qPCR validation of RNA-seq result.

<p>Quantitative PCR for (A) <i>gli1</i>, (B) <i>ptch1</i>, and (C) <i>hsd11b1</i> was performed on Hh ligand treated and Hedgehog ligand and SFE co-treated TRAMPC2 cells. Transcripts concentrations were normalized to control. * indicates p<0.05. In the lower figure, transcripts concentrations of (A) <i>gli1</i>, (B) <i>ptch1</i>, and (C) <i>hsd11b1</i> are represented by quantified sequencing reads, in the form of counts-per-million-reads.</p

Sutherlandia Extract alters genes in TRAMPC2 cells.

<p>(A) Differentially expressed genes in response to Sutherlandia extract treatment. Genes that are related to (B, C, D) are labeled. (B) Gene Ontology analysis of Sutherlandia responsive genes. (C, D) KEGG pathway analysis of Sutherlandia responsive genes.</p

Heat map of Sutherlandia Extract altered Hedgehog-signaling pathway target genes expression.

<p>TRAMPC2 cell were treated with either Hh-CM or co-treated with Hh-CM and 80μg/ml SFE. Over 50% of Hh-responsive genes were repressed by SFE treatment. Gene expression values were represented by Log2 transformed normalized RNA-seq reads (Log2 count-per-million-reads) and color coded.</p

The Role of Estrogen Signaling in a Mouse Model of Inflammatory Bowel Disease: A Helicobacter Hepaticus Model

<div><p>The pathogenesis of inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, is due in part to interactions between the immune system, genetics, the environment, and endogenous microbiota. Gonadal sex hormones (GSH), such as estrogen, are thought to be involved in the development of IBD as variations in disease severity occur during pregnancy, menopause, or oral contraceptives use. In certain strains of mice, infection with <i>Helicobacter hepaticus</i> triggers IBD-like mucosal inflammation that is more severe in female mice than in males, suggesting a role for GSH in this model. To determine the role of estrogen signaling in microbiota-induced intestinal inflammation, estrogen receptor (ER) α and β knock-out (KO) mice, ER agonists, and adoptive transfers were utilized. We demonstrate that, when signaling is limited to ERβ on a non-CD4⁺ cell subset, disease is less severe and this correlates with decreased expression of pro-inflammatory mediators.</p></div

Estrogen treatment decreases disease severity and the expression of pro-inflammatory cytokines and chemokines.

<p>(A) Representative histologic images of ceca from ovariectomized A/J mice sacrificed 90 days after <i>Helicobacter hepaticus</i> inoculation and implantation with a subcutaneous pellet containing either (top) 17β-estradiol (1.5 mg/pellet) or (bottom) placebo. (B) Cecal lesion scores of placebo (circles) and estradiol (squares) treated mice (n = 8, 8 respectively). Each point represents the lesion score for an individual mouse. Lesion score data analyzed with an exact Wilcoxon-Mann-Whitney test; horizontal lines represent medians. (C) Real-time quantitative PCR measurements of cecal cytokine and chemokine mRNA expression levels in mice receiving placebo (circle) or estrogen (square) treatment (n = 8). mRNA expression was normalized to the expression of the housekeeping gene HPRT. All mRNA expression data with an exact Wilcoxon Rank Sum Test; each point represents the expression level for an individual mouse and horizontal lines represent medians. For all data analysis p≤0.05 considered significant and indicated by horizontal capped bars.</p

An Investigation into the Immunomodulatory Activities of <i>Sutherlandia frutescens</i> in Healthy Mice

<div><p><i>Sutherlandia frutescens</i> is a medicinal plant that has been traditionally used in southern Africa for cancers, infections, and inflammatory conditions. We recently published experiments demonstrating that an aqueous extract of <i>S</i>. <i>frutescens</i> possessed potent immune-stimulatory activity. This work was carried out with murine macrophages, an immune cell type that plays a pivotal role in host defense from infection and in shaping host inflammatory and immune responses. Here, we conducted a series of follow-up experiments to explore the impact of consuming <i>S</i>. <i>frutescens</i> on host response to bacterial challenge using healthy mice. We found that feeding mice a diet containing <i>S</i>. <i>frutescens</i> failed to significantly alter host response to systemic infection by either a gram-positive or gram-negative bacterium (i.e., <i>L</i>. <i>monocytogenes</i> and <i>E</i>. <i>coli</i>, respectively). In contrast to the <i>in vitro</i> observations, we found no evidence that <i>S</i>. <i>frutescens</i> consumption stimulated <i>in vivo</i> inflammatory responses; instead, consumption of <i>S</i>. <i>frutescens</i> tended to diminish <i>in vivo</i> inflammatory responses. Several possible reasons for this are discussed.</p></div

<i>S</i>. <i>frutescens</i> (SF) Consumption Failed to Impact Circulating Cytokines and Chemokines in BALB/c Mice 24 hr Following an <i>in vivo</i> Challenge with <i>L</i>. <i>monocytogenes</i>.^<i>a</i>

<p><i>S</i>. <i>frutescens</i> (SF) Consumption Failed to Impact Circulating Cytokines and Chemokines in BALB/c Mice 24 hr Following an <i>in vivo</i> Challenge with <i>L</i>. <i>monocytogenes</i>.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160994#t002fn001" target="_blank">^<i>a</i></a></p

Genotyping PCR Primers, Products, & Annealing Temperatures.

<p>Genotyping PCR Primers, Products, & Annealing Temperatures.</p

Treatment with an ERβ agonist decreases disease severity and the expression of pro-inflammatory cytokines.

<p>Female A/J mice were ovariectomized, administered continuous-release pellets containing a placebo, ERα agonist (PPT) or ERβ agonist (DPN), inoculated with <i>H. hepaticus</i>, and necropsied three months later. (A) Cecal lesion scores of mice treated with a placebo (circles), PPT (squares) or DPN (triangles) pellet (n = 19, 22, and 21 respectively). Points represent the lesion scores of individual mice. Data analyzed by one-way ANOVA with a Student-Newman-Keuls post-hoc test; data presented as group means ± s.d (B) Frequency of mice with severe disease (lesion score ≥6). Data analyzed with chi-squared analysis. (C) Real-time quantitative PCR measurements of cecal mRNA expression levels in mice treated with an ERα agonist (squares), ERβ agonist (triangles), or placebo control (circles) (n = 8, 9, and 9 respectively). The mRNA expression levels of all cytokines are normalized to HPRT expression. Data analyzed with an exact Kruskal-Wallis test followed by Dunn's post-hoc comparisons; each point represents the expression level for an individual mouse and horizontal lines represent medians. For all data analysis p≤0.05 considered significant and indicated by horizontal capped bars.</p

Lack of ER signaling in CD4⁺ cellular populations does not alter cecal inflammation or cytokine and chemokine expression.

<p>(A) Cecal lesion scores in female <i>H. hepaticus-</i>inoculated <i>RAG2</i>^−/− mice adoptively transferred <i>ERα</i>^−/− (squares) or <i>ERβ</i>^−/− (triangles) CD4⁺ lymphocytes (n = 8, 9 respectively). As a control group, <i>RAG</i>^−/− female mice were adoptively transferred wild type CD4⁺ lymphocytes (n = 20)(circles). Points represent the lesion scores of individual mice in each experimental group and horizontal bars indicate median lesion scores; horizontal lines represent medians. (B) Real-time quantitative PCR measurements of cecal cytokine and chemokine mRNA expression levels in <i>RAG2</i>^−/− mice adoptively transferred CD4⁺ lymphocytes deficient for ERα or ERβ expression. The mRNA expression levels of all cytokines were normalized to HPRT mRNA expression. Each point represents the normalized expression of an individual mouse (symbols the same as lesion score data). All data analyzed by an exact Kruskal-Wallis test and horizontal lines represent medians. For all data analysis p≤0.05 considered significant.</p