Tu1622 Glycyrrhizin Decreases HMGB1 Release and Is Effective for the Treatment of Chemical-Induced...
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AGA Abstracts Conclusion: Our data suggest nicotine inhibits chemokine-induced pDC migration through α7nAChR-mediated JAK2 and Caspase-3 activation, and thereby suppresses pDC-mediated antigen presentation in oxazolone colitis. Our results may provide new insights into the development of a therapeutic medicine for ulcerative colitis. Tu1620 Intestinal Alkaline Phosphatase Is an Endogenous Anti-Inflammatory Factor Mussa Mohamed, Konstantinos P. Economopoulos, Palak Patel, Nur Muhammad, Omeed Moaven, Angela K. Moss, Sulaiman R. Hamarneh, Abeba Teshager, Kanakaraju Kaliannan, Seyed M. Abtahi, Sayeda Nasrin Alam, Nondita S. Malo, Qingsong Tao, Madhu S. Malo, Richard A. Hodin Introduction: Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme known to have the ability to detoxify in-vitro many pro-inflammatory bacterial components, including lipopolysaccharides (LPS), lipoteichoic acid (LTA), flagellin, CpG-DNA and uridine diphosphate (UDP). Gastrointestinal tract inflammation and endotoxemia due to elevated bacterial toxic components in the gut and disruption of intestinal permeability play a crucial role in the development and progression of a wide spectrum of diseases. In this study we sought to determine whether the endogenous IAP enzyme functions as an anti-inflammatory factor. Methods: We established a novel intestinal loop model to study the impact of endogenous IAP on the inflammatory activity of different bacterial components within a physiologic in vivo environment. The model was set up in wild type (WT) vs. IAP knockout (KO) mice of approximately 25 grams (n=5 for all groups). In another setting, we applied a fast (48 hours) vs. fed mouse model. Under general anesthesia, a 5 cm segment of proximal jejunum was carefully tied off at the proximal and distal ends, to isolate the loop. Different concentrations of LPS (100 ng/ml), LTA (5 μg/ml), flagellin (100 ng/ml), CpG-DNA (100 μg/ml) or UDP (1 mM) were injected into the loop and the luminal content was collected 2 hours later. Then, the supernatants were applied to RAW264.7 murine macrophage cells in triplicate and incubated overnight. LPS, LTA, Flagellin, CpG-DNA or UDP were directly applied to the cells as positive controls and endotoxin-free water was applied as a negative control. Tumor necrosis factor-alpha (TNF- α) levels were subsequently measured by sand- wich ELISA. Results: All studied bacterial components induced a marked increase in TNF- α levels from the RAW264.7 cells, whereas little TNF- α was seen in the case of endotoxin- free water alone. The luminal contents from the WT mice resulted in significantly lower TNF-α levels compared to the luminal contents from the KO mice for all studied bacterial toxins: LPS (600.9±75.47 vs. 946.2±55.99 pg/ml, p= 0.006), LTA (223.1±62.85 vs. 536.2±54.64 pg/ml, p= 0.005), flagellin (679.9±60.05 vs. 1008.8±61.15 pg/ml, p= 0.005), CpG-DNA (638.7±61.81 vs. 949.6±57.36 pg/ml , p= 0.006) and UDP (212.5±15.77 vs. 312.6±26.60 pg/ml, p= 0.012). Luminal contents from fed mice resulted in lower TNF-a levels compared to fasted mice for LPS (585.4±76.35 vs. 900.2±63.62 pg/ml, p=0.013). Conclusions: IAP detoxifies and prevents the inflammatory effects of LPS, LTA, flagellin, CpG-DNA and UDP in the gut. The loss of IAP expression that occurs with fasting could be responsible for the systemic sepsis syndrome seen in critically ill patients. Tu1621 Anti-Inflammatory Effects of Carbon Monoxide (CO) Liberated From Co- Releasing Molecule (Corm-3) on Trinitrobenzene Sulfonic Acid (TNBS)- Induced Colitis in Mice Wataru Fukuda, Yuji Naito, Tomohisa Takagi, Kazuhiro Katada, Katsura Mizushima, Kentaro Suzuki, Yukiko Uehara, Hideki Horie, Yutaka Inada, Takaya Iida, Toshifumi Tsuji, Hiroyuki Yoriki, Munehiro Kugai, Akifumi Fukui, Tetsuya Okayama, Naohisa Yoshida, Kazuhiro Kamada, Kazuhiko Uchiyama, Ishikawa Takeshi, Osamu Handa, Hideyuki Konishi, Nobuaki Yagi, Satoshi Kokura, Ichikawa Hiroshi, Toshikazu Yoshikawa ,Background and Aims. Carbon monoxide (CO) has been shown to confer anti-inflamma- tory effects in various animal models. We have reported anti-inflammatory effects of CO gas inhalation or DMSO-soluble CO-releasing molecule CORM-2 on experimental colitis models in mice. However, there were some critical issues of CO administration that CO gas inhalation elevated the carboxyhemoglobin (CO-Hb) levels in blood and that CORM-2 was lipid-soluble. Therefore, the aim of this study was to assess the effects of CO liberated from a novel water-soluble CO-releasing molecule CORM-3 on trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice, especially CO-mediated cytokine expressions in anti-CD3/ CD28 stimulated CD4+ T+cells and differentiation of T helper (Th) cells. ,Materials and Methods. To induce colitis, C57BL/6 male mice received an enema of TNBS. CORM-3 (10 mg/kg) was intraperitoneally administered once before and twice daily after the enema of TNBS. The distal colon was removed on the 3rd day after TNBS administration to measure ulcer scores, damage scores of the colon, tissue-associated myeloperoxidase (MPO) activity, and TNF-α, IFN-γ and IL-17A (mRNA and protein) expressions in the colonic mucosa. CD4+ T cells were isolated from the spleen of C57BL/6 male mice by MACS. The isolated CD4+ T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 with or without CORM-3 (50 μM). The cells and supernatants were collected after 24h stimulation and cytokine (TNF-α, IFN-γ, and IL-17A) expressions in anti-CD3/CD28 stimulated CD4+ T+cells were assessed. Furthermore, the effects of CORM-3 on the modulation of Th polariza- tion were investigated by using Naïve CD4+ T cells isolated from mice splenocytes. ,Results. Mucosal ulcer scores and damage scores of the colon were significantly increased in TNBS- induced mice compared to sham-operated mice. Tissue-associated MPO activity and expres- sions of TNF- α, IFN-γ and IL-17A in the colonic mucosa were significantly higher in TNBS-induced mice. These increases were significantly attenuated in CORM-3-treated mice. Productions of TNF-α, IFN-γ and IL-17A in anti-CD3/CD28-stimulated CD4+ T cells were significantly attenuated by CORM-3 (50 μM). Furthermore, decreases in IFN- γ production in Th1 cells and IL-17A production in Th17 cells (differentiation of Th1/Th17 cells) were found in CORM-3-treated mice. ,Conclusion. These findings indicate that CO released from CORM-3 significantly ameliorated inflammatory responses in TNBS-induced colitis possibly in some part via the inhibition of Th1/Th17 cells differentiation, suggesting that CO might be a new therapeutic molecule for inflammatory bowel disease. S-808 AGA Abstracts Tu1622 Glycyrrhizin Decreases HMGB1 Release and Is Effective for the Treatment of Chemical-Induced Murine Colitis Roberta Vitali, Francesca Palone, Anna Negroni, Manuela Costanzo, Anna Dilillo, Marina Aloi, Giulia D'Arcangelo, Salvatore Cucchiara, Laura Stronati Background Inflammatory bowel diseases (IBD) are chronic immune-mediated disorders of the gastrointestinal tract. Since IBD are currently not curable, it is crucial to identify innovative therapies. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that can be released into extracellular milieu in response to stress or damage and subsequently activate the immune system and promote inflammation. HMGB1 has been implicated in several inflammatory and auto-immune disorders. We previously showed that HMGB1 is secreted by inflamed intestinal tissues and is abundantly present in the feces of IBD patients, therefore it was proposed as a novel marker of intestinal mucosal inflammation. Glycyrrhizin, a glycoconjugated triterpene produced by the root of licorice plant Glycyrrhiza glabra, exhibits anti-inflammatory properties and has been shown to importantly inhibit the cytokine activity of HMGB1. Thus, the aim of the present study is to assess the efficacy of a glycyrrhizin- derived compound, the dipotassium glycyrrhizate (DPG), in the treatment of chemical- induced murine colitis. Methods DPG (3-8mg/Kg) was administered to C57BL/6 mice after induction of chemical colitis by DSS. Body weight, clinical score, histological score, colon length/weight were evaluated in treated mice and compared to controls. Expression levels of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and HMGB1 receptors (RAGE, TLR4) were also measured by RT-PCR. Fecal HMGB1 was analyzed by western blot. Results Mice exposed to DPG had a dramatic recovery of body weight (60%), total clinical score, histological score and large intestine length. Moreover, mice treated with DPG showed a significantly decrease in the mRNA levels of pro-inflammatory cytokines and HMGB1 recep- tors (p,0.001). Finally, HMGB1 was found to be abundantly present in the feces of mice with DSS-induced colitis, but this amount was strongly reduced following DPG treatment (p,0.01). Conclusion DPG showed excellent therapeutic properties for the treatment of DSS-induced murine colitis. We believe that these findings may open new perspectives in the setting of alternative strategies for the treatment of human IBD. Tu1623 Development of Fibrosis in the Rat Model of Chronic TNBS Colitis Attenuated by the Selective αVβ3 Rgd Inhibitor Cilengitide Chao Li, John F. Kuemmerle The TGF-β1 gene product, latent TGF- β1, needs regulated cleavage to become active. We recently showed that not only TGF- β1 expression increases but also levels of active TGF- β1 increase in smooth muscle cells of strictures compared to the normal resection margin in the same patient. Latent TGF- β1 binding to the RGD sequence of αVβ3 integrin regulated its activation and was inhibited by the αVβ3 selective RGD inhibitor, cilengitide. Hypothesis: Treatment of rats with the αVβ3 selective RGD inhibitor, cilengitide, will diminish TGF- β1 activation, resulting collagen production and the development of fibrosis from chronic TNBS administration to rats. Methods: Chronic colitis was established in Sprague-Dawley rats using escalating doses of TNBS. Four groups were studied: EtOH vehicle and TNBS treated rats given either 18 mg/kg/d cilengitide ip or PBS alone. Colon sections were used for trichrome staining and immunostaining. Muscle cells of muscularis propria isolated from the colon were used to prepare mRNA and protein lysates. TGF- β1 and collagen I αI expression were measured by qRT-PCR. TGF- β1 levels were measured by ELISA. Direct association of latent TGF- β1 and αVβ3 integrin was determined by proximity ligation hybridization assay (PLA). Collagen was measured by Sircol assay. Fibrosis was measured by muscle thickness and from collagen deposition in digital images with RGD segmentation. Leukocyte infiltration was quantified by myeloperoxidase (MPO) activity assay and CD-11b immunostaining. Cytokine profiling was performed by ELISA. Results: No differences in MPO activity or CD11b staining were seen among the four groups after 42 d of colitis. In contrast TGF-β1 expression increased 8-fold and active TGF- β1 levels increased 2-fold in TNBS treated rats compared to EtOH controls. The increase in TGF- β1 transcripts and active TGF-β1 levels were abolished in cilengitide treated rats. Latent TGF- β1 binding to αVβ3 increased 8 fold in TNBS treated rats and was abolished by cilengitide. After 42 d of TNBS compared to EtOH collagen expression increased 2 fold, collagen deposition increased 35 ng/ml, collagen volume in the muscle layer increased 40%, and muscularis thickness increased 95 μm. Cilengitide inhibited all four parameters in TNBS treated rats 90-100%. Levels of IL-1β, IL-6, TNF- α, IFNγ and TGF- β1 were decreased by cilengitide treatment, whereas levels of IL-4, IL-12 and GM-CSF were increased by cilengitide treatment. Conclu- sions: The αVβ3 selective RGD inhibitor, cilengitide, diminishes TGF- β1 expression and activation, collagen production and development of fibrosis in chronic TNBS colitis. These effects and cilengitide's ability to modify cytokine levels indicate it may be useful to diminish the development of fibrosis in patients with stricturing Crohn's disease. Supported by NIH DK49691 Tu1624 Amelioration of Dextran Sodium Sulfate-Induced Colitis in Mice by Genetic Deletion of KLF4 in the Intestinal Epithelium or by Oral Delivery of Nanoparticles Loaded With KLF4-SiRNA to the Colonic Epithelial Cells Amr Ghaleb, Hamed Laroui, Poonam Rakhya, Didier Merlin, Vincent W. Yang BACKGROUND: KLF4 is a zinc finger transcription factor expressed in the differentiated epithelial cells lining the villus border and surface epithelium of the small and large intestine, respectively. KLF4 inhibits cell proliferation, however, it is a mediator of proinflammatory signaling in macrophages and its over-expression in the esophageal epithelium activates cytokines, leading to inflammation-mediated esophageal squamous cell cancer formation in mice. Intestinal epithelial expression of antioxidants and nuclear factor kappa B (NF- κB) contribute to mucosal barrier integrity and epithelial homeostasis, two key events in the pathogenesis of inflammatory bowel disease (IBD). AIM: To determine whether KLF4 has a proinflammatory activity in vivo and the therapeutic potential of its epithelial inhibition
Transcript of Tu1622 Glycyrrhizin Decreases HMGB1 Release and Is Effective for the Treatment of Chemical-Induced...
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sConclusion: Our data suggest nicotine inhibits chemokine-induced pDC migration throughα7nAChR-mediated JAK2 and Caspase-3 activation, and thereby suppresses pDC-mediatedantigen presentation in oxazolone colitis. Our results may provide new insights into thedevelopment of a therapeutic medicine for ulcerative colitis.
Tu1620
Intestinal Alkaline Phosphatase Is an Endogenous Anti-Inflammatory FactorMussa Mohamed, Konstantinos P. Economopoulos, Palak Patel, Nur Muhammad, OmeedMoaven, Angela K. Moss, Sulaiman R. Hamarneh, Abeba Teshager, Kanakaraju Kaliannan,Seyed M. Abtahi, Sayeda Nasrin Alam, Nondita S. Malo, Qingsong Tao, Madhu S. Malo,Richard A. Hodin
Introduction: Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzymeknown to have the ability to detoxify in-vitro many pro-inflammatory bacterial components,including lipopolysaccharides (LPS), lipoteichoic acid (LTA), flagellin, CpG-DNA and uridinediphosphate (UDP). Gastrointestinal tract inflammation and endotoxemia due to elevatedbacterial toxic components in the gut and disruption of intestinal permeability play a crucialrole in the development and progression of a wide spectrum of diseases. In this study wesought to determine whether the endogenous IAP enzyme functions as an anti-inflammatoryfactor. Methods: We established a novel intestinal loop model to study the impact ofendogenous IAP on the inflammatory activity of different bacterial components within aphysiologic in vivo environment. The model was set up in wild type (WT) vs. IAP knockout(KO) mice of approximately 25 grams (n=5 for all groups). In another setting, we applieda fast (48 hours) vs. fed mouse model. Under general anesthesia, a 5 cm segment of proximaljejunum was carefully tied off at the proximal and distal ends, to isolate the loop. Differentconcentrations of LPS (100 ng/ml), LTA (5 μg/ml), flagellin (100 ng/ml), CpG-DNA (100μg/ml) or UDP (1 mM) were injected into the loop and the luminal content was collected2 hours later. Then, the supernatants were applied to RAW264.7 murine macrophage cellsin triplicate and incubated overnight. LPS, LTA, Flagellin, CpG-DNA or UDP were directlyapplied to the cells as positive controls and endotoxin-free water was applied as a negativecontrol. Tumor necrosis factor-alpha (TNF-α) levels were subsequently measured by sand-wich ELISA. Results: All studied bacterial components induced a marked increase in TNF-α levels from the RAW264.7 cells, whereas little TNF-α was seen in the case of endotoxin-free water alone. The luminal contents from the WT mice resulted in significantly lowerTNF-α levels compared to the luminal contents from the KO mice for all studied bacterialtoxins: LPS (600.9±75.47 vs. 946.2±55.99 pg/ml, p= 0.006), LTA (223.1±62.85 vs.536.2±54.64 pg/ml, p= 0.005), flagellin (679.9±60.05 vs. 1008.8±61.15 pg/ml, p= 0.005),CpG-DNA (638.7±61.81 vs. 949.6±57.36 pg/ml , p= 0.006) and UDP (212.5±15.77 vs.312.6±26.60 pg/ml, p= 0.012). Luminal contents from fed mice resulted in lower TNF-alevels compared to fasted mice for LPS (585.4±76.35 vs. 900.2±63.62 pg/ml, p=0.013).Conclusions: IAP detoxifies and prevents the inflammatory effects of LPS, LTA, flagellin,CpG-DNA and UDP in the gut. The loss of IAP expression that occurs with fasting couldbe responsible for the systemic sepsis syndrome seen in critically ill patients.
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Anti-Inflammatory Effects of Carbon Monoxide (CO) Liberated From Co-Releasing Molecule (Corm-3) on Trinitrobenzene Sulfonic Acid (TNBS)-Induced Colitis in MiceWataru Fukuda, Yuji Naito, Tomohisa Takagi, Kazuhiro Katada, Katsura Mizushima,Kentaro Suzuki, Yukiko Uehara, Hideki Horie, Yutaka Inada, Takaya Iida, Toshifumi Tsuji,Hiroyuki Yoriki, Munehiro Kugai, Akifumi Fukui, Tetsuya Okayama, Naohisa Yoshida,Kazuhiro Kamada, Kazuhiko Uchiyama, Ishikawa Takeshi, Osamu Handa, HideyukiKonishi, Nobuaki Yagi, Satoshi Kokura, Ichikawa Hiroshi, Toshikazu Yoshikawa
,Background and Aims. Carbon monoxide (CO) has been shown to confer anti-inflamma-tory effects in various animal models. We have reported anti-inflammatory effects of COgas inhalation or DMSO-soluble CO-releasing molecule CORM-2 on experimental colitismodels in mice. However, there were some critical issues of CO administration that CO gasinhalation elevated the carboxyhemoglobin (CO-Hb) levels in blood and that CORM-2 waslipid-soluble. Therefore, the aim of this study was to assess the effects of CO liberated froma novel water-soluble CO-releasing molecule CORM-3 on trinitrobenzene sulfonic acid(TNBS)-induced colitis in mice, especially CO-mediated cytokine expressions in anti-CD3/CD28 stimulated CD4+ T+cells and differentiation of T helper (Th) cells. ,Materials andMethods. To induce colitis, C57BL/6 male mice received an enema of TNBS. CORM-3 (10mg/kg) was intraperitoneally administered once before and twice daily after the enema ofTNBS. The distal colon was removed on the 3rd day after TNBS administration to measureulcer scores, damage scores of the colon, tissue-associated myeloperoxidase (MPO) activity,and TNF-α, IFN-γ and IL-17A (mRNA and protein) expressions in the colonic mucosa.CD4+ T cells were isolated from the spleen of C57BL/6 male mice by MACS. The isolatedCD4+ T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 with orwithout CORM-3 (50 μM). The cells and supernatants were collected after 24h stimulationand cytokine (TNF-α, IFN-γ, and IL-17A) expressions in anti-CD3/CD28 stimulated CD4+T+cells were assessed. Furthermore, the effects of CORM-3 on the modulation of Th polariza-tionwere investigated by using Naïve CD4+ T cells isolated frommice splenocytes. ,Results.Mucosal ulcer scores and damage scores of the colon were significantly increased in TNBS-induced mice compared to sham-operated mice. Tissue-associated MPO activity and expres-sions of TNF-α, IFN-γ and IL-17A in the colonic mucosa were significantly higher inTNBS-induced mice. These increases were significantly attenuated in CORM-3-treated mice.Productions of TNF-α, IFN-γ and IL-17A in anti-CD3/CD28-stimulated CD4+ T cells weresignificantly attenuated by CORM-3 (50 μM). Furthermore, decreases in IFN- γ productionin Th1 cells and IL-17A production in Th17 cells (differentiation of Th1/Th17 cells) werefound in CORM-3-treated mice. ,Conclusion. These findings indicate that CO releasedfrom CORM-3 significantly ameliorated inflammatory responses in TNBS-induced colitispossibly in some part via the inhibition of Th1/Th17 cells differentiation, suggesting thatCO might be a new therapeutic molecule for inflammatory bowel disease.
S-808AGA Abstracts
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Glycyrrhizin Decreases HMGB1 Release and Is Effective for the Treatment ofChemical-Induced Murine ColitisRoberta Vitali, Francesca Palone, Anna Negroni, Manuela Costanzo, Anna Dilillo, MarinaAloi, Giulia D'Arcangelo, Salvatore Cucchiara, Laura Stronati
Background Inflammatory bowel diseases (IBD) are chronic immune-mediated disorders ofthe gastrointestinal tract. Since IBD are currently not curable, it is crucial to identify innovativetherapies. High mobility group box 1 (HMGB1) is a DNA-binding nuclear protein that canbe released into extracellular milieu in response to stress or damage and subsequently activatethe immune system and promote inflammation. HMGB1 has been implicated in severalinflammatory and auto-immune disorders. We previously showed that HMGB1 is secretedby inflamed intestinal tissues and is abundantly present in the feces of IBD patients, thereforeit was proposed as a novel marker of intestinal mucosal inflammation. Glycyrrhizin, aglycoconjugated triterpene produced by the root of licorice plant Glycyrrhiza glabra, exhibitsanti-inflammatory properties and has been shown to importantly inhibit the cytokine activityof HMGB1. Thus, the aim of the present study is to assess the efficacy of a glycyrrhizin-derived compound, the dipotassium glycyrrhizate (DPG), in the treatment of chemical-induced murine colitis. Methods DPG (3-8mg/Kg) was administered to C57BL/6 mice afterinduction of chemical colitis by DSS. Body weight, clinical score, histological score, colonlength/weight were evaluated in treated mice and compared to controls. Expression levelsof pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and HMGB1 receptors (RAGE,TLR4) were also measured by RT-PCR. Fecal HMGB1 was analyzed by western blot. ResultsMice exposed to DPG had a dramatic recovery of body weight (60%), total clinical score,histological score and large intestine length. Moreover, mice treated with DPG showed asignificantly decrease in the mRNA levels of pro-inflammatory cytokines and HMGB1 recep-tors (p,0.001). Finally, HMGB1 was found to be abundantly present in the feces of micewith DSS-induced colitis, but this amount was strongly reduced following DPG treatment(p,0.01). Conclusion DPG showed excellent therapeutic properties for the treatment ofDSS-induced murine colitis. We believe that these findings may open new perspectives inthe setting of alternative strategies for the treatment of human IBD.
Tu1623
Development of Fibrosis in the Rat Model of Chronic TNBS Colitis Attenuatedby the Selective αVβ3 Rgd Inhibitor CilengitideChao Li, John F. Kuemmerle
The TGF-β1 gene product, latent TGF-β1, needs regulated cleavage to become active. Werecently showed that not only TGF-β1 expression increases but also levels of active TGF-β1 increase in smooth muscle cells of strictures compared to the normal resection marginin the same patient. Latent TGF-β1 binding to the RGD sequence of αVβ3 integrin regulatedits activation and was inhibited by the αVβ3 selective RGD inhibitor, cilengitide. Hypothesis:Treatment of rats with the αVβ3 selective RGD inhibitor, cilengitide, will diminish TGF-β1 activation, resulting collagen production and the development of fibrosis from chronicTNBS administration to rats. Methods: Chronic colitis was established in Sprague-Dawleyrats using escalating doses of TNBS. Four groups were studied: EtOH vehicle and TNBStreated rats given either 18 mg/kg/d cilengitide ip or PBS alone. Colon sections were usedfor trichrome staining and immunostaining. Muscle cells of muscularis propria isolated fromthe colon were used to prepare mRNA and protein lysates. TGF- β1 and collagen IαIexpression were measured by qRT-PCR. TGF-β1 levels were measured by ELISA. Directassociation of latent TGF-β1 and αVβ3 integrin was determined by proximity ligationhybridization assay (PLA). Collagen was measured by Sircol assay. Fibrosis was measuredby muscle thickness and from collagen deposition in digital images with RGD segmentation.Leukocyte infiltration was quantified by myeloperoxidase (MPO) activity assay and CD-11bimmunostaining. Cytokine profiling was performed by ELISA. Results: No differences inMPO activity or CD11b staining were seen among the four groups after 42 d of colitis. Incontrast TGF-β1 expression increased 8-fold and active TGF-β1 levels increased 2-fold inTNBS treated rats compared to EtOH controls. The increase in TGF- β1 transcripts andactive TGF-β1 levels were abolished in cilengitide treated rats. Latent TGF- β1 binding toαVβ3 increased 8 fold in TNBS treated rats and was abolished by cilengitide. After 42 d ofTNBS compared to EtOH collagen expression increased 2 fold, collagen deposition increased35 ng/ml, collagen volume in the muscle layer increased 40%, and muscularis thicknessincreased 95 μm. Cilengitide inhibited all four parameters in TNBS treated rats 90-100%.Levels of IL-1β, IL-6, TNF-α, IFNγ and TGF-β1 were decreased by cilengitide treatment,whereas levels of IL-4, IL-12 and GM-CSF were increased by cilengitide treatment. Conclu-sions: The αVβ3 selective RGD inhibitor, cilengitide, diminishes TGF- β1 expression andactivation, collagen production and development of fibrosis in chronic TNBS colitis. Theseeffects and cilengitide's ability to modify cytokine levels indicate it may be useful to diminishthe development of fibrosis in patients with stricturing Crohn's disease. Supported byNIH DK49691
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Amelioration of Dextran Sodium Sulfate-Induced Colitis in Mice by GeneticDeletion of KLF4 in the Intestinal Epithelium or by Oral Delivery ofNanoparticles Loaded With KLF4-SiRNA to the Colonic Epithelial CellsAmr Ghaleb, Hamed Laroui, Poonam Rakhya, Didier Merlin, Vincent W. Yang
BACKGROUND: KLF4 is a zinc finger transcription factor expressed in the differentiatedepithelial cells lining the villus border and surface epithelium of the small and large intestine,respectively. KLF4 inhibits cell proliferation, however, it is a mediator of proinflammatorysignaling in macrophages and its over-expression in the esophageal epithelium activatescytokines, leading to inflammation-mediated esophageal squamous cell cancer formation inmice. Intestinal epithelial expression of antioxidants and nuclear factor kappa B (NF- κB)contribute to mucosal barrier integrity and epithelial homeostasis, two key events in thepathogenesis of inflammatory bowel disease (IBD). AIM: To determine whether KLF4 hasa proinflammatory activity in vivo and the therapeutic potential of its epithelial inhibition
