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1.  Fidaxomicin Inhibits Clostridium difficile Toxin A-Mediated Enteritis in the Mouse Ileum 
Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 μM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1β (IL-1β) protein and mRNA expression. Treatment with fidaxomicin (20 μM) or its primary metabolite, OP-1118 (120 μM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1β protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 μM) and metronidazole (20 μM) did not alter toxin A-induced histologic damage and IL-1β protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins.
doi:10.1128/AAC.02783-14
PMCID: PMC4135997  PMID: 24890583
2.  Inhibition of Vasoactive Intestinal Polypeptide (VIP) Induces Resistance to Dextran Sodium Sulfate (DSS)-Induced Colitis in Mice 
VIP is highly expressed in the colon and regulates motility, vasodilatation, and sphincter relaxation. However, its role in the development and progress of colitis is still controversial. Our aim was to determine the participation of VIP on dextran sodium sulfate (DSS)-induced colonic mucosal inflammation using VIP−/− and WT mice treated with VIP antagonists. Colitis was induced in 32 adult VIP−/− and 14 age-matched WT litter-mates by giving 2.5 % DSS in the drinking water. DSS-treated WT mice were injected daily with VIP antagonists, VIPHyb (n=22), PG 97–269 (n=9), or vehicle (n=31). After euthanasia, colons were examined; colonic cytokines mRNA were quantified. VIP−/− mice were remarkably resistant to DSS-induced colitis compared to WT. Similarly, DSS-treated WT mice injected with VIPHyb (1 μM) or PG 97–269 (1 nM) had significantly reduced clinical signs of colitis. Furthermore, colonic expression of IL-1, TNF-α, and IL-6 was significantly lower in VIP−/− and VIPHyb or PG 97–269 compared to vehicle-treated WT. Genetic deletion of VIP or pharmacological inhibition of VIP receptors resulted in resistance to colitis. These data demonstrate a pro-inflammatory role for VIP in murine colitis and suggest that VIP antagonists may be an effective clinical treatment for human inflammatory bowel diseases.
doi:10.1007/s12031-013-0205-3
PMCID: PMC4114158  PMID: 24395090
VIP; Colitis; VIP antagonist: IBD
3.  Identification of a Functional TPH1 Polymorphism Associated with Irritable Bowel Syndrome Bowel Habit Subtypes 
Background & Aims
Alterations in 5-hydroxytryptamine (5-HT) signaling have been implicated as a factor contributing to the altered bowel habit of irritable bowel syndrome (IBS) patients. Tryptophan hydroxylase 1 (TPH1) is the rate–limiting enzyme in enterochromaffin cell 5-HT biosynthesis. We hypothesized that genetic variants affecting TPH1 gene expression might alter intestinal 5-HT bioavailability and subsequently the propensity for distinct bowel habit subtypes in IBS. In this study, we assessed the only common TPH1 proximal promoter variant (-347C/A; rs7130929) and its association with bowel habit predominance in IBS.
Methods
Electrophoretic mobility shift assays were performed to assess whether the -347C/A allele variant affects the DNA-binding of nuclear factors. Genotype distribution was determined for 422 IBS patients subtyped using Rome III criteria and for 495 healthy controls recruited from two university medical centers. Association with bowel habit was tested using a multinomial logistic regression model controlling for race, anxiety, depression, and study site.
Results
Early growth response factor 1 (EGR-1) bound with higher affinity to a site comprising the minor A-allele of SNP -347C/A. TPH1 genotype frequencies did not differ between IBS patients and controls overall. The CC genotype was more prevalent in the IBS-D subtype (47%) than in the IBS-C (25%) and IBS-M (37%) subtypes (P=0.039) after adjusting for race and other covariates. Colonic biopsies from a small cohort of IBS patients from one center were tested for higher TPH1 mRNA expression in samples with CC compared to CA genotype, but the results did not reach statistical significance.
Conclusions
The TPH1 promoter SNP -347C/A differentially binds EGR1, correlates with IBS bowel habit subtypes and possibly colonic TPH1 expression consistent with its role in modulating intestinal 5-HT signaling.
doi:10.1038/ajg.2013.304
PMCID: PMC4067697  PMID: 24060757
Egr1; Sp1; ZBP-89; 5HT; serotonin; SNP
4.  Chronic unpredictable stress regulates visceral adipocyte‐mediated glucose metabolism and inflammatory circuits in male rats 
Physiological Reports  2014;2(5):e00284.
Abstract
Chronic psychological stress is a prominent risk factor involved in the pathogenesis of many complex diseases, including major depression, obesity, and type II diabetes. Visceral adipose tissue is a key endocrine organ involved in the regulation of insulin action and an important component in the development of insulin resistance. Here, we examined for the first time the changes on visceral adipose tissue physiology and on adipocyte‐associated insulin sensitivity and function after chronic unpredictable stress in rats. Male rats were subjected to chronic unpredictable stress for 35 days. Total body and visceral fat was measured. Cytokines and activated intracellular kinase levels were determined using high‐throughput multiplex assays. Adipocyte function was assessed via tritiated glucose uptake assay. Stressed rats showed no weight gain, and their fat/lean mass ratio increased dramatically compared to control animals. Stressed rats had significantly higher mesenteric fat content and epididymal fat pad weight and demonstrated reduced serum glucose clearing capacity following glucose challenge. Alterations in fat depot size were mainly due to changes in adipocyte numbers and not size. High‐throughput molecular screening in adipocytes isolated from stressed rats revealed activation of intracellular inflammatory, glucose metabolism, and MAPK networks compared to controls, as well as significantly reduced glucose uptake capacity in response to insulin stimulation. Our study identifies the adipocyte as a key regulator of the effects of chronic stress on insulin resistance, and glucose metabolism, with important ramifications in the pathophysiology of several stress‐related disease states.
e00284
Our study identifies the adipocyte as a key regulator of the effects of chronic stress on insulin resistance, and glucose metabolism, with important ramifications in the pathophysiology of several stress‐related disease states.
doi:10.14814/phy2.284
PMCID: PMC4098730  PMID: 24819750
Adipocytes; adipokines; fat tissue; insulin resistance; stress
5.  Mechanisms and Metabolic Implications of Regional Differences among Fat Depots 
Cell metabolism  2013;17(5):644-656.
Fat distribution is closely linked to metabolic disease risk. Distribution varies with sex, genetic background, disease state, certain drugs and hormones, development, and aging. Preadipocyte replication and differentiation, developmental gene expression, susceptibility to apoptosis and cellular senescence, vascularity, inflammatory cell infiltration, and adipokine secretion vary among depots, as do fatty-acid handling and mechanisms of enlargement with positive-energy and loss with negative-energy balance. How interdepot differences in these molecular, cellular, and pathophysiological properties are related is incompletely understood. Whether fat redistribution causes metabolic disease or whether it is a marker of underlying processes that are primarily responsible is an open question.
doi:10.1016/j.cmet.2013.03.008
PMCID: PMC3942783  PMID: 23583168
6.  Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes 
Clostridium difficile infection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality. C. difficile mediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blocking C. difficile toxin A- and toxin B-mediated inflammatory responses and histologic damage.
doi:10.1128/AAC.02633-12
PMCID: PMC3697353  PMID: 23629713
7.  Principles and clinical implications of the brain–gut–enteric microbiota axis 
Nature reviews. Gastroenterology & hepatology  2009;6(5):10.1038/nrgastro.2009.35.
While bidirectional brain–gut interactions are well known mechanisms for the regulation of gut function in both healthy and diseased states, a role of the enteric flora—including both commensal and pathogenic organisms—in these interactions has only been recognized in the past few years. The brain can influence commensal organisms (enteric microbiota) indirectly, via changes in gastrointestinal motility and secretion, and intestinal permeability, or directly, via signaling molecules released into the gut lumen from cells in the lamina propria (enterochromaffin cells, neurons, immune cells). Communication from enteric microbiota to the host can occur via multiple mechanisms, including epithelial-cell, receptor-mediated signaling and, when intestinal permeability is increased, through direct stimulation of host cells in the lamina propria. Enterochromaffin cells are important bidirectional transducers that regulate communication between the gut lumen and the nervous system. Vagal, afferent innervation of enterochromaffin cells provides a direct pathway for enterochromaffin-cell signaling to neuronal circuits, which may have an important role in pain and immune-response modulation, control of background emotions and other homeostatic functions. Disruption of the bidirectional interactions between the enteric microbiota and the nervous system may be involved in the pathophysiology of acute and chronic gastrointestinal disease states, including functional and inflammatory bowel disorders.
doi:10.1038/nrgastro.2009.35
PMCID: PMC3817714  PMID: 19404271
8.  The antimicrobial peptide cathelicidin modulates Clostridium difficile-associated colitis and toxin A-mediated enteritis in mice 
Gut  2012;62(9):1295-1305.
Background
Clostridium difficile mediates intestinal inflammation by releasing toxin A (TxA), a potent enterotoxin. Cathelicidins (Camp as gene name, LL-37 peptide in humans and mCRAMP peptide in mice) are antibacterial peptides that also posses anti-inflammatory properties.
Objectives
To determine the role of cathelicidins in models of Clostridium difficile infection and TxA-mediated ileal inflammation and cultured human primary monocytes.
Design
Wild-type (WT) and mCRAMP-deficient (Camp−/−) mice were treated with an antibiotic mixture and infected orally with C difficile. Some mice were intracolonically given mCRAMP daily for 3 days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4 h, while some TxA-treated loops were injected with mCRAMP.
Results
Intracolonic mCRAMP administration to C difficile-infected WT mice showed significantly reduced colonic histology damage, apoptosis, tissue myeloperoxidase (MPO) and tumour necrosis factor (TNF)α levels. Ileal mCRAMP treatment also significantly reduced histology damage, tissue apoptosis, MPO and TNFα levels in TxA-exposed ileal loops. WT and Camp−/− mice exhibited similar intestinal responses in both models, implying that C difficile/TxA-induced endogenous cathelicidin may be insufficient to modulate C difficile/TxA-mediated intestinal inflammation. Both LL-37 and mCRAMP also significantly reduced TxA-induced TNFα secretion via inhibition of NF-κB phosphorylation. Endogenous cathelicidin failed to control C difficile and/or toxin A-mediated inflammation and even intestinal cathelicidin expression was increased in humans and mice.
Conclusion
Exogenous cathelicidin modulates C difficile colitis by inhibiting TxA-associated intestinal inflammation. Cathelicidin administration may be a new anti-inflammatory treatment for C difficile toxin-associated disease.
doi:10.1136/gutjnl-2012-302180
PMCID: PMC3737259  PMID: 22760006
9.  Adipose Tissue and IBD Pathogenesis 
Inflammatory Bowel Diseases  2012;18(8):1550-1557.
Creeping fat has long been recognized as an indicator of Crohn’s disease activity. Although most patients with Crohn’s Disease (CD) have normal or low BMI, the ratio of intra-abdominal fat to total abdominal fat is far greater than that of controls. The obesity epidemic has instructed us on the inflammatory nature of hypertrophic adipose tissue and similarities between mesenteric depots in obese and CD patients can be drawn. However, several important physiological differences exist between these two depots as well. While the molecular basis of the cross-talk between mesenteric adipose and the inflamed intestine in CD is largely unknown, novel evidence implicate neuropeptides along with adipocyte-derived paracrine mediators (adipokines) as potential targets for future investigations and highlight adipose tissue physiology as a potential important determinant in the course of IBD.
doi:10.1002/ibd.22893
PMCID: PMC3374883  PMID: 22407798
10.  Neurotensin stimulates expression of early growth response gene-1 and EGF receptor through MAP kinase activation in human colonic epithelial cells 
Neurotensin (NT) is a highly expressed gastrointestinal (GI) neuropeptide, which modulates GI motility, secretion and cell growth as well as intestinal inflammation. Since EGF receptor is highly expressed in human colon cancer cells, we sought to examine whether NT stimulation contributes to the EGFR overexpression using nontransformed colonocyte NCM460 cells. The results show that NT treatment caused a significant increase in EGFR protein expression and gene transcription. Pretreatment with MAP kinase pathway inhibitor PD98059 blocked NT-induced EGFR expression. As the EGFR promoter has a functional Egr-1 site, previously shown to mediate its transcription in response to hypoxia, we examined the role of Egr-1 in the NT response. We first show that NT stimulated Egr-1 expression, which can be inhibited by PD98059. We also determined whether NT increases Egr-1 binding to its site within the EGFR promoter. The data indicate that NT enhanced the amount of Egr-1 binding to the EGFR Egr-1 site and that this binding was significantly decreased by PD98059. To verify that Egr-1 mediated NT-induced EGFR transcription, Egr-1 siRNA was used to knock down its expression. The data show that transfection of Egr-1 siRNA significantly inhibited NT-stimulated EGFR transcription. Together, our results suggest that NT can stimulate MAP kinase-mediated Egr-1 and EGFR gene expression in human colonocytes. Our results may be relevant to the mechanisms by which NT participates in the development of colon cancer.
doi:10.1002/ijc.22407
PMCID: PMC3685406  PMID: 17230532
neurotensin; early growth response gene-1; EGF receptor
12.  Antimicrobial Peptides and Colitis 
Current pharmaceutical design  2013;19(1):40-47.
Antimicrobial peptides (AMPs) are important components of innate immunity. They are often expressed in response to colonic inflammation and infection. Over the last several years, the roles of several antimicrobial peptides have been explored. Gene expression of many AMPs (beta defensin HBD2-4 and cathelicidin) is induced in response to invasion of gut microbes into the mucosal barrier. Some AMPs are expressed in a constitutive manner (alpha defensin HD 5-6 and beta defensin HBD1), while others (defensin and bactericidal/permeability increasing protein BPI) are particularly associated with Inflammatory Bowel Disease (IBD) due to altered defensin expression or development of autoantibodies against Bactericidal/permeability increasing protein (BPI). Various AMPs have different spectrum and strength of antimicrobial effects. Some may play important roles in modulating the colitis (cathelicidin) while others (lactoferrin, hepcidin) may represent biomarkers of disease activity. The use of AMPs for therapeutic purposes is still at an early stage of development. A few natural AMPs were shown to be able to modulate colitis when delivered intravenously or intracolonically (cathelicidin, elafin and SLPI) in mouse colitis models. New AMPs (synthetic or artificial non-human peptides) are being developed and may represent new therapeutic approaches against colitis. This review discusses the latest research developments in the AMP field with emphasis in innate immunity and pathophysiology of colitis.
PMCID: PMC3662473  PMID: 22950497
Antimicrobial peptides; colitis; infection; microflora; protein; Crohn’s disease; ulcerative colitis
13.  The neurotensin receptor-1 promotes tumor development in a sporadic but not an inflammation-associated mouse model of colon cancer 
Neurotensin receptor-1 (NTR-1) is overexpressed in colon cancers and colon cancer cell lines, Signaling through this receptor stimulates proliferation of colonocyte-derived cell lines and promotes inflammation and mucosal healing in animal models of colitis. Given the causal role of this signaling pathway in mediating colitis and the importance of inflammation in cancer development we tested the effects of NTR-1 in mouse models of inflammation-associated and sporadic colon cancer using NTR-1 deficient (Ntsr1−/−) and wild type (Ntsr1+/+) mice. In mice treated with azoxymethane (AOM) to model sporadic cancer, NTR-1 had a significant effect on tumor development with Ntsr1+/+ mice developing over 2-fold more tumors than Ntsr1−/− mice (p = 0.04). There was no effect of NTR-1 on the number of aberrant crypt foci or tumor size, suggesting that NT/NTR-1 signaling promotes the conversion of precancerous cells to adenomas. Interestingly, NTR-1 status did not affect tumor development in an inflammation-associated cancer model where mice were treated with AOM followed by two cycles of 5% dextran sulphate sodium (DSS). In addition, colonic molecular and histopathologic analyses were performed shortly after a single cycle of DSS. NTR-1 status did not affect colonic myeloperoxidase activity or histopathologic scores for damage and inflammation. However, Ntsr1−/− mice were more resistant to DSS-induced mortality (p = 0.01) and had over 2-fold higher colonic expression levels of Il6 and Cxcl2 (p < 0.04), cytokines known to promote tumor development. These results represent the first direct demonstration that targeted disruption of the Ntsr1 gene reduces susceptibility to colon tumorigenesis.
doi:10.1002/ijc.26208
PMCID: PMC3288327  PMID: 21630261
Neurotensin receptor-1; azoxymethane; dextran sulphate sodium; aberrant crypt foci; colon cancer
14.  Diet-Induced Obesity Promotes Colon Tumor Development in Azoxymethane-Treated Mice 
PLoS ONE  2013;8(4):e60939.
Obesity is an important risk factor for colon cancer in humans, and numerous studies have shown that a high fat diet enhances colon cancer development. As both increased adiposity and high fat diet can promote tumorigenesis, we examined the effect of diet-induced obesity, without ongoing high fat diet, on colon tumor development. C57BL/6J male mice were fed regular chow or high fat diet for 8 weeks. Diets were either maintained or switched resulting in four experimental groups: regular chow (R), high fat diet (H), regular chow switched to high fat diet (RH), and high fat diet switched to regular chow (HR). Mice were then administered azoxymethane to induce colon tumors. Tumor incidence and multiplicity were dramatically smaller in the R group relative to all groups that received high fat diet at any point. The effect of obesity on colon tumors could not be explained by differences in aberrant crypt foci number. Moreover, diet did not alter colonic expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, interleukin-1β, and interferon-γ, which were measured immediately after azoxymethane treatment. Crypt apoptosis and proliferation, which were measured at the same time, were increased in the HR relative to all other groups. Our results suggest that factors associated with obesity – independently of ongoing high fat diet and obesity – promote tumor development because HR group animals had significantly more tumors than R group, and these mice were fed the same regular chow throughout the entire carcinogenic period. Moreover, there was no difference in the number of aberrant crypt foci between these groups, and thus the effect of obesity appears to be on subsequent stages of tumor development when early preneoplastic lesions transition into adenomas.
doi:10.1371/journal.pone.0060939
PMCID: PMC3616169  PMID: 23560112
15.  Sustained TL1A (TNFSF15) expression on both lymphoid and myeloid cells leads to mild spontaneous intestinal inflammation and fibrosis  
TL1A is a member of the TNF superfamily, and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, patients with certain TNFSF15 variants over-express TL1A and have a higher risk of developing strictures in the small intestine. Consistently, mice with sustained Tl1a expression in either lymphoid or myeloid cells develop spontaneous ileitis and increased intestinal collagen deposition. Transgenic (Tg) mice with constitutive Tl1a expression in both lymphoid and myeloid cells were generated to assess their in vivo consequence. Constitutive expression of Tl1a in both lymphoid and myeloid cells showed increased spontaneous ileitis and collagen deposition than WT mice. T cells with constitutive expression of Tl1a in both lymphoid and myeloid cells were found to have a more activated phenotype, increased gut homing marker CCR9 expression, and enhanced Th1 and Th17 cytokine activity than WT mice. Although no differences in T cell activation marker, Th1 or Th17 cytokine activity, ileitis, or collagen deposition were found between constitutive Tl1a expression in lymphoid only, myeloid only, or combined lymphoid and myeloid cells. Double hemizygous Tl1a-Tg mice appeared to have worsened ileitis and intestinal fibrosis. Our findings confirm that TL1A–DR3 interaction is involved in T cell-dependent ileitis and fibrosis.
doi:10.1556/EuJMI.3.2013.1.2
PMCID: PMC3638202  PMID: 23638306
transgenic, ileitis, fibrosis, mucosal inflammation
16.  Clostridial toxins 
Gut Microbes  2012;3(1):35-41.
The current global outbreak of Clostridium difficile infection exemplifies the major public health threat posed by clostridial glucosylating toxins. In the western world, C. difficile infection is one of the most prolific causes of bacterial-induced diarrhea and potentially fatal colitis. Two pathogenic enterotoxins, TcdA and TcdB, cause the disease. Vancomycin and metronidazole remain readily available treatment options for C. difficile infection, but neither is fully effective as is evident by high clinical relapse and fatality rates. Thus, there is an urgent need to find an alternative therapy that preferentially targets the toxins and not the drug-resistant pathogen. Recently, we addressed these critical issues in a Nature Medicine letter, describing a novel host defense mechanism for subverting toxin virulence that we translated into prototypic allosteric therapy for C. difficile infection. In this addendum article, we provide a continued perspective of this antitoxin mechanism and consider the broader implications of therapeutic allostery in combating gut microbial pathogenesis.
doi:10.4161/gmic.19250
PMCID: PMC3337123  PMID: 22356854
C. difficile; allostery; dietary supplement; inositol phosphate; S-nitrosylation; toxin
17.  Oncogenic Lin28A and Lin28B inhibit let-7 microRNA biogenesis by distinct mechanisms 
Cell  2011;147(5):1066-1079.
Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUTase4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a TUTase-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts is restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors while Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function, and have implications for the development of new strategies for cancer therapy.
doi:10.1016/j.cell.2011.10.039
PMCID: PMC3227872  PMID: 22118463
Lin-28; Lin28A; Lin28B; let-7; microRNA (miRNA); TUTase; Zcchc11; TUTase4; TUT4; Cancer
18.  Cathelicidin Signaling via the Toll-Like Receptor Protects Against Colitis in Mice 
Gastroenterology  2011;141(5):1852-1863.e3.
Background & Aims
Cathelicidin (encoded by Camp) is an anti-microbial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease; we investigated its signaling mechanisms.
Methods
Quantitative, real-time, reverse transcription PCR, bacterial 16S PCR, immunofluorescence, and small interfering (si)RNA analyses were performed. Colitis was induced in mice using sodium dextran sulfate (DSS); levels of cathelicidin were measured in human primary monocytes.
Results
Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis, compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to E coli DNA induced expression of Camp mRNA, which required signaling by ERK; expression was reduced by siRNAs against toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9 −/− mice with DSS-induced colitis. Compared with wild-type mice, Camp −/− mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice.
Conclusions
Cathelicidin protects against colitis induction in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9–ERK signaling by bacterial DNA. This pathway might be involved in pathogenesis of ulcerative colitis.
doi:10.1053/j.gastro.2011.06.079
PMCID: PMC3199285  PMID: 21762664
Cramp; LL-37; IBD; mouse models; endogenous inhibitors; immune regulation
19.  The effects of acute and chronic psychological stress on bladder function in a rodent model 
Urology  2011;78(4):967.e1-967.e7.
Purpose
Psychological stress plays a role in the exacerbation of functional lower urinary tract disorders such as painful bladder syndrome and overactive bladder. To better understand the mechanism underlying this relationship, we characterized changes in micturition, anxiety-related behavior, and bladder pathology in rats exposed to repeated water avoidance (WA) stress.
Methods
Twenty-four Wistar rats were subjected to WA stress or sham. Immediately following acute (day 1) and chronic (day 10) stress or sham, rats were placed in a metabolic cage for a 2-hour voiding behavior assessment. Voiding parameters were compared to baseline values obtained prior to stress. Four animals from each group were sacrificed on day 10 and bladders harvested for histologic and gene expression studies. The remaining 8 animals per group underwent repeated voiding assessment every 3 days for 1 month followed by 10 days of repeat WA stress or sham. Bladder histology and gene expression were studied.
Results
Rats exposed to WA stress developed a significant increase in micturition frequency and decrease in latency to void, voiding interval and volume of first void compared to sham and baseline. Alterations in micturition persisted for approximately 1 month. Stressed rats showed increased fecal pellet excretion and anxiety-like behavior. Additionally, bladder specimens from stressed animals revealed increased angiogenesis, and increased total and activated mast cells.
Conclusions
In rats, repeated psychological stress results in lasting alterations in micturition frequency, interval, and volume. This rodent model may represent a valid tool for studying syndromes characterized by increased urinary frequency.
doi:10.1016/j.urology.2011.06.041
PMCID: PMC3190050  PMID: 21868072
Water avoidance stress; overactive bladder; painful bladder syndrome/interstitial cystitis; animal model; urinary frequency
20.  Activation of Corticotropin-Releasing Factor Receptor 2 Mediates the Colonic Motor Coping Response to Acute Stress in Rodents 
Gastroenterology  2011;140(5):1586-96.e6.
Background & Aims
Corticotropin-releasing factor receptor-1 (CRF1) mediates the stress-induced colonic motor activity. Less is known about the role of CRF2 in the colonic response to stress.
Methods
We studied colonic contractile activity (CCA) in rats and CRF2-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute, partial-restraint stress (PRS), and/or intraperitoneal (IP) injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF1 and CRF2 using immunohistochemical and immunoblot analyses. We measured phosphorylation of ERK1/2 by CRF ligands in primary cultures of LMMP-neurons (PC-LMMPn) and cAMP production in HEK-293 cells transfected with CRF1 and/or CRF2.
Results
In rats, a selective agonist of CRF2 (urocortin 2) reduced CRF-induced defecation (>50%), CCA, and Fos expression in the colonic LMMP. A selective antagonist of CRF2 (astressin2-B) increased these responses. Urocortin 2 reduced PRS-induced CCA in wild-type and CRF-overexpressing mice, whereas disruption of CRF2 increased PRS-induced CCA and CRF-induced defecation. CRF2 co-localized with CRF1 and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of ERK in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF1 (NBI35965) or astressin2-B, respectively. The EC50 for the CRF-induced cAMP response was 8.6 nM in HEK-293 cells that express only CRF1; this response was suppressed 10-fold in cells that express CRF1 and CRF2.
Conclusions
In colon tissues of rodents, CRF2 activation inhibits CRF1 signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF2 function impairs colonic coping responses to stress.
doi:10.1053/j.gastro.2011.01.039
PMCID: PMC3150547  PMID: 21277852
colonic contraction; myenteric neurons; nNOS; stress response
21.  The Insect Peptide Coprisin Prevents Clostridium difficile-Mediated Acute Inflammation and Mucosal Damage through Selective Antimicrobial Activity▿ 
Antimicrobial Agents and Chemotherapy  2011;55(10):4850-4857.
Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the α-helical region of it (LLCIALRKK) that had antimicrobial activity (J.-S. Hwang et al., Int. J. Pept., 2009, doi:10.1155/2009/136284). Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.
doi:10.1128/AAC.00177-11
PMCID: PMC3186999  PMID: 21807975
22.  Serum and Colonic Mucosal Immune Markers in Irritable Bowel Syndrome 
OBJECTIVES
Low-grade colonic mucosal inflammation has been postulated to have an important role in the pathophysiology of irritable bowel syndrome (IBS). The objectives of this study were (i) to identify serum and tissue-based immunological and neuroendocrine markers associated with mucosal inflammation in male (M) and female (F) patients with non-post-infectious IBS (non-PI-IBS) compared with healthy controls and (ii) to assess possible correlations of such markers with IBS symptoms.
METHODS
Sigmoid mucosal biopsies were obtained from 45 Rome II positive IBS patients without a history of PI-IBS (26 F, 35.5% IBS-C, 33.3% IBS-D, 31.1% IBS-A/M) and 41 healthy controls (22 F) in order to measure immunological markers (serum cytokine levels, colonic mucosal mRNA levels of cytokines, mucosal immune cell counts) and neuroendocrine markers associated with mucosal inflammation (corticotropin releasing factor- and neurokinin (NK)-related ligands and receptors, enterochromaffin cells). Symptoms were measured using validated questionnaires.
RESULTS
Of all the serum and mucosal cytokines measured, only interleukin-10 (IL-10) mRNA expression showed a group difference, with female, but not male, patients showing lower levels compared with female controls (18.0 ± 2.9 vs. 29.5 ± 4.0, P = 0.006). Mucosal mRNA expression of NK-1 receptor was significantly lower (1.15 ± 0.19 vs. 2.66 ± 0.56, P = 0.008) in female, but not male, patients compared with healthy controls. No other significant differences were observed.
CONCLUSIONS
Immune cell counts and levels of cytokines and neuropeptides that are associated with inflammation were not significantly elevated in the colonic mucosa of non-PI-IBS patients, and did not correlate with symptoms. Thus, these findings do not support that colonic mucosal inflammation consistently has a primary role in these patients. However, the finding of decreased IL-10 mRNA expression may be a possible biomarker of IBS and warrants further investigation.
doi:10.1038/ajg.2011.423
PMCID: PMC3297737  PMID: 22158028
23.  Efficacy and safety of the probiotic Saccharomyces boulardii for the prevention and therapy of gastrointestinal disorders 
Several clinical trials and experimental studies strongly suggest a place for Saccharomyces boulardii as a biotherapeutic agent for the prevention and treatment of several gastrointestinal diseases. S. boulardii mediates responses resembling the protective effects of the normal healthy gut flora. The multiple mechanisms of action of S. boulardii and its properties may explain its efficacy and beneficial effects in acute and chronic gastrointestinal diseases that have been confirmed by clinical trials. Caution should be taken in patients with risk factors for adverse events. This review discusses the evidence for efficacy and safety of S. boulardii as a probiotic for the prevention and therapy of gastrointestinal disorders in humans.
doi:10.1177/1756283X11428502
PMCID: PMC3296087  PMID: 22423260
efficacy; gastrointestinal disorders; probiotic; Saccharomyces boulardii; safety
24.  Host S-nitrosylation inhibits clostridial small molecule–activated glucosylating toxins 
Nature Medicine  2011;17(9):1136-1141.
The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins1–4. Virulence is dependent on the autoactivation of a toxin cysteine protease5–9, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6)10–17. Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins18,19. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.
doi:10.1038/nm.2405
PMCID: PMC3277400  PMID: 21857653
25.  The Corticotropin Releasing Hormone Family of Peptides Regulates Intestinal Angiogenesis 
Gastroenterology  2010;138(7):2457-2467.e5.
BACKGROUND & AIMS
The corticotrophin-releasing hormone (CRH) family of peptides modulates intestinal inflammation and the CRH receptor 2 (CRHR2) suppresses postnatal angiogenesis in mice. We investigated the functions of CRHR1 and CRHR2 signaling during intestinal inflammation and angiogenesis.
METHODS
The activities of CRHR1 and CRHR2 were disrupted by genetic deletion in mice or with selective antagonists. A combination of in vivo, ex vivo, and in vitro measures of angiogenesis were used to determine their activity. CRHR1−/− mice and CRHR2−/− mice with dextran sodium sulfate-induced colitis were analyzed in comparison with wild-type littermates (controls).
RESULTS
Colitis was significantly reduced in mice in which CRHR1 activity was disrupted by genetic deletion or with an antagonist, determined by analyses of survival rate, weight loss, histological scores, and cytokine production. Inflammation was exacerbated in mice in which CRHR2 activity was inhibited by genetic deletion or with an antagonist, compared with controls. The inflamed intestines of CRHR1−/− mice had reduced microvascular density and expression of vascular endothelial growth factor (VEGF)-A, whereas the intestines of CRHR2−/− mice had increased angiogenesis and VEGF-A levels. An antagonist of VEGFR2 activity alleviated colitis in CRHR2−/− mice. Ex vivo aortic vessel outgrowth was reduced when CRHR1 was deficient but increased when CRHR2 was deficient. The CRHR1 preferred agonist CRH stimulated tube formation, proliferation, and migration of cultured intestinal microvascular endothelial cells by phosphorylating Akt whereas the specific CRHR2 agonist Urocortin III had opposite effects.
CONCLUSION
CRHR1 promotes intestinal inflammation, as well as endogenous and inflammatory angiogenesis whereas CRHR2 inhibits these activities.
doi:10.1053/j.gastro.2010.02.055
PMCID: PMC2883634  PMID: 20206175
neuropeptide; inflammatory bowel disease; PI3K; HIMECs

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