Accumulating evidence to support the safe and efficacious use of a proprietary blend of capsaicinoids in mediating risk factors for obesity.

Obesity is a significant public health concern, and finding safe and effective means for combating this condition is needed. This study investigates the safety and efficacy of supplementation of a blend of capsaicinoids on weight gain, fat mass, and blood chemistry in a high-fat diet (HFD) model of obesity in mice and on adipocyte differentiation and gene expression in 3T3-L1 preadipocytes. High-fat diet (HFD)-fed mice were treated with a proprietary capsaicinoid concentrate (Capsimax®; OmniActive Health Technologies Ltd., India) and compared to orlistat (ORL) and normal chow-fed mice (NC). Mice fed a high-fat diet showed significantly lower weight gain upon Capsimax® (CAP) administration than their HFD counterparts and similar to that observed with ORL animals. In addition, CAP decreased the high-fat diet-induced increases in adipose tissue and epididymal fat pad mass and hypertrophy after 52 days of treatment. Both the CAP and ORL groups had increased plasma concentrations of leptin. CAP extracts decreased triacylglycerol content in 3T3-L1 preadipocytes and decreased markers of adipogenesis including peroxisome proliferator-activated receptor (PPAR-É£) and fatty acid-binding protein 4 (FABP4). Expression of genes involved in lipogenesis such as stearoyl-CoA desaturase (SCD) and fatty acid synthase (FSN) was decreased by CAP in a dose-dependent manner. Thermogenic genes and markers of brown adipose tissue including uncoupling protein 1 (UCP1) and PR domain-containing 16 (Prdm16) were induced by CAP in the preadipocyte cells. These in vivo and in vitro data support that this proprietary capsaicinoid concentrate reduces weight gain and adiposity at least in part through decreasing lipogenesis and increasing thermogenesis.

Molecular profiling demonstrates modulation of immune cell function and matrix remodeling during luteal rescue†.

The corpus luteum (CL) is essential for maintenance of pregnancy in all mammals and luteal rescue, which occurs around day 16-19 in the cow, is necessary to maintain luteal progesterone production. Transcriptomic and proteomic profiling were performed to compare the day 17 bovine CL of the estrous cycle and pregnancy. Among mRNA and proteins measured, 140 differentially abundant mRNA and 24 differentially abundant proteins were identified. Pathway analysis was performed using four programs. Modulated pathways included T cell receptor signaling, vascular stability, cytokine signaling, and extracellular matrix remodeling. Two mRNA that were less in pregnancy were regulated by prostaglandin F2A in culture, while two mRNA that were greater in pregnancy were regulated by interferon tau. To identify mRNA that could be critical regulators of luteal fate, the mRNA that were differentially abundant during early pregnancy were compared to mRNA that were differentially abundant during luteal regression. Eight mRNA were common to both datasets, including mRNA related to regulation of steroidogenesis and gene transcription. A subset of differentially abundant mRNA and proteins, including those associated with extracellular matrix functions, were predicted targets of differentially abundant microRNA (miRNA). Integration of miRNA and protein data, using miRPath, revealed pathways such as extracellular matrix-receptor interactions, abundance of glutathione, and cellular metabolism and energy balance. Overall, this study has provided a comprehensive profile of molecular changes in the corpus luteum during maternal recognition of pregnancy and has indicated that some of these functions may be miRNA-regulated.

Replication Study: Systematic identification of genomic markers of drug sensitivity in cancer cells.

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Vanden Heuvel et al., 2016), that described how we intended to replicate selected experiments from the paper 'Systematic identification of genomic markers of drug sensitivity in cancer cells' (Garnett et al., 2012). Here we report the results. We found Ewing's sarcoma cell lines, overall, were more sensitive to the PARP inhibitor olaparib than osteosarcoma cell lines; however, while the effect was in the same direction as the original study (Figure 4C; Garnett et al., 2012), it was not statistically significant. Further, mouse mesenchymal cells transformed with either the EWS-FLI1 or FUS-CHOP rearrangement displayed similar sensitivities to olaparib, whereas the Ewing's sarcoma cell line SK-N-MC had increased olaparib sensitivity. In the original study, mouse mesenchymal cells transformed with the EWS-FLI1 rearrangement and SK-N-MC cells were found to have similar sensitivities to olaparib, whereas mesenchymal cells transformed with the FUS-CHOP rearrangement displayed a reduced sensitivity to olaparib (Figure 4E; Garnett et al., 2012). We also studied another Ewing's sarcoma cell line, A673: A673 cells depleted of EWS-FLI1 or a negative control both displayed similar sensitivities to olaparib, whereas the original study reported a decreased sensitivity to olaparib when EWS-FLI1 was depleted (Figure 4F; Garnett et al., 2012). Differences between the original study and this replication attempt, such as the use of different sarcoma cell lines and level of knockdown efficiency, are factors that might have influenced the outcomes. Finally, where possible, we report meta-analyses for each result.

Changes in Myeloid Lineage Cells in the Uterus and Peripheral Blood of Dairy Heifers During Early Pregnancy.

Establishment of pregnancy requires interaction between the developing conceptus and the uterine mucosal immune system. Myeloid lineage cells (macrophages and dendritic cells) are key mediators of pregnancy in rodents and humans but relatively little is known regarding their role and distribution during early pregnancy in ruminants. We tested the hypothesis that myeloid lineage cell number, distribution, and function are altered during early pregnancy in dairy heifers. Dairy heifers were inseminated using sperm from a single bull (Day 0), and uteri and blood were collected at slaughter on Days 17 and 20 of pregnancy to investigate the response of myeloid lineage cells to the presence of a conceptus. Responses were compared to noninseminated heifers on Day 17 of the estrous cycle. Peripheral blood and uterine-derived immune cells were isolated magnetically and examined using flow cytometry. Immunohistochemical analysis was used to evaluate the spatial distribution of myeloid lineage cells in the endometrium and quantitative polymerase chain reaction was conducted to quantify abundance of mRNA transcripts associated with myeloid lineage cell function. Transcripts for major histocompatibility complex (MHC) II, cluster of differentiation (CD) 80, CD86, CD163, and indoleamine 2,3-dioxygenase (IDO) 1 were greater in endometrium of pregnant compared to cyclic heifers. Immunofluorescence analysis revealed increased labeling for MHCII and SIRPA in pregnant compared to cyclic heifers. There were approximately 50% more CD14+CD11c+ cells in the peripheral circulation of pregnant compared to cyclic heifers. A greater number of myeloid lineage cells were observed during early pregnancy, and this increase was most pronounced in and around the shallow glands. Furthermore, expression of molecules associated with a tolerogenic or alternatively activated phenotype of these cells also increased in pregnant heifers. The results support the hypothesis that myeloid lineage cells with a tolerogenic phenotype are involved in establishment of pregnancy in dairy heifers.

Changes in MicroRNA Expression During Maturation of the Bovine Corpus Luteum: Regulation of Luteal Cell Proliferation and Function by MicroRNA-34a.

The corpus luteum (CL) develops from the remnants of the ovulatory follicle and produces progesterone, required for maintenance of pregnancy in mammals. The differentiation of granulosal and thecal cells into luteal cells is accompanied by hypertrophy and hyperplasia of cells. As the CL matures, growth ceases and in ruminants, the tissue acquires the ability to undergo regression in response to prostaglandin F2alpha. The regulators of this transition are poorly understood. MicroRNA, which are posttranscriptional regulators of tissue development and function, are expressed in the CL. However, the pattern of their expression and their function during the transition from developing to functional CL is not known. The objectives of this study were to profile the expression of miRNA in developing versus mature bovine CL and determine effects of miRNA on bovine luteal cell survival and function. Knockdown of Drosha in midcycle (MC) luteal cells decreased progesterone and increased luteal cell apoptosis in the presence or absence of proinflammatory cytokines. Microarray analysis demonstrated that a greater number of miRNA were expressed in MC compared to D4 CL. Ingenuity pathway analysis (IPA) predicted that D4-specific miRNA regulate pathways related to carbohydrate metabolism, while MC-specific miRNA regulate pathways related to cell cycle and apoptosis signaling. Both predictions are consistent with a switch in the CL from a growing phase to a maintenance phase. One of the MC specific miRNA, miR-34a, was selected for further analysis. Increased concentrations of miR-34a in MC luteal cells resulted in decreased luteal cell proliferation, increased progesterone production, and inhibition of Notch1 and YY1 translation, but had no effect on luteal cell apoptosis. In conclusion, these data support a role for miRNA in general, and miR-34a in particular, in luteal formation and function.

Regulating life or death: potential role of microRNA in rescue of the corpus luteum.

The role of miRNA in tissue biology has added a new level of understanding of gene regulation and function. The corpus luteum (CL) is a transitory endocrine gland; the dynamic nature of the CL makes it a candidate for regulation by miRNA. Rescue of the CL from luteolysis is essential for the maintenance of pregnancy in all eutherian mammals. Using next generation sequencing, we profiled miRNA expression in the bovine CL during maternal recognition of pregnancy. We identified 590 luteal miRNA, of which 544 were known and 46 were novel miRNAs. Fifteen (including 3 novel) miRNAs were differentially expressed between CL of pregnant vs. cyclic animals. Target analysis of the differentially expressed miRNA resulted in genes involved in regulating apoptosis and immune response, providing evidence that miRNAs regulate the intracellular pathways that lead to either luteal regression or survival.

Progesterone effects on lymphocytes may be mediated by membrane progesterone receptors.

Luteal cell-induced proliferation of T lymphocytes devoid of the nuclear progesterone receptor (PGR) is inhibited by progesterone. Functional effects of progesterone on bovine lymphocytes and the expression of membrane progesterone receptors (mPRs) alpha (PAQR7), beta (PAQR8), gamma (PAQR5), and progesterone receptor membrane component 1 (PGRMC1) mRNA were analyzed in corpus luteum (CL) and lymphocytes. Progesterone and a cell-impermeable progesterone conjugate caused a dose-dependent decrease in IL2 receptor α-subunit (IL2RA) mRNA and an increase in interleukin 2 (IL2) mRNA concentrations in cultured PBMCs. In luteal tissues, concentrations of PAQR7 and PAQR8 mRNA were lower in CL collected on day 11 compared with day 18, whereas PGRMC1 and PGR mRNA were greater on day 11 than on day 18. The mRNA of all three PAQRs and PGRMC1 were detected in bovine T lymphocytes, but not in B cells/monocytes. Progesterone increased intracellular Ca(++) and reduced the phosphorylation of zeta-chain-associated protein kinase 70 (Zap70). A specific, saturable, and single progesterone binding site with a steroid specificity characteristic of mPRs was demonstrated by saturation and competitive binding assays using T lymphocyte membranes, and PAQR7 receptors were localized on the plasma membranes by immunofluorescence. Thus, progesterone induces specific and rapid functional effects on T lymphocytes in the absence of PGR. The mPRs are potential intermediaries of the cell-surface actions of progesterone because they are expressed in lymphocytes, the actions of progesterone are mimicked by a cell-impermeable form of progesterone, and specific, saturable progesterone binding, which is characteristic of mPRs, is present on lymphocyte membranes.

Epidermal α6ß4 integrin stimulates the influx of immunosuppressive cells during skin tumor promotion.

BACKGROUND: Induction of α6ß4 integrin in the differentiated epidermal cell layers in skin is a hallmark of human cutaneous squamous cell carcinoma (SCC) pathogenesis and stimulates chemically induced SCC formation in Invα6ß4 transgenic mice, which exhibit persistent expression of α6ß4 in the suprabasal epidermal layers. However, the molecular basis for the support of SCC development by suprabasal α6ß4 is not fully understood. OBJECTIVE: We examined the relevance for suprabasal α6ß4 expression in the epidermis for the recruitment of immunosuppressive leukocytes during the early stages of tumor promotion. METHODS: In this study, we made use of the Invα6ß4 transgenic mouse model, which exhibits expression of α6ß4 integrin in the suprabasal layers of the epidermis driven by the involucrin promoter. First, we examined protein lysates from Invα6ß4 transgenic skin using a pro-inflammatory cytokine array panel. Next, we immunofluorescence labeling of murine skin sections was employed to immunophenotype tumor promoter-treated Invα6ß4 transgenic skin. Finally, a macrophage colony stimulating factor (M-CSF) neutralizing antibody strategy was administered to resolve Invα6ß4 transgenic skin inflammation. RESULTS: Employing the Invα6ß4 transgenic mouse model, we show that suprabasal α6ß4 integrin expression selectively alters the profile of secreted pro-inflammatory molecules by epidermal cells, in particular CXCL5 and M-CSF, in response to acute tumor promoter treatment. The induction of CXCL5 and M-CSF in Invα6ß4 transgenic epidermis was shortly followed by an exacerbated influx of CD200R(+) myeloid-derived suppressor cells (MDSCs), which co-expressed the M-CSF receptor, and FoxP3(+) Treg cells compared to wild-type mice. As a result, the levels of activated CD4(+) T lymphocytes were dramatically diminished in Invα6ß4 transgenic compared to wild-type skin, whereas similar levels of lymphocyte activation were observed in the peripheral blood. Finally, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CD200R(+) infiltrative cells and epidermal proliferation were suppressed in Invα6ß4 mice treated with M-CSF neutralizing antibodies. CONCLUSIONS: We conclude that aberrant expression of α6ß4 integrin in post-mitotic epidermal keratinocytes stimulates a pro-tumorigenic skin microenvironment by augmenting the influx of immunosuppressive granular cells during tumor promotion.

Inflammatory responses in epithelia: endotoxin-induced IL-6 secretion and iNOS/NO production are differentially regulated in mouse mammary epithelial cells.

BACKGROUND: IL-6 is a pro-inflammatory cytokine that signals via binding to a soluble or membrane bound receptor, while nitric oxide (NO), an oxidative stress molecule, diffuses through the cell membrane without a receptor. Both mediators signal through different mechanisms, yet they are dependent on NFκB. We proposed that both mediators are co-induced and co-regulated in inflamed mammary epithelial cells. METHODS: SCp2 mammary epithelial cells were treated with bacterial endotoxin (ET) for different time periods and analyzed for induction of IL-6 secretion and NO production by ELISA and Griess reaction, respectively. The expression of IL-6 and induced NO synthase (iNOS) was assayed by real time PCR and/or western immunoblots, and the activation of NFκB was assayed by immunobinding assay. To investigate the role of mammary cell microenvironment (cell-substratum or interaction of mammary epithelial cell types; critical to mammary development, function, and disease) in modulation of the inflammatory response, SCp2 cells were cultured with or without extracellular matrix (EHS) or in coculture with their myoepithelial counterpart (SCg6), and assayed for ET-induced IL-6 and NO. RESULTS: Endotoxin induced NFκB activation at 1 h after ET application. IL-6 secretion and NO production were induced, but with unexpected delay in expression of mRNA for iNOS compared to IL-6. NFκB/p65 activation was transient but NFκB/p50 activation persisted longer. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in IL-6 and iNOS regulation via NFκB. SCp2 cells in coculture with SCg6 but not in presence of EHS dramatically induced IL-6 secretion even in the absence of ET. ET-induced NO production was blunted in SCp2/SCg6 cocultures compared to that in SCp2 alone. CONCLUSIONS: The differential regulation of IL-6 and iNOS together with the differential activation of different NFκB dimers suggest that IL-6 and iNOS are regulated by different NFκB dimers, and differentially regulated by the microenvironment of epithelial cells. The understanding of innate immune responses and inflammation in epithelia and linkage thereof is crucial for understanding the link between chronic inflammation and cancer in epithelial tissues such as the mammary gland.

Regulation of ultraviolet B radiation-mediated activation of AP1 signaling by retinoids in primary keratinocytes.

The main cause of skin cancer and photo-aging is chronic exposure to ultraviolet B (UVB) radiation. Such damage can be ameliorated by retinoid treatment. UVB-radiation-induced skin carcinogenesis is associated with the induction of activator protein 1 (AP1) signaling and factors, namely FOS and JUN family members. We investigated the effects of several retinoids, all-trans-retinoic acid (tRA), 9-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl)-retinamide (HPR), on UVB-induced damage in primary mouse keratinocytes. In addition, the interplay between UVB radiation, retinoid receptors, and AP1 signaling was assessed using Western blot analysis and ribonuclease protection and gene reporter assays. Exposure of keratinocytes to UVB radiation caused a down-regulation of the retinoid receptor protein levels in a proteasome-mediated manner. In contrast, FOS and JUN proteins were transiently induced shortly after exposure to UVB radiation. Retinoid treatment caused a dose-dependent reduction in the levels of retinoid receptor proteins. When irradiated cells were treated with retinoids, no significant effects on AP1 protein expression were noted. Interestingly, pretreatments with tRA and cRA, but not HPR, suppressed UVB-radiation-induced AP1 activity by more than 50%, whereas post-treatment failed to produce similar effects. Our findings indicate that the inhibition of AP1 activity by retinoids explains, at least in part, the chemopreventive potential of retinoids in UV-radiation-associated epidermal damage.

Protective effect of vitamin E on ultraviolet B light-induced damage in keratinocytes.

Ultraviolet (UV) B radiation is the most common environmental factor in the pathogenesis of skin cancer. Exposure of human skin to UVB radiation leads to the depletion of cutaneous antioxidants, the activation of nuclear factor kappa B (NF-kappaB), and programmed cell death (apoptosis). Although antioxidant supplementation has been shown to prevent UVB-induced photooxidative damage, its effect on components of cell signaling pathways leading to gene expression has not been clearly established. In the present study, the effect of the antioxidant vitamin, alpha-tocopherol (alpha-T), and its acetate analog, alpha-tocopherol acetate (alpha-TAc), on UVB-induced damage in primary and neoplastic mouse keratinocytes was investigated. The ability of both vitamins to modulate UVB-induced apoptosis and activation of the transcription factor NF-kappaB were studied. Treatment of normal and neoplastic mouse epidermal keratinocytes (308 cells) with 30-60 mJ/cm(2) UVB markedly decreased viable cell number and was accompanied by DNA fragmentation. When both vitamins were applied to cells at times before and after UVB radiation, a significant increase in the percentage of viable cells and concomitant decrease in the number of apoptotic cells was noted, with vitamin pretreatment providing a better protection than posttreatment. Simultaneous posttreatment of irradiated cells with alpha-TAc abolished the cytotoxic effects of UVB and restored cell viability to control levels. In addition, simultaneous posttreatment of irradiated cells with alpha-T reduced the number of apoptotic cells by half, indicating a synergistic effect of two such treatments compared with any single one. Flow cytometry analysis indicated that vitamin treatment suppressed both an increase in pre-G0 cells and a decrease in cycling cells by UVB exposure. In addition, NF-kappaB activation was detected 2 h after UV exposure and was maintained for up to 8 h. Pretreatment with vitamins significantly inhibited NF-kappaB activation at 4 and 8 h. These results indicate that vitamin E and its acetate analog can modulate the cellular response to UVB partly through their action on NF-kappaB activation. Thus, these antioxidant vitamins are potential drugs for the protection from or the reduction of UVB-associated epidermal damage.