secretion and gene expression. This suggests that the acute effect of TNF-α to increase circulating leptin levels in vivo may be indirect. TNF-α at higher concentrations increases glucose uptake, but does not increase the conversion of glucose to lactate. Therefore, TNF-α appears to induce a dissociation between adipocyte glucose metabolism and leptin production.

Effect of dietary fish oil on development and selected functions of murine inflammatory macrophages.

Inflammatory macrophages from mice fed diets containing menhaden fish oil (MFO) have a reduced capacity for cytotoxicity of mastocytoma cells upon activation with interferon‐γ (IFNγ) and lipopolysaccharide due to an altered responsiveness to IFNγ. In an effort to elucidate further how dietary MFO effects macrophage function, we have studied the maturation of inflammatory macrophages from mice fed MFO compared with mice fed safflower oil (SFO) using several processes that serve as markers of the activational state. No significant differences in the recruitment or percentage of peritoneal exudate cells as macrophages after thioglycollate injection and no differences in spreading, binding, or phagocytosis of sheep erythrocytes or phagocytosis of yeast by inflammatory macrophages were observed when the dietary groups were compared. However, MFO macrophages had an altered capacity for peroxide release when stimulated with unopsonized zymosan (10–200 μg/ml). Furthermore, to elucidate how MFO feeding could alter IFNγ‐induced responses of inflammatory macrophages, we assessed phorbol‐12‐myristate‐13‐acetate‐induced hydrogen peroxide production and expression of class II MHC determinants (la). There were no differences between macrophages from mice fed the two diets with respect to the production of peroxide when they were preincubated with 0.1–10 U/ml of IFNγ. However, MFO macrophages had greater peroxide production after enhancement with 100 U/ml of IFNγ. With respect to la induction, the percentage of macrophages responding to IFNγ was not altered by diet, and there were no differences in expression of la induced by 24 hr exposure to IFNγ. Thus the differential effect of MFO compared with SFO is probably mediated not by an alteration in the maturation of inflammatory macrophages but rather through the alteration of IFNγ‐induced functions such as peroxide production.

Eicosanoids and linoleate-enhanced growth of mouse mammary tumor cells

Metastatic mouse mammary tumor cell line 4526 was used to determine whether linoleate (LN)-derived cyclooxygenase metabolites were involved in the mechanism of LN-enhanced 4526 tumor growth. Unstimulated line 4526 cells converted LN to both PGE1 and PGE2 in serum free medium (SFM). However, neither prostaglandin (PG) influenced growth, while db-cGMP, but not db-cAMP, stimulated growth to the same extent as LN. Cyclooxygenase inhibitors stimulated growth while suppressing PG synthesis. Lipoxygenase inhibitors decreased growth in a dose dependent manner. Supplemental LN had no effect on cyclooxygenase inhibition while the IC50s for lipoxygenase inhibition were increased several fold. These results indicate that lipoxygenase products rather than cyclooxygenase metabolites play a major role in LN-stimulated growth of line 4526 cells.