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1.  Leptin antagonist ameliorates chronic colitis in IL-10−/− mice 
Immunobiology  2013;218(12):1439-1451.
Background
Although the etiology of two major forms of inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) are unknown and evidence suggests that chronic intestinal inflammation is caused by an excessive immune response to mucosal antigens. Previous studies support the role for TGF-β1 through 3 in the initiation and maintenance of tolerance via the induction of regulatory T cells (Tregs) to control intestinal inflammation. Leptin, a satiety hormone produced primarily by adipose tissue, has been shown to increase during colitis progression and is believed to contribute to disease genesis and/or progression.
Aim
We investigated the ability of a pegylated leptin antagonist (PG-MLA) to ameliorate the development of chronic experimental colitis.
Results
Compared to vehicle control animals, PG-MLA treatment of mice resulted in an (1) attenuated clinical score; (2) reversed colitis-associated pathogenesis including a decrease in body weight; (3) reduced systemic and mucosal inflammatory cytokine expression; (4) increased insulin levels and (5) enhanced systemic and mucosal Tregs and CD39+ Tregs in mice with chronic colitis. The percentage of systemic and mucosal TGF-β1, -β2 and -β3 expressing CD4+ T cells were augmented after PG-MLA treatment. The activation of STAT1 and STAT3 and the expression of Smad7 were also reduced after PG-MLA treatment in the colitic mice. These findings clearly suggest that PG-MLA treatment reduces intestinal Smad7 expression, restores TGF-β1-3 signaling and reduces STAT1/STAT3 activation that may increase the number of Tregs to ameliorate chronic colitis.
Conclusion
This study clearly links inflammation with the metabolic hormone leptin suggesting that nutritional status influences immune tolerance through the induction of functional Tregs. Inhibiting leptin activity through PG-MLA might provide a new and novel therapeutic strategy for the treatment of IBD.
doi:10.1016/j.imbio.2013.04.020
PMCID: PMC3778116  PMID: 23726523
Inflammation; Inflammatory bowel disease (IBD); Leptin; Antagonist; Pegylated; Ulcerative Colitis (UC); Crohn’s Disease (CD)
2.  The Emerging Role of Leptin Antagonist as Potential Therapeutic Option for Inflammatory Bowel Disease 
Inflammatory bowel disease (IBD) is a chronic relapsing immune-mediated inflammatory disorder that affects millions of people around the world. Leptin is a satiety hormone produced primarily by adipose tissue and acts both centrally and peripherally. Leptin has been shown to play a major role in regulating metabolism, which increases during IBD progression. Leptin mediates several physiological functions including elevated blood pressure, tumorogenesis, cardiovascular pathologies and enhanced immune response in many autoimmune diseases. Recent development of a leptin mutant antagonist that blocks leptin activity raises great hope and opens up new possibilities for therapy in many autoimmune diseases including IBD. To this end, preliminary data from an ongoing study in our laboratory on pegylated leptin antagonist mutant L39A/D40A/F41A (PEG-MLA) treatment shows an inhibition of chronic colitis in IL-10−/− mice. PEG-MLA effectively attenuates the overall clinical scores, reverses colitis-associated pathogenesis including a decrease in body weight, and decreases systemic leptin level. PEG-MLA induces both central and peripheral leptin deficiency by mediating the cellular immune response. In summary, after blocking leptin activity, the correlative outcome between leptin-mediated cellular immune response, systemic leptin levels, and amount of adipose tissue together may provide new strategies for therapeutic intervention in autoimmune diseases, especially for intestinal inflammation.
doi:10.3109/08830185.2013.809071
PMCID: PMC4159716  PMID: 23841494
Crohn’s disease (CD); inflammation; inflammatory bowel disease (IBD); leptin antagonist; pegylated leptin; ulcerative colitis (UC)
3.  Alternative Medicines as Emerging Therapies for Inflammatory Bowel Diseases 
Inflammatory bowel disease (IBD) can be divided into two major categories, ulcerative colitis (UC) and Crohn disease (CD). While the main cause(s) of IBD remain unknown, a number of interventional and preventive strategies have been proposed for use against CD and UC. Many reports have focused on the use of alternative natural medicines as potential therapeutic interventions in IBD patients with minimal side effects. While the use of alternative medicines may be effective in IBD patients that are refractory to corticosteroids or thiopurins, alternative treatment strategies are limited and require extensive clinical testing before being optimized for use in patients.
doi:10.3109/08830185.2011.642909
PMCID: PMC4138959  PMID: 22251008
bromelain; Crohn disease (CD); curcumin; inflammation; inflammatory bowel disease (IBD); polyphenols; pomegranate; resveratrol; rutin; ulcerative colitis (UC)
4.  Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients 
Glioma is the most common and believed to be one of the most aggressive tumors of the central nervous system (CNS) in humans. Very little information is available on the etiology and pathogenesis of these tumors to date. A significant gap remains in our current understanding of the molecular pathways involved in the genesis, progression and clinical behavior of these tumors. Recently, several single nucleotide polymorphisms (SNPs) have been identified in cytokine gene sequences, particularly within the promoter region of these genes, and have been shown to be associated with the development of different types of brain tumors. The present study investigates the association of C-33T SNP in the interleukin-4 (IL-4) gene with systemic IL-4 level and the S503P SNP in the IL-4R gene with the incidence of glioma.
Blood samples were collected from 100 histologically confirmed adult patients with glioma, and 30 apparently healthy individuals from the same area. DNA was extracted from each blood sample, and the IL-4 and IL-4R genes were amplified using polymerase chain reaction (PCR) with gene-specific primers. Systemic IL-4 concentration was assessed in serum samples from each participant by enzyme-linked immunosorbent assay (ELISA). We observed a negative association between the homozygous genotype (CC) of the SNP C-33T of the IL-4 gene with the incidence of glioma (OR=0.19, 95% CI=0.035-1.02), while the T allele of the SNP demonstrated a significant protective association against glioma. Similarly, the heterozygous (CT) and homozygous mutant (CC) of the SNP S503P of the IL-4R gene demonstrated a significant association with glioma development (OR=0.405, 95% CI=0.17-0.969 and OR=0.147, 95% CI=0.036-0.6 respectively), while the C allele exhibited a highly significant association with protection from glioma formation.
These findings suggest that the T allele of the SNP C-33T in the IL-4 gene and the C allele of the SNP S503P in IL-4R may have a protective role against glioma development.
doi:10.7150/ijms.9412
PMCID: PMC4147641  PMID: 25170298
Single nucleotide polymorphism (SNPs); interleukin-4; interleukin-4R; brain tumor; and Glioma
5.  The Severity of Experimental Autoimmune Cystitis Can be Ameliorated by Anti-CXCL10 Ab Treatment 
PLoS ONE  2013;8(11):e79751.
Background
Interstitial cystitis (IC), more recently called painful bladder syndrome (PBS) is a complex disease associated with chronic bladder inflammation that primarily affects women. Its symptoms include frequent urinary urgency accompanied by discomfort or pain in the bladder and lower abdomen. In the United States, eight million people, mostly women, have IC/PBS. New evidence that autoimmune mechanisms are important in the pathogenesis of IC/PBS triggered interest.
Methodology/Principal Findings
SWXJ mice immunized with a homogenate of similar mice’s urinary bladders develop an autoimmune phenotype comparable to clinical IC with functional and histological alterations confined to the urinary bladder. Using the murine model of experimental autoimmune cystitis (EAC), we found that serum levels of CXCR3 ligand and local T helper type 1 (Th1) cytokine are elevated. Also, IFN-γ-inducible protein10 (CXCL10) blockade attenuated overall cystitis severity scores; reversed the development of IC; decreased local production of CXCR3 and its ligands, IFN-γ, and tumor necrosis factor-α (TNF-α); and lowered systemic levels of CXCR3 ligands. Urinary bladder CD4+ T cells, mast cells, and neutrophils infiltrates were reduced following anti-CXCL10 antibody (Ab) treatment of mice. Anti-CXCL10 Ab treatment also reversed the upregulated level of CXCR3 ligand mRNA at urinary bladder sites. The decreased number and percentage of systemic CD4+ T cells in EAC mice returned to normal after anti-CXCL10 Ab treatment.
Conclusion/Significance
Taken together, our findings provide important new information about the mechanisms underlying EAC pathogenesis, which has symptoms similar to those of IC/PBS. CXCL10 has the potential for use in developing new therapy for IC/PBS.
doi:10.1371/journal.pone.0079751
PMCID: PMC3836899  PMID: 24278169
7.  Novel Vaccine Adjuvants 
BioMed Research International  2013;2013:835105.
doi:10.1155/2013/835105
PMCID: PMC3819826
8.  Resveratrol suppresses colitis and colon cancer associated with colitis 
Resveratrol is a naturally occurring polyphenol that exhibits pleiotropic health beneficial effects including anti-inflammatory, cardio- and cancer-protective activities. It is recognized as one of the more promising natural molecules in the prevention and treatment of chronic inflammatory and autoimmune disorders. Ulcerative Colitis (UC) is an idiopathic, chronic inflammatory disease of the colon associated with a high colon cancer risk. Here, we used a Dextran Sulfate Sodium (DSS) mouse model of colitis, which resembles human UC pathology. Resveratrol mixed in food ameliorates DSS-induced colitis in mice in a dose-dependent manner. Resveratrol significantly improves inflammation score, down regulates the percentage of neutrophils in the mesenteric lymph nodes and lamina propiria, and modulates CD3+ T cells that express tumor necrosis factor-alpha and interferon gamma. Markers of inflammation and inflammatory stress (p53 and p53-Phospho-Serine 15), are also down regulated by resveratrol. Since chronic colitis drives colon cancer risk, we carried out experiments to determine the chemopreventive properties of resveratrol. Tumor incidence is reduced from 80% in mice treated with Azoxymethane (AOM) + DSS to 20% in AOM + DSS + Resveratrol (300 p.p.m.) treated mice. Tumor multiplicity also decreased with resveratrol treatment. AOM + DSS treated mice had 2.4 ± 0.7 tumors per animal compared with AOM + DSS + 300 p.p.m. resveratrol, which had 0.2 ± 0.13 tumors per animal. The current study indicates that resveratrol is a useful, non-toxic complementary and alternative strategy to abate colitis and potentially colon cancer associated with colitis.
doi:10.1158/1940-6207.CAPR-09-0117
PMCID: PMC2853724  PMID: 20332304
Inflammation; Resveratrol; Colitis; Colon Cancer
9.  Cannabinoids Receptor-2 (CB2) agonist ameliorates colitis in IL-10−/− mice by attenuating the activation of T cells and promoting their apoptosis 
Toxicology and applied pharmacology  2011;258(2):256-267.
Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Recent studies have shown that the cannabinoid system may play a critical role in mediating protection against intestinal inflammation. However, the effect of cannabinoid receptors induction after chronic colitis progression has not been investigated. Here, we investigate the effect of cannabinoid receptor-2 (CB2) agonist, JWH-133, after chronic colitis in IL-10−/− mice. JWH-133 effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis and decrease in body weight in IL-10−/− mice. After JWH-133 treatment, the percentage of CD4+ T cells, neutrophils, mast cells, natural killer (NK1.1) cells, and activated T cells in the LP of colitis mice declined after JWH-133 treatment in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLN). JWH-133 was also effective in ameliorating dextran sodium sulphate (DSS)-induced colitis. In this model, JWH-133 reduced the number and percentage of macrophages and IFN-γ expressing cells that were induced during colitis progression. Treatment with aminoalkylindole 6-iodopravadoline (AM630), a CB2 receptor antagonist, reversed the colitis protection provided by JWH-133 treatment. Also, activated T cells were found to undergo apoptosis following JWH-133 treatment both in-vivo and in-vitro. These findings suggest that JWH-133 mediates its effect through CB2 receptors, and ameliorates chronic colitis by inducing apoptosis in activated T cells, reducing the numbers of activated T cells, suppressing induction of mast cells, NK cells, and neutrophils at sites of inflammation in the LP. These results support the idea that the CB2 receptor agonists may serve as a therapeutic modality against IBD.
doi:10.1016/j.taap.2011.11.005
PMCID: PMC4117838  PMID: 22119709
Cannabinoid-2 receptors; JWH 133; Colitis; inflammatory bowel disease
10.  MicroRNA let-7e is associated with the pathogenesis of experimental autoimmune encephalomyelitis 
European journal of immunology  2012;43(1):104-114.
MicroRNAs (miRNAs) play important roles in the regulation of immune responses. There is evidence that miRNAs also participate in the pathogenesis of multiple sclerosis (MS), but how the miRNAs regulate the pathogenesis of MS is still under investigation. The identification of new members of the miRNA family associated with the pathogenesis of MS could facilitate early diagnosis and treatment. Here we show that the level of miRNA let-7e is significantly upregulated in experimental autoimmune encephalomyelitis (EAE), an animal model of MS using miRNA array and quantitative real-time PCR. The expression of let-7e was mainly in CD4+ T cells and infiltrated mononuclear cells of central nervous system, and highly correlated with the development of EAE. We found that let-7e silencing in vivo inhibited encephalitogenic Th1 and Th17 cells and attenuated EAE, with reciprocal increase of Th2 cells; overexpression of let-7e enhanced Th1 and Th17 cells and aggravated EAE. We also identified IL-10 as one of the functional targets of let-7e. Together, we propose that let-7e is a new miRNA involved in the regulation of encephalitogenic T-cell differentiation and the pathogenesis of EAE.
doi:10.1002/eji.201242702
PMCID: PMC3650085  PMID: 23079871
MicroRNA; mir-let-7e; EAE/MS; Th1/Th2 cells; Cell differentiation
11.  Nuclease Activity via Self-Activation and Anticancer Activity of Mononuclear Copper(II) Complex: Novel Role of Tertiary Butyl Group in the Ligand Frame 
Inorganic Chemistry  2012;51(6):3343-3345.
Copper complex [Cu(tBuPhimp)(Cl)] (1) derived from tridentate ligand tBuPhimpH having N2O donors was synthesized and molecular structure was determined. Phenoxyl radical complex was generated in solution at room temperature using Ce(IV) salt. Nuclease activity and anticancer activity of 1 was investigated. Roles of tert-butyl group and singlet oxygen in DNA cleavage activity were also discussed.
doi:10.1021/ic2016676
PMCID: PMC3307853  PMID: 22372979
12.  Clinical and biological significance of CXCR5 expressed by prostate cancer specimens and cell lines 
Chemokines and chemokine receptors, has been shown to be involved in metastatic process of prostate cancer (PCa). In this study, we have show that primary PCa tissues and cell lines (LNCaP and PC3) express CXCR5, a specific chemokine receptor for the CXCL13. Expression of CXCR5 was significantly higher (P < 0.001) in PCa cases than compared to normal match (NM) tissues. CXCR5 intensity correlated (R2 = 0.97) with Gleason score. While prostate tumor tissues with Gleason scores ≥ 7, displayed predominantly nuclear CXCR5 expression patterns, PCa specimens with Gleason scores ≤ 6 showed predominantly membrane and cytoplasmic expression patterns that were comparable to benign prostatic hyperplasia (BPH). Similar to tissue expression, PCa cell lines expressed significantly more CXCR5 than normal prostatic epithelial cells (PrECs) and CXCR5 expression was distributed among intracellular and extracellular compartments. Functional in vitro assays showed higher migratory and invasive potentials toward CXCL13, an effect that was CXCR5-mediated. In both PCa cell lines, CXCL13 treatment increased the expression of collagenase-1 or matrix metalloproteinase-1 (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromalysin-2 (MMP-10), and stromelysin-3 (MMP-11). These data demonstrate the clinical and biological relevance of the CXCL13-CXCR5 pathway and its role in PCa cell invasion and migration.
doi:10.1002/ijc.24574
PMCID: PMC3600527  PMID: 19610059
chemokine; prostate cancer; tissue expression
13.  Serum CXCL13 positively correlates with prostatic disease, prostate-specific antigen and mediates prostate cancer cell invasion, integrin clustering and cell adhesion 
Cancer letters  2009;283(1):29-35.
Chemokines and corresponding receptor interactions have been shown to be involved in prostate cancer (PCa) progression and organ-specific metastasis. We have recently shown that PCa cell lines and primary prostate tumors express CXCR5, which correlates with PCa grade. In this study, we present the first evidence that CXCL13, the only ligand for CXCR5, and IL-6 were significantly elevated in PCa patient serum compared to serum from subjects with benign prostatic hyperplasia (BPH), or high-grade prostatic intraepithelial neoplasia (HGPIN) as well as normal healthy donors (NHD). Serum CXCL13 levels significantly (p < 0.0001) correlated with serum prostate-specific antigen (PSA), whereas serum IL-6 levels significantly (p < 0.0003) correlated with CXCL13 serum levels. CXCL13 was found to be a better predictor of PCa than PSA. In addition, CXCL13 was highly expressed by human bone marrow endothelial (HBME) cells and osteoblasts (OBs), but not osteoclasts (OCs), following treatment with physiologically relevant levels of interleukin-6 (IL-6). We further demonstrate that CXCL13, produced by IL-6-treated HBME cells, was able to induce PCa cell invasion in a CXCR5-dependent manner. CXCL13-mediated PCa cell αvβ3-integrin clustering and adhesion to HBME cells was abrogated by CXCR5 blockade. These results demonstrate that the CXCL13-CXCR5 axis is significantly associated with PCa progression.
doi:10.1016/j.canlet.2009.03.022
PMCID: PMC3600557  PMID: 19375853
chemokine; prostate; integrin; adhesion; invasion
14.  CXCR4-gp120-IIIB interactions induce caspase-mediated apoptosis of prostate cancer cells and inhibit tumor growth 
Molecular cancer therapeutics  2009;8(1):178-184.
CXC chemokine receptor 4 (CXCR4) has been implicated in prostate cancer metastasis and this receptor also acts as a coreceptor for HIV-1 120-kDa glycoprotein variant IIIB (gp120-IIIB). The interaction between CXCR4 and gp120-IIIB has been shown to mediate apoptosis of both immune and endothelial cells. In this study, we have examined the effects of gp120-IIIB on hormone-refractory prostate cancer cells (PC3 and DU145) in vitro and tumor growth in vivo. Normal prostatic epithelial (PrEC) and prostate cancer cell lines were treated with gp120-IIIB with or without anti-CXCR4 antibody. Caspase expression was evaluated by real-time PCR and active caspase assays. Apoptosis was determined by flow cytometry. gp120-IIIB treatment correlated with active caspase-3 and -9 expression and apoptosis of prostate cancer cells but not PrEC cells. This effect was significantly inhibited after CXCR4 blockade. PC3 and DU145 tumor-bearing mice received intraperitoneal injections of gp120-IIIB and controls received bovine serum albumin in PBS. PC3 and DU145 tumor sizes were measured over time and excised tumors were evaluated for CD44, CD34, lymphatic endothelial cell marker LYVE-1, active caspase-3, and active caspase-9 expression by immunohistochemistry. The tumor size in mice receiving gp120-IIIB was significantly smaller than compared with tumors in control mice. This regression was associated with significant decreases in CD44, CD34, and LYVE-1 and increases in active caspase-3 and -9 expression. These results suggest that gp120-IIIB induced apoptosis in prostate cancer cells and reduced tumor-associated lymphoendothelial cells.
doi:10.1158/1535-7163.MCT-08-0643
PMCID: PMC3600559  PMID: 19139127
15.  Role of resveratrol-induced CD11b+ Gr-1+ myeloid derived suppressor cells (MDSCs) in the reduction of CXCR3+ T cells and amelioration of chronic colitis in IL-10−/− mice 
Brain, behavior, and immunity  2011;26(1):72-82.
Resveratrol, a naturally occurring polyphenol has received significant attention as a potent anti-inflammatory agent. Inflammatory bowel disease (IBD) is a chronic intestinal inflammation caused by hyperactivated effector immune cells that produce pro-inflammatory cytokines. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population characterized by the co-expression of CD11b+ and Gr-1+ and have long been known for their immunosuppressive function. We report that resveratrol effectively attenuated overall clinical scores as well as various pathological markers of colitis in IL-10−/− mice by down regulating Th1 responses. Resveratrol lessened the colitis-associated decrease in body weight and increased levels of serum amyloid A (SAA), CXCL10 and colon TNF-α, IL-6, RANTES, IL-12 and IL-1β concentrations. After resveratrol treatment, the percentage of CXCR3 expressing T cells was decreased in the spleen, mesenteric lymph nodes (MLN), and intestinal lamina propria (LP). However, the percentage and absolute numbers of CD11b+ and Gr-1+cells in the lamina propria (LP) and spleen were increased after resveratrol treatment as compared with the vehicle treatment. Co-culture of resveratrol induced CD11b+ Gr-1+ cells with T cells, attenuated T cell proliferation, and most importantly reduced IFN-γ and GM-CSF production by LP derived T cells from vehicle treated IL-10−/− mice with chronic colitis. The current study suggests that administration of resveratrol into IL-10−/− mice induces immunosuppressive CD11b+ Gr-1+ MDSCs in the colon, which correlates with reversal of established chronic colitis, and down regulation of mucosal and systemic CXCR3+ expressing effector T cells as well as inflammatory cytokines in the colon. The induction of immunosuppressive CD11b+ Gr-1+ cells by resveratrol during colitis is unique, and suggests an as-yet-unidentified mode of anti-inflammatory action of this plant polyphenol.
doi:10.1016/j.bbi.2011.07.236
PMCID: PMC3506001  PMID: 21807089
Inflammation; Resveratrol; Colitis; CXCR3; CD11b+ and Gr-1+ and MDSCs
16.  Resveratrol (trans-3,5,4'-trihydroxystilbene) suppresses EL4 tumor growth by induction of apoptosis involving reciprocal regulation of SIRT1 and NF-κB 
Molecular nutrition & food research  2011;55(8):1207-1218.
Scope
Understanding the molecular mechanisms through which natural products and dietary supplements exhibit anticancer properties is crucial and can lead to drug discovery and chemoprevention. The current study sheds new light on the mode of action of Resveratarol (RES), a plant-derived polyphenolic compound, against EL-4 lymphoma growth.
Methods and results
Immuno-compromised NOD/SCID mice injected with EL-4 tumor cells and treated with RES (100 mg/kg body weight) showed delayed development and progression of tumor growth and increased mean survival time. RES caused apoptosis in EL4 cells through activation of aryl hydrocarbon receptor (AhR) and upregulation of Fas and FasL expression in vitro. Blocking of RES-induced apoptosis in EL4 cells by FasL mAb, cleavage of caspases and PARP, and release of cytochorme c, demonstrated the participation of both extrinsic and intrinsic pathways of apoptosis. RES also induced upregulation of SIRT1 and downregulation of NF-kB in EL4 cells. SiRNA-mediated down regulation of SIRT1 in EL4 cells increased the activation of NF-kB but decreased RES-mediated apoptosis, indicating the critical role of SIRT1 in apoptosis via blocking activation of NF-kB.
Conclusion
These data suggest that RES-induced SIRT1 upregulation promotes tumor cell apoptosis through negative regulation of NF-kB, leading to suppression of tumor growth.
doi:10.1002/mnfr.201000576
PMCID: PMC3516994  PMID: 21520490
Cancer; Lymphoma; Resveratrol; AhR; Fas; FasL; Apoptosis; Extrinsic pathway; Intrinsic pathway
17.  CD49f and CD61 identify Her2/neu-induced mammary tumor initiating cells that are potentially derived from luminal progenitors and maintained by the integrin-TGFβ signaling 
Oncogene  2011;31(21):2614-2626.
HER2/Neu is overexpressed in 20-30% of breast cancers and associated with aggressive phenotypes and poor prognosis. For deciphering the role of HER2/Neu in breast cancer, mouse mammary tumor virus (MMTV)-Her2/neu transgenic mice that develop mammary tumors resembling human HER2-subtype breast cancer have been established. Several recent studies have revealed that HER2/Neu is overexpressed in and regulates self renewal of breast tumor initiating cells (TICs). However, in the MMTV-Her2/neu transgenic mouse model, the identity of TICs remains elusive, despite previous studies showing supportive evidence for existence of TICs in Her2/neu-induced mammary tumors. Through systematic screening and characterization, we identified surface markers CD49f, CD61 and ESA were aberrantly overexpressed in Her2-overexpressing mammary tumor cells. Analysis of these markers as well as CD24 detected anomalous expansion of the luminal progenitor population in preneoplastic mammary glands of Her2/neu-transgenic mice, indicating that aberrant luminal progenitors originated Her2-induced mammary tumors. The combined markers, CD49f and CD61, further delineated the CD49fhighCD61high-sorted fraction as a TIC-enriched population, which displayed increased tumorsphere formation ability, enhanced tumorigenicity both in vitro and in vivo and drug resistance to pacitaxel and doxorubicin. Moreover, the TIC-enriched population manifested increased TGFβ signaling and exhibited gene expression signatures of stemness, TGFβ signaling and Epithelial-to-Mesenchymal Transition. Our findings that self-renewal and clonogenicity of TICs were suppressed by pharmacologically inhibiting the TGFβ signaling further indicate that the TGFβ pathway is vital for maintenance of the TIC population. Finally, we showed that the integrin β3 (CD61) signaling pathway was required for sustaining active TGFβ signaling and self-renewal of TICs. We for the first time developed a technique to highly enrich TICs from mammary tumors of Her2/neu-transgenic mice, unraveled their properties and identified the cooperative integrin β3-TGFβ signaling axis as a potential therapeutic target for HER2-induced TICs.
doi:10.1038/onc.2011.439
PMCID: PMC3260386  PMID: 21996747
Tumor Initiating Cells; Her2/neu; mammary tumor; CD49f; CD61; ESA
18.  Prenatal Exposure to TCDD Triggers Significant Modulation of microRNA Expression Profile in the Thymus That Affects Consequent Gene Expression 
PLoS ONE  2012;7(9):e45054.
Background
MicroRNAs (miRs) are a class of small RNAs that regulate gene expression. There are over 700 miRs encoded in the mouse genome and modulate most of the cellular pathways and functions by controlling gene expression. However, there is not much known about the pathophysiological role of miRs. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), an environmental contaminant is well known to induce severe toxicity (acute and chronic) with long-term effects. Also, in utero exposure of fetus to TCDD has been shown to cause thymic atrophy and alterations in T cell differentiation. It is also relevant to understand “the fetal basis of adult disease” hypothesis, which proposes that prenatal exposure to certain forms of nutritional and environmental stress can cause increased susceptibility to clinical disorders later in life. In the current study, therefore, we investigated the effects of prenatal exposure to TCDD on miR profile in fetal thymocytes and searched for their possible role in causing thymic atrophy and alterations in the expression of apoptotic genes.
Methodology/Principal Findings
miR arrays of fetal thymocytes post exposure to TCDD and vehicle were performed. Of the 608 mouse miRs screened, 78 miRs were altered more than 1.5 fold and 28 miRs were changed more than 2 fold in fetal thymocytes post-TCDD exposure when compared to vehicle controls. We validated the expression of several of the miRs using RT-PCR. Furthermore, several of the miRs that were downregulated contained highly complementary sequence to the 3′-UTR region of AhR, CYP1A1, Fas and FasL. Also, the Ingenuity Pathway Analysis software and database was used to analyze the 78 miRs that exhibited significant expression changes and revealed that as many as 15 pathways may be affected.
Conclusions/Significance
These studies revealed that TCDD-mediated alterations in miR expression may be involved in the regulation of its toxicity including cancer, hepatic injury, apoptosis, and cellular development.
doi:10.1371/journal.pone.0045054
PMCID: PMC3443208  PMID: 23024791
19.  Resveratrol Prevents Endothelial Cells Injury in High-Dose Interleukin-2 Therapy against Melanoma 
PLoS ONE  2012;7(4):e35650.
Immunotherapy with high-dose interleukin-2 (HDIL-2) is an effective treatment for patients with metastatic melanoma and renal cell carcinoma. However, it is accompanied by severe toxicity involving endothelial cell injury and induction of vascular leak syndrome (VLS). In this study, we found that resveratrol, a plant polyphenol with anti-inflammatory and anti-cancer properties, was able to prevent the endothelial cell injury and inhibit the development of VLS while improving the efficacy of HDIL-2 therapy in the killing of metastasized melanoma. Specifically, C57BL/6 mice were injected with B16F10 cells followed by resveratrol by gavage the next day and continued treatment with resveratrol once a day. On day 9, mice received HDIL-2. On day 12, mice were evaluated for VLS and tumor metastasis. We found that resveratrol significantly inhibited the development of VLS in lung and liver by protecting endothelial cell integrity and preventing endothelial cells from undergoing apoptosis. The metastasis and growth of the tumor in lung were significantly inhibited by HDIL-2 and HDIL-2 + resveratrol treatment. Notably, HDIL-2 + resveratrol co-treatment was more effective in inhibiting tumor metastasis and growth than HDIL-2 treatment alone. We also analyzed the immune status of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) and FoxP3+CD4+ regulatory T cells (Treg). We found that resveratrol induced expansion and suppressive function of MDSC which inhibited the development of VLS after adoptive transfer. However, resveratrol suppressed the HDIL-2-induced expansion of Treg cells. We also found that resveratrol enhanced the susceptibility of melanoma to the cytotoxicity of IL-2-activated killer cells, and induced the expression of the tumor suppressor gene FoxO1. Our results suggested the potential use of resveratrol in HDIL-2 treatment against melanoma. We also demonstrated, for the first time, that MDSC is the dominant suppressor cell than regulatory T cell in the development of VLS.
doi:10.1371/journal.pone.0035650
PMCID: PMC3331985  PMID: 22532866
20.  Activation of Aryl Hydrocarbon Receptor (AhR) Leads to Reciprocal Epigenetic Regulation of FoxP3 and IL-17 Expression and Amelioration of Experimental Colitis 
PLoS ONE  2011;6(8):e23522.
Background
Aryl hydrocarbon receptor (AhR), a transcription factor of the bHLH/PAS family, is well characterized to regulate the biochemical and toxic effects of environmental chemicals. More recently, AhR activation has been shown to regulate the differentiation of Foxp3+ Tregs as well as Th17 cells. However, the precise mechanisms are unclear. In the current study, we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR ligand, on epigenetic regulation leading to altered Treg/Th17 differentiation, and consequent suppression of colitis.
Methodology/Principal Findings
Dextran sodium sulphate (DSS) administration induced acute colitis in C57BL/6 mice, as shown by significant weight loss, shortening of colon, mucosal ulceration, and increased presence of CXCR3+ T cells as well as inflammatory cytokines. Interestingly, a single dose of TCDD (25 µg/kg body weight) was able to attenuate all of the clinical and inflammatory markers of colitis. Analysis of T cells in the lamina propria (LP) and mesenteric lymph nodes (MLN), during colitis, revealed decreased presence of Tregs and increased induction of Th17 cells, which was reversed following TCDD treatment. Activation of T cells from AhR+/+ but not AhR -/- mice, in the presence of TCDD, promoted increased differentiation of Tregs while inhibiting Th17 cells. Analysis of MLN or LP cells during colitis revealed increased methylation of CpG islands of Foxp3 and demethylation of IL-17 promoters, which was reversed following TCDD treatment.
Conclusions/Significance
These studies demonstrate for the first time that AhR activation promotes epigenetic regulation thereby influencing reciprocal differentiation of Tregs and Th17 cells, and amelioration of inflammation.
doi:10.1371/journal.pone.0023522
PMCID: PMC3156147  PMID: 21858153
21.  Stem cells as potential therapeutic targets for inflammatory bowel disease 
The rates of incidence and prevalence of Crohn’s disease and ulcerative colitis, the two major forms of inflammatory bowel disease (IBD), are rising. Estimates indicate >1 million new cases of IBD in the United States annually. The conventional therapies available for IBD range from anti-inflammatory drugs to immunosuppressive agents, but these therapies generally fail to achieve satisfactory results due to their side effects. Interest in a new therapeutic option, that is, biological therapy, has gained much momentum recently due to its focus on different stages of the inflammatory process. Stem cell (SC) research has become a new direction for IBD therapy due to our recent understanding of cell populations involved in the pathogenic process. To this end, hematopoietic and mesenchymal stem cells are receiving more attention from IBD investigators. The intestinal environment, with its crypts and niches, supports incoming embryonic and hematopoietic stem cells and allows them to engraft and differentiate. The above findings suggest that, in the future, SC-based therapy will be a promising alternative to conventional therapy for IBD. In this review, we discuss SCs as potential therapeutic targets for future treatment of IBD.
PMCID: PMC2900153  PMID: 20515838
Inflammatory bowel disease; Crohn’s disease; Ulcerative Colitis; Stem cells; Review
22.  Circadian Disruption, Per3, and Human Cytokine Secretion 
Integrative cancer therapies  2009;8(4):329-336.
Circadian disruption has been linked with inflammation, an established cancer risk factor. Per3 clock gene polymorphisms have also been associated with circadian disruption and with increased cancer risk. Patients completed a questionnaire and provided a blood sample prior to undergoing a colonoscopy (n = 70). Adjusted mean serum cytokine concentrations (IL-6, TNF-alpha, gamma-INF, IL-I ra, IL-I-beta, VEGF) were compared among patients with high and low scores for fatigue (Multidimensional Fatigue Inventory), depressive symptoms (Beck Depression Inventory II), or sleep disruption (Pittsburgh Sleep Quality Index), or among patients with different Per3 clock gene variants. Poor sleep was associated with elevated VEGF, and fatigue-related reduced activity was associated with elevated TNF-alpha concentrations. Participants with the 4/5 or 5/5 Per3 variable tandem repeat sequence had elevated IL-6 concentrations compared to those with the 4/4 genotype. Biological processes linking circadian disruption with cancer remain to be elucidated. Increased inflammatory cytokine secretion may playa role.
doi:10.1177/1534735409352029
PMCID: PMC2959170  PMID: 19926609
circadian rhythm; clock gene; cytokine; inflammation
23.  Taming the beast within: resveratrol suppresses colitis and prevents colon cancer 
Aging (Albany NY)  2010;2(4):183-184.
PMCID: PMC2881508  PMID: 20436227
Inflammation; complementary and alternative medicine; SIRT-1; NF-kappaB; inflammatory bowel disease
24.  Helper T Cell Epitope-Mapping Reveals MHC-Peptide Binding Affinities That Correlate with T Helper Cell Responses to Pneumococcal Surface Protein A 
PLoS ONE  2010;5(2):e9432.
Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA) is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA) would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC) class II. We also characterized spleen- and cervical lymph node (CLN)-derived helper T lymphocyte (HTL) cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA199–246) consistently caused the greatest IFN-γ, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4+ T cells isolated from S. pneumonia strain EF3030-challeged F1 (B6×BALB/c) mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA199–246 also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th) cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.
doi:10.1371/journal.pone.0009432
PMCID: PMC2828482  PMID: 20195541
25.  1-(2-Bromo­benz­yl)-3-isopropyl­benz­imid­azolin-2-one 
In the structure of the title compound, C17H17BrN2O, the central phenyl and imidazol-2-one rings are coplanar (dihedral angle between planes of 0.73 (11)°). The angles subtended by the substituents on the N atoms of the imidazol-2-one ring range from 109.71 (14)° to 128.53 (15) due to steric hindrance of these substituents with the phenyl H atoms. The carbonyl O and Br both make two weak C—H⋯O and C—H⋯Br inter­actions with two adjacent mol­ecules, thus forming an three-dimensional array.
doi:10.1107/S1600536809042871
PMCID: PMC2971215  PMID: 21578426

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