TRAF3 loss-of-function reveals the noncanonical NF-κB pathway as a therapeutic target in diffuse large B cell lymphoma.

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.

Butyrate and tributyrin reduce LPS-induced inflammatory cytokine production from human visceral fat.

INTRODUCTION: The current obesity crisis has resulted in many people with excess adipose tissue suffering from chronic inflammation. This inflammation is largely due to the release of cytokines and chemokines from visceral fat. The aim of this study was to identify potential anti-inflammatory agents that might alleviate obesity-induced chronic inflammation. METHODS: To identify agents that might alleviate this obesity-induced chronic inflammation we have developed a simple protocol for incubating intact pieces of human visceral adipose tissue in 35 mm tissue culture plates, in the presence of low-dose lipopolysaccharide (LPS) and co-incubating these samples with potential anti-inflammatory agents. RNA-Seq analysis was performed to identify enriched gene expression signatures among the most significantly differentially expressed genes. RESULTS: From this screen, we have identified the short-chain fatty acid (SCFA) sodium butyrate and its triacylglyceride form, tributyrin, as effective agents, significantly reducing the production of LPS-induced inflammatory cytokines and chemokines from all adipose tissue samples tested. As well, these agents appear to be non-toxic at the concentrations tested. RNA-Seq analysis has revealed that IL36γ is one of the most upregulated genes in response to LPS and one of the most downregulated when sodium butyrate is added to human fat samples stimulated with LPS. IL-36γ ELISAs confirmed this holds true at the protein level as well. CONCLUSIONS: These studies suggest that the short-chain fatty acid, sodium butyrate, and its triacylglyceride form, tributyrin, might alleviate the chronic inflammation that is associated with many individuals with obesity.

A low-carbohydrate diet containing soy protein and fish oil reduces breast but not prostate cancer in C3(1)/Tag mice.

We recently showed that a low-carbohydrate (CHO) diet containing soy protein and fish oil dramatically reduces lung nodules in a mouse model of lung cancer when compared to a Western diet. To explore the universality of this finding, we herein compared this low-CHO diet to a Western diet on in preventing breast and prostate cancer using a mouse model that expresses the SV40 large T-antigen specifically in breast epithelia in females and prostate epithelia in males. We found that breast cancer was significantly reduced with this low-CHO diet and this correlated with a reduction in plasma levels of glucose, insulin, IL-6, TNFα and prostaglandin E2 (PGE2). This also corresponded with a reduction in the Ki67 proliferation index within breast tumors. On the other hand, this low-CHO diet did not reduce the incidence of prostate cancer in the male mice. Although it reduced both blood glucose and insulin to the same extent as in the female mice, there was no reduction in plasma IL-6, TNFα or PGE2 levels, or in the Ki67 proliferation index in prostate lesions. Based on immunohistochemistry studies with antibodies to 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), carnitine palmitoyltransferase Ia (CPT1a) and fatty acid synthase (FAS), it is likely that this difference in response of the two cancer types to this low-CHO diet reflects differences in the glucose dependence of breast and prostate cancer, with the former being highly dependent on glucose for energy and the latter being more dependent on fatty acids.

Low carbohydrate diets containing soy protein and fish oil slow the growth of established NNK-induced lung tumors.

We recently found that a diet composed of 15% of total calories as carbohydrate (CHO), primarily as amylose, 35% soy protein and 50% fat, primarily as fish oil (FO) (15%Amylose/Soy/FO) was highly effective at preventing lung nodule formation in a nicotine-derived nitrosamine ketone (NNK)-induced lung cancer model. We asked herein whether adopting such a diet once cancers are established might also be beneficial. To test this, NNK-induced lung nodules were established in mice on a Western diet and the mice were then either kept on a Western diet or switched to various low CHO diets. Since we previously found that sedentary mice develop more lung nodules than active mice, we also compared the effect of exercise in this cancer progression model. We found that switching to a 15%Amylose/Soy/FO diet reduced lung nodules and slowed tumor growth with both 'active' and 'sedentary' mice. Ki67, cleaved caspase 3 and Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling assays suggested that the efficacy of the 15%Amylose/Soy/FO in lowering tumor nodule count and size was not due to a reduction in tumor cell proliferation, but to an increase in apoptosis. The 15%Amylose/Soy/FO diet also significantly lowered liver fatty acid synthase and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 expression, pointing to a global metabolic switch from glycolysis to fatty acid oxidation. Mice fed the 15%Amylose/Soy/FO diet also had significantly reduced plasma levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor α. These results suggest that the 15%Amylose/Soy/FO diet may slow tumor growth by suppressing proinflammatory cytokines, inducing a metabolic switch away from glycolysis and inducing apoptosis in tumors.

The effect of diet and exercise on tobacco carcinogen-induced lung cancer.

In previous studies, we found that low-carbohydrate (CHO) diets reduced the incidence of tumors in mice genetically predisposed to cancer. However, because >90% of human cancers arise via carcinogen-induced somatic mutations, we investigated, herein, the role that different types and levels of CHO, protein and lipid play in lung cancer induced by the tobacco-specific carcinogen, nicotine-derived nitrosamine ketone (NNK) in A/J mice. We found lowering CHO levels significantly reduced lung nodules and blood glucose levels. We also found that soy protein was superior to casein and that coconut oil was ineffective at reducing lung nodules. Diets containing amylose or inulin (at 15% of total calories), soy protein (at 35%) and fat (at 50%, 30% being fish oil) were the most effective at reducing lung nodules. These fish oil-containing diets increased plasma levels of the ketone body, ß-hydroxybutyrate, while reducing both insulin and 8-isoprostane in plasma and bronchoalveolar interleukin-12 and lung PGE2 levels. After only 2 weeks on this diet, the levels of γ-H2AX were significantly reduced, 24 hours after NNK treatment. Housing these mice in two-tiered rat cages with exercise wheels led to similar mouse weights on the different diets, whereas keeping mice in standard mouse cages led to both significant weight differences between the low-CHO, soy protein, fish oil diet and Western diet and substantially more lung nodules than in the two-tiered cages. Our results suggest that low-CHO, soy protein, fish oil-containing diets, together with exercise, may reduce the incidence of lung cancer.

Tocopherol isoforms (α-, γ-, and δ-) show distinct capacities to control Nrf-2 and NfκB signaling pathways that modulate inflammatory response in Caco-2 intestinal cells.

We recently showed that α-, γ-, and δ-tocopherols (Toc) were isoform dependent in modulating an inflammatory response in differentiated human Caco-2 intestinal cells. Here, we aim to investigate the relative capacity of Toc isoforms to modify the stress-activated NfκB and Nrf-2 signaling pathways that regulate the expression of pro-inflammatory cytokines and antioxidant enzymes, respectively, in this well-established in vitro model of the small intestine The modulation of IFNγ/phorbol myristate acetate (PMA)-induced inflammatory responses, determined by the expression of IL8 mRNA and protein, corresponded to the extent by which different Toc isoforms altered intracellular oxidative status in Caco-2 cells. α Toc was more effective at suppressing IFNγ/PMA-induced NfκB activation than γ-Toc, while δ-Toc was ineffective. On the other hand, only δ-Toc and to a lesser extent γ-Toc promoted IFNγ/PMA-induced Nrf-2 activation. Up-regulation of Nrf-2 by δ-Toc coincided with a decrease in GSH/GSSG ratio, thus pointing to pro-oxidant activity of δ-Toc isoform in IFNγ/PMA-stimulated Caco-2 cells. The induction of oxidative stress in IFNγ/PMA-treated cells by δ-Toc was lowered (P < 0.05) in the presence of ascorbic acid. Ascorbic acid also enabled a greater suppression of IL8 secretion than when cells were treated with δ-Toc isoform alone. Our findings show that δ-Toc uniquely promoted oxidative stress which translated to Toc isoform-specific modulation of the stress-activated Nrf-2 and NfκB signaling pathway and an influence on IL8 expression.

A ketogenic diet rich in fish oil is superior to other fats in preventing NNK-induced lung cancer in A/J mice.

Given that ketogenic diets (KDs) are extremely high in dietary fat, we compared different fats in KDs to determine which was the best for cancer prevention. Specifically, we compared a Western and a 15% carbohydrate diet to seven different KDs, containing either Western fats or fats enriched in medium chain fatty acids (MCTs), milk fat (MF), palm oil (PO), olive oil (OO), corn oil (CO) or fish oil (FO) for their ability to reduce nicotine-derived nitrosamine ketone (NNK)-induced lung cancer in mice. While all the KDs tested were more effective at reducing lung nodules than the Western or 15% carbohydrate diet, the FO-KD was most effective at reducing lung nodules. Correlating with this, mice on the FO-KD had low blood glucose and the highest ß-hydroxybutyrate level, lowest liver fatty acid synthase/carnitine palmitoyl-1a ratio and a dramatic increase in fecal Akkermansia. We found no liver damage induced by the FO-KD, while the ratio of total cholesterol/HDL was unchanged on the different diets. We conclude that a FO-KD is superior to KDs enriched in other fats in reducing NNK-induced lung cancer, perhaps by being the most effective at skewing whole-body metabolism from a dependence on glucose to fats as an energy source.

Modulation of NF-κB and Nrf2 control of inflammatory responses in FHs 74 Int cell line is tocopherol isoform-specific.

The present study investigates the relative ability of α-, γ-, and δ-tocopherol (Toc) to modulate cell signaling events that are associated with inflammatory responses in fetal-derived intestinal (FHs 74 Int) cells. Secretion of the proinflammatory cytokine IL-8 in FHs 74 Int cells was stimulated in the following order: α-Toc<γ-Toc<δ-Toc. A similar proinflammatory response was observed when inflammation was induced in FHs 74 Int cells. Modulation of IL-8 expression by Toc corresponded to an isoform-specific modulation of NF-κB and nuclear factor-erythroid 2-related factor 2 (Nrf2) cell signaling pathways involved in expression of proinflammatory cytokines and antioxidant enzymes, respectively. δ-Toc and, to a lesser extent, γ-Toc activated NF-κB and Nrf2 signaling, as indicated by the greater nuclear translocation of transcription factors. Activation of NF-κB signaling by γ- and δ-Toc was accompanied by upregulation of NF-κB target genes, such as IL-8 and prostaglandin-endoperoxide synthase 2, with and without a prior IFNγ-PMA challenge. Nevertheless, γ- and δ-Toc, particularly δ-Toc, concurrently downregulated glutamate-cysteine ligase, a Nrf2 target gene that encodes for glutathione biosynthesis. This observation was substantiated by confirmation that γ- and δ-Toc were effective at decreasing glutamate-cysteine ligase protein expression and cellular glutathione content. Downregulation of glutathione content in fetal intestinal cells corresponded to induction of apoptosis-mediated cytotoxicity. In conclusion, γ- and δ-Toc are biologically active isoforms of vitamin E and show superior bioactivity to α-Toc in modulating cell signaling events that contribute to a proinflammatory response in fetal-derived intestinal cells.

Differences in vitamin E and C profile between infant formula and human milk and relative susceptibility to lipid oxidation.

The vitamin E isoforms and vitamin (vit) C content of infant formulas were compared to human milk and related to relative susceptibilities to lipid peroxidation. We report that a highly distinct vitamin E and C profile exists between formula and human milk. Whileα-tocopherol (α-Toc) is the dominant vit E isoform in human milk, formula contains a substantial amount of α-Toc and δ-Toc that was greater than the level found in human milk (12- and 32-fold, respectively). Vitamin C was also two- fold higher in infant formula compared to human milk. Despite the higher vitamin E and C content, we also observed higher rates of lipid oxidation in the formula when compared to human milk. Storing human milk for one day at refrigeration temperatures did not produce hexanal in human milk, but this storage resulted in an increase in hexanal in formulas. We conclude that the higher concentrations of γ-Toc and δ-Toc in infant formulas did not provide similar protection from lipid oxidation as human milk. We also observed that vit C content was reduced during storage in both infant formula and human milk, which did not occur with the Toc isoforms.

Development of a novel human triculture model of non-alcoholic fatty liver disease and identification of berberine as ameliorating steatosis, oxidative stress and fibrosis.

Objectives: Non-alcoholic fatty liver disease (NAFLD) and its progression to non-alcoholic steatohepatitis (NASH) and hepatocarcinoma is a serious and growing problem. However, the development of new therapies is severely hindered by a lack of high-throughput assays for drug testing. Methods: We have developed a simple transwell assay comprised of HepG2 hepatocytes, hepatic LX-2 stellate cells, and differentiated THP-1 cells. The cells were incubated with an activating mixture containing the NASH-associated risk factors, glucose, insulin, free fatty acids (FFAs), and lipopolysaccharide (LPS) for 72 h. We compared different combinations of culture conditions to obtain a model system that recapitulates the main features of NAFLD/NASH, i.e., increased steatosis, reactive oxygen species (ROS), secretion of pro-inflammatory cytokines/chemokines, and presence of fibrosis. To confirm the usefulness of the optimized model system, we screened for compounds that inhibit steatosis in the hepatocytes and evaluated the most effective compound in the triculture model system. Results: The activating mixture stimulated HepG2 cells in this triculture to accumulate more fat and produce higher levels of reactive oxygen species (ROS) than HepG2 cells in monocultures. As well, higher levels of inflammatory cytokines and chemokines (IL-8, IL-6, MIP-1α, etc.) were produced in this triculture compared to monocultures. In addition, in all LX-2 monocultures and cocultures, exposure to the activating mixture increased markers of fibrosis. A major strength of our triculture system is that it makes possible the simultaneous monitoring of 4 main features of NASH, i.e., steatosis, oxidative stress, inflammation and fibrosis. Screening potential modulators that may reduce steatosis in HepG2 cells revealed the protective effects of the isoalkaloid, berberine. Tested using this novel triculture assay, treatment with 5 µM berberine decreased steatosis and ROS in HepG2 hepatocytes, reduced inflammatory cytokine production and inhibited collagen production from LX-2 cells. Conclusion: This simple triculture model recapitulates the main features of NAFLD/NASH and should be useful for high-throughput preclinical drug discovery. In this model, berberine showed promising results in decreasing steatosis and ROS and protection against fibrosis.

The Antioxidant Capacity of Breast Milk and Plasma of Women with or without Gestational Diabetes Mellitus.

Women with gestational diabetes (GD) have reduced antioxidant capacity; however, the relationship between maternal diet, maternal biochemical capacity, breast milk concentration, and infant intake has not been adequately explored in the literature. An exploration of underlying mechanism(s) is warranted, particularly for nutrient antioxidants impacted by maternal intake. These nutrients may provide a means for modifying maternal and infant antioxidant capacity. Oxygen radical absorbance capacity (ORAC), alpha-tocopherol, ascorbic acid, and beta-carotene concentrations were measured in breast milk of women with and without GD. Plasma, three-day diet records, and breast milk were collected at 6 to 8 weeks postpartum. Student's t-test was used to compare breast milk ORAC, nutrient antioxidant concentration and plasma ORAC between women with and without GD. Pearson correlations were used to determine associations among antioxidant concentrations in breast milk and dietary antioxidant intake. Breast milk antioxidant concentrations were associated with maternal intake of beta-carotene (r = 0.629, p = 0.005). Breast milk and plasma ORAC and antioxidant vitamin concentrations were not significantly different between GD and NG women. Breast milk ORAC associated with breast milk alpha-tocopherol for NG (r = 0.763, p = 0.010), but not GD women (r = 0.385, p = 0.35), and with breast milk ascorbic acid for GD (r = 0.722, p = 0.043) but not NG women (r = 0.141, p = 0.70; interaction p = 0.041). In GD participants, breast milk ORAC was significantly associated with plasma ORAC (r = 0.780, p = 0.039). ORAC and antioxidant vitamin concentrations in breast milk in women with GD were comparable to women with NG; however, the relationships between breast milk ORAC and vitamin concentrations differed in GD versus NG women for alpha-tocopherol and ascorbic acid.

A low carbohydrate diet high in fish oil and soy protein delays inflammation, hematopoietic stem cell depletion, and mortality in miR-146a knock-out mice.

Since our previous studies found a low carbohydrate (CHO) diet containing soy protein and fish oil (15%Amylose/Soy/FO) significantly reduced lung and breast cancer in mice we asked herein if this low CHO diet could also delay the onset of myeloid malignancies. To test this we employed a miR-146a knock-out (KO) mouse model and found the 15%Amylose/Soy/FO diet increased their median lifespan by 8.5 month, compared to these mice on a Western diet. This was associated with increased lymphocytes and reduced monocytes, granulocytes, blood glucose and insulin levels. Inflammatory cytokine/chemokine studies carried out with 6-month-old mice, before any signs of illness, revealed the 15%Amylose/Soy/FO diet significantly reduced pro-inflammatory cytokines. This low CHO diet also led to an increase in plasma ß-hydroxybutyrate and in liver fatty acid synthase levels. This, together with higher liver carnitine palmitoyltransferase I levels suggested that the 15%Amylose/Soy/FO diet was causing a systemic metabolic shift from glucose to fatty acids as an energy source. Lastly, we found the 15%Amylose/Soy/FO diet resulted in significantly higher numbers of primitive hematopoietic stem cells (HSCs) in the bone marrow of 6-month-old mice than those fed a Western diet. Taken together, these results suggest a 15%Amylose/Soy/FO diet reduces chronic inflammation and increases fatty acid oxidation and that this, in turn, may prevent HSC proliferation and exhaustion, thereby delaying myeloid malignancy-induced death of miR-146a KO mice. We suggest a low CHO diet containing soy protein and fish oil could be beneficial in reducing the risk of myeloid malignancies in patients with low miR-146a levels.

Omega 3 supplementation reduces C-reactive protein, prostaglandin E2 and the granulocyte/lymphocyte ratio in heavy smokers: An open-label randomized crossover trial.

Objectives: Given the current controversy concerning the efficacy of omega 3 supplements at reducing inflammation, we evaluated the safety and efficacy of omega 3 on reducing inflammation in people with a 6-year lung cancer risk >1.5% and a C reactive protein (CRP) level >2 mg/L in a phase IIa cross-over study. Materials and methods: Forty-nine healthy participants ages 55 to 80, who were still smoking or had smoked in the past with ≥30 pack-years smoking history, living in British Columbia, Canada, were randomized in an open-label trial to receive 2.4 g eicosapentaenoic acid (EPA) + 1.2 g docosahexaenoic acid (DHA)/day for 6 months followed by observation for 6 months or observation for 6 months first and then active treatment for the next 6 months. Blood samples were collected over 1 year for measurement of plasma CRP, plasma and red blood cell (RBC) membrane levels of EPA, DHA and other fatty acids, Prostaglandin E2 (PGE2), Leukotriene B4 (LTB4) and an inflammatory marker panel. Results: Twenty one participants who began the trial within the active arm completed the trial while 20 participants who started in the control arm completed the study. Taking omega 3 resulted in a significant decrease in plasma CRP and PGE2 but not LTB4 levels. Importantly, the effect size for the primary outcome, CRP values, at the end of the intervention relative to baseline was medium (Cohen's d = 0.56). DHA, but not EPA levels in RBC membranes inversely correlated with PGE2 levels. Omega 3 also led to a significant reduction in granulocytes and an increase in lymphocytes. These high-dose omega 3 supplements were well tolerated, with only minor gastrointestinal symptoms in a subset of participants. Conclusion: Omega 3 fatty acids taken at 3.6 g/day significantly reduce systemic inflammation with negligible adverse health effects in people who smoke or have smoked and are at high risk of lung cancer.ClinicalTrials.gov, NCT number: NCT03936621.

Tryptophan from human milk induces oxidative stress and upregulates the Nrf-2-mediated stress response in human intestinal cell lines.

Chemical screening of digested human milk protein using the oxygen radical absorbance capacity (ORAC(FL)) antioxidant assay confirmed the presence of a peptide fraction (PF23) with high antioxidant activity [5.53 mmol Trolox equivalents (TE)/g] that contained tryptophan as a main component. We evaluated the effects of both PF23 and tryptophan alone on the modulation of oxidative stress in cultured intestinal cells using a dichlorofluorescein diacetate probe. Despite the high ORAC(FL) value, PF23 enhanced (P < 0.05) 2, 2'-azobis (2-amidinopropane) dihydrochloride (peroxyl radical generator)-induced intracellular oxidation in the Caco-2 human adenocarcinoma cell line, suggesting prooxidant activity. Compared to selected peptide fractions with relatively lower ORAC(FL) values, PF23 induced oxidative stress more than all other peptide fractions tested (P < 0.05) and contained more tryptophan than the others (P < 0.05). Similar prooxidant activity was observed for tryptophan when it was added to culture medium for both the Caco-2 cells and FHs 74 Int primary fetal enterocytes, while also exhibiting a high ORAC(FL) value (9.69 mmol TE/g). The effect of tryptophan that involves activation of the Nrf-2 pathway and transcription of antioxidant enzymes was therefore investigated in FHs 74 Int cells. Exposure of infant intestinal cells to tryptophan resulted in Nrf-2 activation and an increase in the gene transcript level of glutathione peroxidase 2. We conclude that tryptophan-induced oxidative stress associated with tryptophan-containing milk peptides induces an adaptive response that involves the activation of the antioxidant responsive signaling pathway in intestinal cells.

The Pros and Cons of Low Carbohydrate and Ketogenic Diets in the Prevention and Treatment of Cancer.

Ketogenic diets are low carbohydrate (CHO), high fat diets that are currently very popular for weight loss. Since cancer cells typically consume far more glucose than normal cells, low CHO diets are currently being considered as possible therapeutic regimens to manage cancer. However, our understanding of the safety and efficacy of such CHO-restricted diets in the prevention and treatment of cancer is still in its infancy. In this perspective we provide an overview of the current state of knowledge regarding the use of low CHO diets in the prevention and treatment of cancer. We also highlight the gaps in our knowledge regarding the potential usefulness of low CHO diets in cancer. While pre-clinical rodent studies have provided convincing evidence that CHO restriction may be effective in reducing cancer growth, there has not been sufficient attention given to the effect of these low CHO diets, that are often high in fats and low in soluble fiber, on inflammation. This is important, given that different fats have distinct effects on inflammation. As well, we demonstrate that short chain fatty acids, which are produced via the fermentation of fiber by our gut microbiome, have more anti-inflammatory properties than ß-hydroxybutyrate, a ketone body produced during nutritional ketosis that is touted to have anti-inflammatory activity. Since chronic inflammation is strongly associated with cancer formation, defining the type of fats in low CHO diets may contribute to our understanding of whether these diets may work simply by reducing glucose bioavailability, or via modulation of inflammatory responses.

The effect of smoking on chronic inflammation, immune function and blood cell composition.

Smoking is the number one risk factor for cancer mortality but only 15-20% of heavy smokers develop lung cancer. It would, therefore, be of great benefit to identify those at high risk early on so that preventative measures can be initiated. To investigate this, we evaluated the effects of smoking on inflammatory markers, innate and adaptive immune responses to bacterial and viral challenges and blood cell composition. We found that plasma samples from 30 heavy smokers (16 men and 14 women) had significantly higher CRP, fibrinogen, IL-6 and CEA levels than 36 non-smoking controls. Whole blood samples from smokers, incubated for 7 h at 37 °C in the absence of any exogenous stimuli, secreted significantly higher levels of IL-8 and a number of other cytokines/chemokines than non-smokers. When challenged for 7 h with E. coli, whole blood samples from smokers secreted significantly lower levels of many inflammatory cytokines/chemokines. However, when stimulated with HSV-1, significantly higher levels of both PGE2 and many cytokines/chemokines were secreted from smokers' blood samples than from controls. In terms of blood cell composition, red blood cells, hematocrits, hemoglobin levels, MCV, MCH, MCHC, Pct and RDW levels were all elevated in smokers, in keeping with their compromised lung capacity. As well, total leukocytes were significantly higher, driven by increases in granulocytes and monocytes. In addition, smokers had lower NK cells and higher Tregs than controls, suggesting that smoking may reduce the ability to kill nascent tumor cells. Importantly, there was substantial person-to person variation amongst smokers with some showing markedly different values from controls and others showing normal levels of many parameters measured, indicating the former may be at significantly higher risk of developing lung cancer.

Exploratory examination of inflammation state, immune response and blood cell composition in a human obese cohort to identify potential markers predicting cancer risk.

Obesity has reached epidemic proportions and is often accompanied by elevated levels of pro-inflammatory cytokines that promote many chronic diseases, including cancer. However, not all obese people develop these diseases and it would be very helpful to identify those at high risk early on so that preventative measures can be instituted. We performed an extensive evaluation of the effects of obesity on inflammatory markers, on innate and adaptive immune responses, and on blood cell composition to identify markers that might be useful in distinguishing those at elevated risk of cancer. Plasma samples from 42 volunteers with a BMI>35 had significantly higher CRP, PGE2, IL-1RA, IL-6 and IL-17 levels than 34 volunteers with normal BMIs. Of the cytokines and chemokines tested, only IL-17 was significantly higher in men with a BMI>35 than women with a BMI>35. As well, only IL-17 was significantly higher in those with a BMI>35 that had type 2 diabetes versus those without type 2 diabetes. Whole blood samples from participants with a BMI>35, when challenged with E. coli, produced significantly higher levels of IL-1RA while HSV-1 challenge resulted in significantly elevated IL-1RA and VEGF, and a non-significant increase in G-CSF and IL-8 levels. T cell activation of PBMCs, via anti-CD3 plus anti-CD28, resulted in significantly higher IFNγ production from volunteers with a BMI>35. In terms of blood cells, red blood cell distribution width (RDW), monocytes, granulocytes, CD4+T cells and Tregs were all significantly higher while, natural killer (NK) and CD8+ T cells were all significantly lower in the BMI>35 cohort, suggesting that obesity may reduce the ability to kill nascent tumor cells. Importantly, however, there was considerable person-to-person variation amongst participants with a BMI>35, with some volunteers showing markedly different values from controls and others showing normal levels of many parameters measured. These person-to-person variations may prove useful in identifying those at high risk of developing cancer.

Tryptophan released from mother's milk has antioxidant properties.

Bioactive factors in human milk (HM) are crucial to the health of newborns, especially preterm infants. These compounds assist in reducing the oxidative stress that may occur as a result of combined exposure to supplemental oxygen and immature physiologic defenses. To identify the components in HM that contribute to its greater resistance to oxidative stress compared with infant formulae, enzymatic hydrolysates of HM were prepared, ultrafiltered, separated, and analyzed for antioxidant potential. The antioxidant activity [microM Trolox equivalent (TE/g)] of nondigested milk, whole digested milk, and derived ultrafiltrates were 80.4 +/- 13.3, 159.0 +/- 5.6, and 127.4 +/- 3.1, respectively. An HPLC fraction denoted as fraction 23 (5274 +/- 630 microM TE/g) was obtained and its constituents identified as tryptophan (Trp), peptides HNPI, and PLAPQA. Scavenging activity was not observed for PLAPQA, whereas moderate activity was associated with HNPI (144 +/- 10.7 microM TE/g) and very high activity to Trp (7986 +/- 468 microM TE/g). Trp addition to HM and two infant formulas significantly increased formulae antioxidant properties. Trp appeared to be a powerful free radical scavenger naturally present in HM. Its antioxidant effects and potential application in the diets of infants, particularly preterm, must be examined further.

UV Light-inactivated HSV-1 Stimulates Natural Killer Cell-induced Killing of Prostate Cancer Cells.

Herein we demonstrate that ultraviolet light-inactivated Herpes Simplex Virus-1 (UV-HSV-1) stimulates peripheral blood mononuclear cells (PBMCs) to lyse both androgen-sensitive and androgen-independent prostate cancer (PrCA) cell lines, but not the benign prostatic hyperplastic epithelial cell line, BPH-1, and is 1000-10,000-fold more potent at stimulating this killing than ultraviolet light-inactivated Vesicular Stomatitis Virus, adenovirus, reovirus or cytomegalovirus. Among PBMCs, natural killer (NK) cells appear to be a major cell type involved in this killing and UV-HSV-1 appears to directly and potently stimulate NK cell expression of CD69, degranulation, cytokine production, and migration to IL-8 in PC3 conditioned medium. We also found that UV-HSV-1 stimulates glycolysis in PBMCs and NK cells, and that 2-deoxyglucose and the protein kinase C inhibitor, Go6976, and the NFκB inhibitor, Bay 11-7082, all abrogate UV-HSV-1 activated killing of PC3 cells by PBMCs and NK cells. Using neutralizing anti-Toll-like receptor 2 (TLR2) we found that UV-HSV-1, like HSV-1, activates NK cells via TLR2. Taken together, these results are consistent with Toll-like receptor 2 ligands on UV-HSV-1 stimulating TLR2 on NK cells to activate protein kinase C, leading to enhanced glycolysis and NFκB activation, both of which play a critical role in this anti-PrCA innate immune response. Importantly, UV-HSV-1 synergizes with IL-15 to increase the cytolytic activity of PBMCs against PC3 cells and there was considerable donor-to-donor variation in killing ability. These results support the preclinical development of UV-HSV-1 as an adjuvant, in combination with IL-15, for cell infusions of healthy, preselected NK cells to treat PrCA.

Evaluation of viability assays for anthocyanins in cultured cells.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a widely accepted cytotoxicity assay which can produce inaccurate results due to possible interference with the antioxidant property of anthocyanins. Alternative methods to the MTT assay, such as BrdU (DNA-based) and CellTiter-Glo (ATP-based) assays were evaluated to assess anthocyanin cytotoxicity, derived from blackberry in LNCaP, MCF-7 and MDA-MB-453 cell lines. The standard cell counting method was the reference assay. Greater correlation of cell viability values following anthocyanin exposure was obtained from multiple cell lines with the alternative assays when compared with cell counting. MTT and cell counting results were not always correlated, albeit this was a function of cell type. In particular, poor correlations between cell counting and MTT procedures used to assess cytotoxicity of anthocyanins were observed in the MDA-MB-453 cell lines. Comparison of cytotoxicity derived from alternative assays and the MTT assays with the cell counting method was dependent on the assay procedure and the cell type. The LC(50) of blackberry crude extract ranged from 0.4 to 9.4 mg/mL between assays and across all cell lines, whereas a semi-purified anthocyanin extract was not cytotoxic. Cytotoxicity evaluation of polyphenolic-rich extracts using BrdU and CellTiter-Glo assays as alternatives to the MTT method is recommended.