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Mo2019 Extracorporeal Shock Wave Therapy Modulates Inflammation and Prevents Gastrointestinal Ileus After Tissue Trauma and Postoperative Ileus
Mo2019
Direct Effects of Infliximab (IFX) on Intestinal Mucosa: Exploring Mechanisms of Mucosal Healing Franco Scaldaferri, Loris Riccardo Lopetuso, Valentina Petito, Viviana Gerardi, Vincenzo Arena, Egidio Stigliano, Alfredo Papa, Valerio Cufino, Arianna Amato, Giovanni Cammarota, Alessandro Sgambato, Antonio Gasbarrini
Extracorporeal Shock Wave Therapy Modulates Inflammation and Prevents Gastrointestinal Ileus After Tissue Trauma and Postoperative Ileus Vlado Antonic, Alexander Stojadinovic, Mina J. Izadjoo, Anthony J. Bauer Introduction: Over the past 20 years the effects of ESWT has been studied on cells and tissues demonstrating its pro-angiogenic, anti-inflammatory and proliferative effects. However, there is a paucity of data published on the systemic modulatory effects of shock wave. Aim: We sought to determine the systemic effects of ESWT in two injury models: peripheral tissue trauma (TBX) and postoperative ileus (POI). Methods: The safety and feasibility of abdominal ESWT (applied dorsally and ventrally; 600 impulses, 4Hz, EFD:0.03 mJ/mm2, DermaGold, Tissue Regenerative Technologies, Woodstock, GA) was evaluated in adult male mice. Animals were randomly designated to one of the following TBX (12.5% of body weight minced syngeneic muscle and bone dorsally implanted subcutaneously into recipient mice) experimental groups: 1) naïve control (NC), 2) abdominal ESWT 24h pre-conditioning, 3) TBX, 4) abdominal ESWT 24 hr preconditioning+TBX, and 5) TBX+ESWT 4h post implantation. The POI (surgical manipulation of small intestine=SM) groups: 1) naïve controls (NC), 2) abdominal ESWT 24h preconditioning, 3) SM, and 4) abdominal ESWT 24h preconditioning+SM. Twenty-four hours after TBX or SM, mice were given per os FITCdextran (FD70kD MW) and were sacrificed 75min later to generate gastrointestinal transit distribution histograms and calculated geometric centers (GC). Serum and wound fluid were analyzed to determine the levels of 8 pro-inflammatory cytokines (IFN-γ, IL-10, IL-12 p70, IL-1β, IL-6, keratinocyte cytokine (KC), TNF-α, and GM-CSF). Mann-Whitney U test of ranks was used for comparison of cytokine concentrations. P <0.05 was considered significant. Results: TBX: GI transit GC analysis showed a significant protective effect of ESWT preconditioning on the TBX-induced ileus: naïve controls (10.49±0.088); ESWT 24h preconditioning (10.79±0.026), TBX (3.83±0.076); TBX+ESWT preconditioning (6.54±0.195); TBX+ESWT 4h post implantation (4.41±0.040). Comparison of wound fluid cytokine levels did not differ significantly between groups (P>0.05). There was a significant increase in serum IL10 (p=0.002), IL-6 (p<0.001), KC (p<0.001), and TNF-α (p=0.047) in the group with ESWT 24h preconditioning. POI: Gastrointestinal transit GC analysis showed a significant protective effect of ESWT preconditioning on SM-induced POI: naïve controls (10.23±0.083), ESWT 24h preconditioning (10.13±0.074), POI only (3.71±0.144) and POI+ESWT preconditioning (8.10±0.436), respectively. Statistical analysis of the serum samples harvested at 24 hrs showed significant differences between groups in INF-γ (p=0.036). Conclusions: Defocused shock waves can be safely administered to the abdomen without causing intestinal damage. ESWT preconditioning has protective effects on motility in both TBX and POI models by modulating the inflammatory response.
BACKGROUND Mucosal healing is one of the biggest issue on the management of IBD. Although clinical use of these drugs in achieving mucosal healing is a reality, the mechanisms of it are still not clear. Aim of our study is to identify the effect of IFX on intestinal epithelial cells in order to induce mucosal healing, by exploring whether IFX is really acting at intestinal mucosal level and whether IFX acts on «wound healing» process of intestinal epithelial cells, using In Vitro and In Vivo approach. METHODS Human intestinal mucosal biopsies from active IBD patients were cultured for 48 h with or without IFX (50 ng/ml), and supernatants were assessed for cytokines content and histology performed. Effect of IFX was explored in C57BL/6 mice exposed for 7 days to 2,5 % of DSS. IFX was administered iv (5 mg/Kg) at day 5 or by daily enema (300 mg/200 ml) for 3 days starting at day 5. At day 9 mice were sacrificed and histology was taken. A scratch assay was performed on CT26 and Caco 2 cell line monolayer. Precice scratches were obtained by seading cells in micro-chamber formed by culture inserts. Cells were exposed for 24 h to IFX (50 ng/ml) and/or TNF-α (100 U/ml), or to supernatants of PBMC or fibroblast treated or not for 24 h with IFX and/ or TNFα. Seriated pictures were taken for 24 h following the stimulation and analized by computer software. RESULTS Human biopsies exposed to IFX showed a decrease in TNFa content, as well as of innate and adaptive immunity cytokines. Furthermore at histology IFX-treated biopsies showed a a decreased immune cells infiltration. In Vivo, IFX iv or given as enema ameliorated the severity of colitis in mice, by lowering the disease activity index (DAI) and the loss of body weight. At day 9 a reduction of intestinal inflammatory infiltrate was observed in treated mice. At scratch assay, TNF-a slightly decreased the healing process, while IFX alone or in combination did not. TNF-a exposed PBMC supernatants decreased epithelial healing; also TNF-a fibroblast supernatants slowed the healing process but adding IFX the healing process was restored. CONCLUSIONS Taken together our preliminary observations suggest that IFX acts locally at mucosa levels, as shown by the decrease in inflammatory infiltrate and cytokines contents in human biopsies and the effect on murine colitis when given intrarectally. The effect of IFX is only partially dependent on direct effect on epithelial cells. IFX ameliorates epithelial cell healing by blocking the detrimental capacity of TNF-a pre-treated fibroblast on intestinal epithelial cells. More data are necessary to unravel molecular pathways of these observations. Understanding mechanisms of action of IFX may clarify mechanisms of loss of responses to it as well as new targets for potential new therapies.
Mo2020 Mo2018 Mucin Cys Domain Strengthens the Mucus Barrier During Experimental Intestinal Inflammation Valérie Gouyer, Laurent Dubuquoy, Christel Neut, Pierre Desreumaux, Frédéric Gottrand, Jean-Luc Desseyn
Vitamin D Deficiency in Utero Through Adulthood Results in an Inflammation-Prone Colonic Gene Expression Profile in Healthy CD-1 Mice While IL-10 Knock-out Mice are Not Responsive Raha Jahani, Andrea J. Glenn, Kristina A. Fielding, Reinhold Vieth, Wendy E. Ward, Elena Comelli
Introduction The mucus layer prevents contact between bacteria of the luminal content and the host. Thickening of the mucus epithelial barrier is believed to be important in the initiation and in the perpetuation of inflammatory bowel disease. The central region of the polymerizing mucin MUC2, the main mucin of the intestinal gel mucus, is interrupted by a CYS-rich peptide (CYS domain) found in two copies. This domain is highly conserved and found in multiple copies in mucins of many organisms. CYS domains are involved in reversible hydrophobic interactions between them. We hypothesized that the CYS domain plays a major function in the protection of the epithelium and may have a therapeutic role in intestinal inflammation. Methods A transgenic (Tg) mouse strain secreting into the intestinal lumen a string of 12 CYS domains tagged with the eGFP sequence has been created. Tg expression was studied by immunohistochemistry (IHC), Western blot and using a confocal fibered endomicroscopy (Cellvizio). Mice were challenge by DSS, with an adherentinvasive E. coli (AIEC) found frequently in patients with Crohn's disease and with the rodent pathogenic bacteria C. rodentium. Results Tg mice secreted the transgene in the intestinal lumen. The mucus blanket was modified in Tg mice as expected with morphological changes of mucus granules in goblet cells, an increase number of PAS-positive cells in the ileum (1.3 fold; p=0.03), no modification of Muc2 expression and an increase of the mucus thickness with an intense Muc2 staining by IHC in the mucus blanket. Moreover, biochemical analysis of the mucus revealed that Muc2 was more fucosylated in Tg mice. Microbiota analysis using bacteria culture and mass spectrometry analysis showed that Tg mice harbored a higher load of lactobacilli (2.5 log; p=0.016) than their wild-type (Wt) littermates with an alteration of the lactobacilli composition in the colon and in the ileum. Tg mice were more protected during a pseudo-chronic DSS protocol (2.5% DSS for 5 days followed by H2O for 7 days) with a better histological score for the Tg mice (p=0.015) and a higher mitotic index (p=0.019). There was a decrease of bacteria translocation in liver and spleen of Tg mice vs Wt mice when mice were challenged by gavage with the AIEC strain (p= 0.019 and p=0.05, respectively). We found a lower weight/length ratio of the colon of Tg mice (p=0.019) after a C. rodentium challenge indicating that the tissue is less inflamed at 10 days post-infection with a lower load of the pathogen in the stools of Tg mice (2 log; p=0.038). Conclusion The delivery of a string of 12 CYS domains naturally found in two copies in Muc2 reinforces the mucus barrier and limits or stops colonization and translocation of pathogens. Supported by the French IBD Association AFA, the French Foundation FRM and the US Broad foundation BMRP
Background: Vitamin D signalling through its nuclear hormone receptor VDR has emerged as a pluripotent regulator of biological functions far beyond its canonical roles in calcium metabolism. In the realm of gastrointestinal physiology, many genes integral to the intestinal barrier, such as mucins, tight junctions, and antimicrobial peptides contain vitamin D responsive elements, attesting that their expression may be directly regulated by vitamin D. In addition, vitamin D is an important factor in immune cell development and immunomodulation. Vitamin D and VDR are involved in the development of specific intestinal intraepithelial lymphocytes and their deficiency has been implicated in exacerbation of symptoms in inflammatory bowel disease (IBD). Objective: The aim of this study was to investigate effects of exposure to low or supplemental levels of vitamin D from early life to adulthood on vitamin D status and intestinal gene expression signature in both healthy (CD-1) and IBD (IL-10 KO) mouse models. Methods: CD-1 and 129/SvEv IL-10 KO were randomized to a diet containing 25 IU (low level) or 5,000 IU (supplemental level) of vitamin D3/kg of diet in utero until necropsy at 3 months when serum and colon samples were obtained from the male offspring (n=15-23/group). Colonic inflammation in the IL-10 KO mice was assessed histologically. Serum 25(OH) vitamin D levels and colonic gene expression profiles in the CD-1 and IL-10 KO mice were determined by chemiluminescence immunoassay and whole genome microarray, respectively. Results: There was no significant difference in the colonic inflammatory score among the low vitamin D and supplemented IL-10 KO mice. The serum 25(OH)D levels were significantly higher in supplemented CD-1 (120.1 nmol/L ±3.1 ) and IL-10 KO mice (93.8 nmol/L ±2.7) in comparison to their low vitamin D CD-1 (13.8 nmol/ L ±0.78 ) and IL-10 KO (23.0 nmol/L ±2.2) counterparts. A total of 104 genes, not including VDR, were differentially expressed between CD-1 mice on low and supplemented vitamin D diets. In particular, expression of genes encoding for extracellular matrix proteins and immunoglobulins were increased and those involved in detoxification processes were decreased in low vitamin D compared to supplemented CD-1 mice. The colonic gene expression profiles of IL-10 KO mice were not altered by their vitamin D status. Conclusions: Continuous dietary vitamin D supplementation from conception onwards determines higher serum 25(OH)D levels in both healthy and IL-10 KO mice in adulthood, suggesting that vitamin D uptake is not impaired in IBD. While vitamin D deficiency in healthy CD-1 mice resulted in an inflammation-prone gene expression profile, the IL-10 KO mice were not responsive to vitamin D; therefore, vitamin D may be preventative in healthy animals but not in this genetic model of IBD.
S-721
AGA Abstracts
AGA Abstracts
Mo2017
Direct Effects of Infliximab (IFX) on Intestinal Mucosa: Exploring Mechanisms of Mucosal Healing Franco Scaldaferri, Loris Riccardo Lopetuso, Valentina Petito, Viviana Gerardi, Vincenzo Arena, Egidio Stigliano, Alfredo Papa, Valerio Cufino, Arianna Amato, Giovanni Cammarota, Alessandro Sgambato, Antonio Gasbarrini
Extracorporeal Shock Wave Therapy Modulates Inflammation and Prevents Gastrointestinal Ileus After Tissue Trauma and Postoperative Ileus Vlado Antonic, Alexander Stojadinovic, Mina J. Izadjoo, Anthony J. Bauer Introduction: Over the past 20 years the effects of ESWT has been studied on cells and tissues demonstrating its pro-angiogenic, anti-inflammatory and proliferative effects. However, there is a paucity of data published on the systemic modulatory effects of shock wave. Aim: We sought to determine the systemic effects of ESWT in two injury models: peripheral tissue trauma (TBX) and postoperative ileus (POI). Methods: The safety and feasibility of abdominal ESWT (applied dorsally and ventrally; 600 impulses, 4Hz, EFD:0.03 mJ/mm2, DermaGold, Tissue Regenerative Technologies, Woodstock, GA) was evaluated in adult male mice. Animals were randomly designated to one of the following TBX (12.5% of body weight minced syngeneic muscle and bone dorsally implanted subcutaneously into recipient mice) experimental groups: 1) naïve control (NC), 2) abdominal ESWT 24h pre-conditioning, 3) TBX, 4) abdominal ESWT 24 hr preconditioning+TBX, and 5) TBX+ESWT 4h post implantation. The POI (surgical manipulation of small intestine=SM) groups: 1) naïve controls (NC), 2) abdominal ESWT 24h preconditioning, 3) SM, and 4) abdominal ESWT 24h preconditioning+SM. Twenty-four hours after TBX or SM, mice were given per os FITCdextran (FD70kD MW) and were sacrificed 75min later to generate gastrointestinal transit distribution histograms and calculated geometric centers (GC). Serum and wound fluid were analyzed to determine the levels of 8 pro-inflammatory cytokines (IFN-γ, IL-10, IL-12 p70, IL-1β, IL-6, keratinocyte cytokine (KC), TNF-α, and GM-CSF). Mann-Whitney U test of ranks was used for comparison of cytokine concentrations. P <0.05 was considered significant. Results: TBX: GI transit GC analysis showed a significant protective effect of ESWT preconditioning on the TBX-induced ileus: naïve controls (10.49±0.088); ESWT 24h preconditioning (10.79±0.026), TBX (3.83±0.076); TBX+ESWT preconditioning (6.54±0.195); TBX+ESWT 4h post implantation (4.41±0.040). Comparison of wound fluid cytokine levels did not differ significantly between groups (P>0.05). There was a significant increase in serum IL10 (p=0.002), IL-6 (p<0.001), KC (p<0.001), and TNF-α (p=0.047) in the group with ESWT 24h preconditioning. POI: Gastrointestinal transit GC analysis showed a significant protective effect of ESWT preconditioning on SM-induced POI: naïve controls (10.23±0.083), ESWT 24h preconditioning (10.13±0.074), POI only (3.71±0.144) and POI+ESWT preconditioning (8.10±0.436), respectively. Statistical analysis of the serum samples harvested at 24 hrs showed significant differences between groups in INF-γ (p=0.036). Conclusions: Defocused shock waves can be safely administered to the abdomen without causing intestinal damage. ESWT preconditioning has protective effects on motility in both TBX and POI models by modulating the inflammatory response.
BACKGROUND Mucosal healing is one of the biggest issue on the management of IBD. Although clinical use of these drugs in achieving mucosal healing is a reality, the mechanisms of it are still not clear. Aim of our study is to identify the effect of IFX on intestinal epithelial cells in order to induce mucosal healing, by exploring whether IFX is really acting at intestinal mucosal level and whether IFX acts on «wound healing» process of intestinal epithelial cells, using In Vitro and In Vivo approach. METHODS Human intestinal mucosal biopsies from active IBD patients were cultured for 48 h with or without IFX (50 ng/ml), and supernatants were assessed for cytokines content and histology performed. Effect of IFX was explored in C57BL/6 mice exposed for 7 days to 2,5 % of DSS. IFX was administered iv (5 mg/Kg) at day 5 or by daily enema (300 mg/200 ml) for 3 days starting at day 5. At day 9 mice were sacrificed and histology was taken. A scratch assay was performed on CT26 and Caco 2 cell line monolayer. Precice scratches were obtained by seading cells in micro-chamber formed by culture inserts. Cells were exposed for 24 h to IFX (50 ng/ml) and/or TNF-α (100 U/ml), or to supernatants of PBMC or fibroblast treated or not for 24 h with IFX and/ or TNFα. Seriated pictures were taken for 24 h following the stimulation and analized by computer software. RESULTS Human biopsies exposed to IFX showed a decrease in TNFa content, as well as of innate and adaptive immunity cytokines. Furthermore at histology IFX-treated biopsies showed a a decreased immune cells infiltration. In Vivo, IFX iv or given as enema ameliorated the severity of colitis in mice, by lowering the disease activity index (DAI) and the loss of body weight. At day 9 a reduction of intestinal inflammatory infiltrate was observed in treated mice. At scratch assay, TNF-a slightly decreased the healing process, while IFX alone or in combination did not. TNF-a exposed PBMC supernatants decreased epithelial healing; also TNF-a fibroblast supernatants slowed the healing process but adding IFX the healing process was restored. CONCLUSIONS Taken together our preliminary observations suggest that IFX acts locally at mucosa levels, as shown by the decrease in inflammatory infiltrate and cytokines contents in human biopsies and the effect on murine colitis when given intrarectally. The effect of IFX is only partially dependent on direct effect on epithelial cells. IFX ameliorates epithelial cell healing by blocking the detrimental capacity of TNF-a pre-treated fibroblast on intestinal epithelial cells. More data are necessary to unravel molecular pathways of these observations. Understanding mechanisms of action of IFX may clarify mechanisms of loss of responses to it as well as new targets for potential new therapies.
Mo2020 Mo2018 Mucin Cys Domain Strengthens the Mucus Barrier During Experimental Intestinal Inflammation Valérie Gouyer, Laurent Dubuquoy, Christel Neut, Pierre Desreumaux, Frédéric Gottrand, Jean-Luc Desseyn
Vitamin D Deficiency in Utero Through Adulthood Results in an Inflammation-Prone Colonic Gene Expression Profile in Healthy CD-1 Mice While IL-10 Knock-out Mice are Not Responsive Raha Jahani, Andrea J. Glenn, Kristina A. Fielding, Reinhold Vieth, Wendy E. Ward, Elena Comelli
Introduction The mucus layer prevents contact between bacteria of the luminal content and the host. Thickening of the mucus epithelial barrier is believed to be important in the initiation and in the perpetuation of inflammatory bowel disease. The central region of the polymerizing mucin MUC2, the main mucin of the intestinal gel mucus, is interrupted by a CYS-rich peptide (CYS domain) found in two copies. This domain is highly conserved and found in multiple copies in mucins of many organisms. CYS domains are involved in reversible hydrophobic interactions between them. We hypothesized that the CYS domain plays a major function in the protection of the epithelium and may have a therapeutic role in intestinal inflammation. Methods A transgenic (Tg) mouse strain secreting into the intestinal lumen a string of 12 CYS domains tagged with the eGFP sequence has been created. Tg expression was studied by immunohistochemistry (IHC), Western blot and using a confocal fibered endomicroscopy (Cellvizio). Mice were challenge by DSS, with an adherentinvasive E. coli (AIEC) found frequently in patients with Crohn's disease and with the rodent pathogenic bacteria C. rodentium. Results Tg mice secreted the transgene in the intestinal lumen. The mucus blanket was modified in Tg mice as expected with morphological changes of mucus granules in goblet cells, an increase number of PAS-positive cells in the ileum (1.3 fold; p=0.03), no modification of Muc2 expression and an increase of the mucus thickness with an intense Muc2 staining by IHC in the mucus blanket. Moreover, biochemical analysis of the mucus revealed that Muc2 was more fucosylated in Tg mice. Microbiota analysis using bacteria culture and mass spectrometry analysis showed that Tg mice harbored a higher load of lactobacilli (2.5 log; p=0.016) than their wild-type (Wt) littermates with an alteration of the lactobacilli composition in the colon and in the ileum. Tg mice were more protected during a pseudo-chronic DSS protocol (2.5% DSS for 5 days followed by H2O for 7 days) with a better histological score for the Tg mice (p=0.015) and a higher mitotic index (p=0.019). There was a decrease of bacteria translocation in liver and spleen of Tg mice vs Wt mice when mice were challenged by gavage with the AIEC strain (p= 0.019 and p=0.05, respectively). We found a lower weight/length ratio of the colon of Tg mice (p=0.019) after a C. rodentium challenge indicating that the tissue is less inflamed at 10 days post-infection with a lower load of the pathogen in the stools of Tg mice (2 log; p=0.038). Conclusion The delivery of a string of 12 CYS domains naturally found in two copies in Muc2 reinforces the mucus barrier and limits or stops colonization and translocation of pathogens. Supported by the French IBD Association AFA, the French Foundation FRM and the US Broad foundation BMRP
Background: Vitamin D signalling through its nuclear hormone receptor VDR has emerged as a pluripotent regulator of biological functions far beyond its canonical roles in calcium metabolism. In the realm of gastrointestinal physiology, many genes integral to the intestinal barrier, such as mucins, tight junctions, and antimicrobial peptides contain vitamin D responsive elements, attesting that their expression may be directly regulated by vitamin D. In addition, vitamin D is an important factor in immune cell development and immunomodulation. Vitamin D and VDR are involved in the development of specific intestinal intraepithelial lymphocytes and their deficiency has been implicated in exacerbation of symptoms in inflammatory bowel disease (IBD). Objective: The aim of this study was to investigate effects of exposure to low or supplemental levels of vitamin D from early life to adulthood on vitamin D status and intestinal gene expression signature in both healthy (CD-1) and IBD (IL-10 KO) mouse models. Methods: CD-1 and 129/SvEv IL-10 KO were randomized to a diet containing 25 IU (low level) or 5,000 IU (supplemental level) of vitamin D3/kg of diet in utero until necropsy at 3 months when serum and colon samples were obtained from the male offspring (n=15-23/group). Colonic inflammation in the IL-10 KO mice was assessed histologically. Serum 25(OH) vitamin D levels and colonic gene expression profiles in the CD-1 and IL-10 KO mice were determined by chemiluminescence immunoassay and whole genome microarray, respectively. Results: There was no significant difference in the colonic inflammatory score among the low vitamin D and supplemented IL-10 KO mice. The serum 25(OH)D levels were significantly higher in supplemented CD-1 (120.1 nmol/L ±3.1 ) and IL-10 KO mice (93.8 nmol/L ±2.7) in comparison to their low vitamin D CD-1 (13.8 nmol/ L ±0.78 ) and IL-10 KO (23.0 nmol/L ±2.2) counterparts. A total of 104 genes, not including VDR, were differentially expressed between CD-1 mice on low and supplemented vitamin D diets. In particular, expression of genes encoding for extracellular matrix proteins and immunoglobulins were increased and those involved in detoxification processes were decreased in low vitamin D compared to supplemented CD-1 mice. The colonic gene expression profiles of IL-10 KO mice were not altered by their vitamin D status. Conclusions: Continuous dietary vitamin D supplementation from conception onwards determines higher serum 25(OH)D levels in both healthy and IL-10 KO mice in adulthood, suggesting that vitamin D uptake is not impaired in IBD. While vitamin D deficiency in healthy CD-1 mice resulted in an inflammation-prone gene expression profile, the IL-10 KO mice were not responsive to vitamin D; therefore, vitamin D may be preventative in healthy animals but not in this genetic model of IBD.
S-721
AGA Abstracts
AGA Abstracts
Mo2017