- Email: [email protected]
CX601, Allogeneic Expanded Adipose-Derived Mesenchymal Stem Cells (EASC), for Complex Perianal Fistulas in Crohn's Disease: Long-Term Results from a Phase III Randomized Controlled Trial
987
CX601, ALLOGENEIC EXPANDED ADIPOSE-DERIVED MESENCHYMAL STEM CELLS (EASC), FOR COMPLEX PERIANAL FISTULAS IN CROHN'S DISEASE: LONG-TERM RESULTS FROM A PHASE III RANDOMIZED CONTROLLED TRIAL Julian Panes, Damian Garcia-Olmo, Gert A. Van Assche, Jean Frederic Colombel, Walter Reinisch, Daniel C. Baumgart, Maria Nachury, Marc Ferrante, Lili Kazemi-Shirazi, Jeancharles Grimaud, Fernando de la Portilla, Eran Goldin, Marie Paule Richard, Ignacio Tagarro, Silvio Danese
EGFR-ACTIVATED NOTCH SIGNALING CONTRIBUTES TO PATHOGENESIS OF MÉNÉTRIER'S DISEASE Won Jae Huh, Robert J. Coffey Background and Aims: Ménétrier's disease is a rare acquired protein-losing hypertrophic gastropathy characterized by giant gastric rugal folds, decreased acid secretion, increased gastric mucus production, and hypoalbuminemia due to protein loss in the gastric mucosa. Microscopically, it is characterized by massive foveolar hyperplasia, oxyntic gland atrophy, and repatterning of cell specification. We previously reported transgenic mice overexpressing the EGF receptor (EGFR) ligand, TGF-α, in the stomach phenocopy Ménétrier's disease, that TGF-α is overexpressed in the stomach of Ménétrier's disease patients, and that the EGFR neutralizing monoclonal antibody (mAb), cetuximab, is the first effective medical therapy for Ménétrier's disease. In a proteomic analysis of gastric tissue of Ménétrier's disease patients before and after cetuximab treatment, we observed decreased levels of the Notch ligand, Jagged1, after cetuximab treatment. The aim of this study was to examine whether Notch signaling is a downstream target of EGFR signaling and whether it contributes to the pathogenesis of Ménétrier's disease. Methods: We tested whether Notch signaling is activated in the stomach of TGF-α transgenic mice and whether Notch activation is reduced by MM151, a cocktail of EGFR mAbs that blocks mouse EGFR, and/ or dibenzazepine (DBZ), a γ-secretase inhibitor, as determined by Hes-1 immunofluorescent staining. We also examined the level of foveolar hyperplasia and proliferation, and the number of parietal and chief cells in the gastric mucosa of these mice using markers: UEA1 (foveolar pit cell), Ki-67 (proliferation), H+/K+ ATPase (parietal cell), and gastric intrinsic factor (chief cell). Results: Nuclear Hes-1 expression was upregulated in the pit, isthmus, and neck compartments in the stomach of TGF-α transgenic mice compared to wild-type mice. Number of nuclear Hes-1 positive cells were decreased by MM-151 and/ or DBZ treatment in TGF-α transgenic mice. Also, MM-151 and/ or DBZ treatment led to decreased foveolar hyperplasia and proliferation, and increased numbers of parietal and chief cells in the gastric mucosa of TGF-α transgenic mice. Conclusions: These results show that Notch signaling is activated in TGF-α transgenic mice and activated Notch signaling contributes, at least in part, to the pathogenesis of Ménétrier's disease. Moreover, these findings suggest that blockade of Notch signaling may be a novel therapeutic strategy for Ménétrier's disease.
Background Complex perianal fistulas are a common complication of Crohn's disease (CD), which are challenging to treat, as they are associated with a high rate of treatment failure. The objectives of this study were to assess the long-term safety and efficacy of Cx601, added on to current therapies, in CD patients with treatment-refractory complex perianal fistulas. Methods This was a phase III, double-blind, parallel group multicenter study in which patients with inactive or mildly-active luminal CD (CDAI≤220) and complex active perianal fistulas were randomized (1:1) to Cx601 (single injection of 120 million eASC to all tracts + standard of care) or control (placebo + standard of care). Patients underwent fistula curettage and seton placement (if indicated) at least 2 weeks before study treatment, and could continue current treatment (immunosuppressants and/or anti-TNFs) without dose or regimen modification; antibiotics were allowed for up to four weeks. Pre-specified efficacy endpoints evaluated at week 52 in the mITT population (randomized, treated and ≥1 postbaseline efficacy assessment) were combined remission (CR, closure of all treated external openings that were draining at baseline assessed clinically, and absence of collections >2 cm of the treated perianal fistulas confirmed by blinded central MRI reading) and clinical remission (closure of all treated external openings that were draining at baseline assessed clinically). Sustained CR at week 52 after CR at week 24 was also evaluated. Results 107 patients were randomized to Cx601 and 105 to control; 70 patients in the Cx601 and 61 in the control group completed the 52-week follow-up. As shown in the Table, the beneficial effect of Cx601 on CR and clinical remission at week 24 was sustained at week 52. A significantly greater proportion of Cx601- vs control-treated patients achieved CR and clinical remission at week 52. Among patients with CR at week 24, a greater proportion of those treated with Cx601 vs control had no relapse at week 52 (75.0% vs 55.9%). The frequency and type of treatment-related adverse events were similar in the two groups (Cx601: 20.4%; control: 26.5%), the most common of which were anal abscess and proctalgia. Conclusions A single administration of Cx601 was safe and effective for the treatment of complex perianal fistulas in CD patients, with efficacy and tolerability sustained for up to one year.
988 ZONULIN TRANSGENIC MOUSE MODEL SHOWS ALTERED SMALL INTESTINAL PERMEABILITY AND INCREASED MORBIDITY AND MORTALITY IN THE DSS MODEL OF COLITIS Craig Sturgeon, Jinggang Lan, Alessio Fasano Background: Increased intestinal permeability has been proposed to be an integral element, along with genetic makeup and environmental triggers in the pathogenies of chronic inflammatory diseases (CID). We identified zonulin to be a master regular of intercellular tight junctions. It has been linked by our group and others to the development of several CIDs. We identified human zonulin as pre-haptoglobin(HP)-2. HP has two alleles, HP1 and HP2, with HP2 arising from a duplication event which only occurs in humans. Aims: To study the role of zonulin (alias preHP2)-mediated small intestinal permeability in the pathogenesis and development of CIDs. Methods: HP2-2 zonulin transgenic mice, the transgenic zonulin HP2 mouse model created through genetic engineering to be homologous to human HP2, was subjected to dextran-sodium-sulfate (DSS) treatment and compared to C57Bl/6 (WT) HP1-1 mice. Mice were treated with DSS for 7 days and followed for 4-7 days recovery. Intestinal permeability was measured both in vivo, by serum FITC-dextran, and ex vivo by mounting small intestinal tissue in microsnapwells and measuring cascade-blue-dextran passage. To mechanistically link the clinically outcome of DSS treatment in zonulin transgenic HP2 mice, animals were treated with the zonulin inhibitor AT1001 in addition to DSS. Results: We observed a mortality rate of 70% at 14 days post DSS treatment in HP2 mice compared to 0% in WT(p=0.0002). Additionally, DSS-treated HP2 animals showed increased loss of body weight (p<0.0001. In vivo HP2 mice had increased small intestinal permeability at baseline compared to WT (p<0.01) which was exacerbated by DSS treatment (p<0.05). Ex vivo measurement of intestinal permeability showed similar changes with HP2 mice showing increased permeability compared to WT after DSS in both the duodenum (p<0.01) and jejunum (p<0.05). These changes in permeability were accompanied by upregulation of HP2 gene expression by 4-fold in the duodenum (p<0.01) and 8-fold in the jejunum (p<0.05) compared to untreated HP2 mice. Treatment with AT1001 prevented the DSSinduced increased small intestinal permeability both in vivo (p<0.01) and ex vivo in both the duodenum (p<0.01), jejunum (p<0.05), and colon (p<0.05). In addition, the AT1001 completely reverted mortality due to the recovery of weight loss after DSS withdrawn. Conclusions: Combined, these results suggest that compared to C57Bl/6 WT mice, zonulin transgenic HP2 mice treated with DSS showed a more pronounced functional impairment of small intestinal permeability, a more severe decrease in body weight, and ~70% mortality rate (compared to 0% of WT mice). These data show that increased small intestinal permeability can be linked to inflammation in the colon and zonulin dependent gut barrier impairment is an early step leading to the break of tolerance with subsequent development of CIDs.
*97.5% CI; **95.0% CI
986 DEFICITS IN ENTEROCYTE APICAL TRANSPORTERS ASSOCIATED WITH LOSS OF MYOSIN VB Amy C. Engevik, Victoria G. Weis, Byron C. Knowles, Cameron Schlegel, Nadia A. Ameen, Hermann Koepsell, Nicholas C. Zachos, Mark Donowitz, James R. Goldenring Background: Microvillus Inclusion Disease (MVID) is a rare form of congenital diarrhea resulting from inactivating mutations in Myosin Vb (MYO5B). Treatment options for patients with MVID are extremely limited and carry a high risk of complications and mortality. Thus, despite the rarity of MVID new treatment strategies are necessary to improve patient outcome. Currently, the mechanism driving MVID diarrhea is unknown. We hypothesize that loss of MYO5B results in aberrant expression of key apical enterocyte membrane transporters that promote the absorption of nutrients and water. Methods: Our lab has generated a mouse model of MVID with a germline deletion of MYO5B (MYO5B KO) to elucidate the mechanism by which loss of MYO5B results in untreatable diarrhea. Duodenum tissue was collected from neonatal MYO5B KO mice and heterozygous or wildtype littermates (control). Immunofluorescence staining and quantitative real time PCR (qPCR) were performed to determine the localization of apical transporters and the relative gene expression of transporters respectively in intestinal tissue of MYO5B KO and control mice. Results: Phalloidin conjugates identified apical F-actin in the intestinal brush border of both MYO5B KO and control mice. Similar to MVID patients, numerous F-actin positive inclusions were observed in MYO5B KO enterocytes. To assess the localization of key transporters known to regulate water absorption, we performed immunofluorescence for the sodium hydrogen exchanger isoform 3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), sodium glucose co-transporter (SGLT1) and downregulated in adenoma (DRA). Additionally we assessed NHE regulatory factor 1 (NHERF1), a component known to anchor ion transporters in the plasma membrane. Control mice showed apical localization of NHE3, CFTR, SGLT1, DRA and NHERF1. In contrast MYO5B KO mice exhibited NHE3 and CFTR in F-actin positive inclusions, with some basolateral expression of NHE3. Likewise, SGLT1 was present in intracellular inclusions and had decreased apical expression in MYO5B KO mice. MYO5B KO mice also had decreased apical expression of DRA. Alkaline phosphatase 1, an anchored ectoenzyme highly expressed in the brush border membrane of duodenal epithelial cells, was present in control mice, but absent in MYO5B KO mice. qPCR showed decreased expression of NHE3, DRA and GLUT2 and increased expression of SGLT1 in the intestinal tissue of MYO5B KO mice compared to control mice. Enteroids generated from MYO5B KO mice recapitulated in vivo findings with the presence of F-actin positive inclusions and subapical expression of SGLT1 and CFTR. Conclusions: Our data suggest that loss of MYO5B results in altered expression of transporters known to regulate water and nutrient absorption. These data implicate intestinal ion transporter deficits as a likely explanation for the pathogenesis of MVID.
S-187
AGA Abstracts
AGA Abstracts
985
CX601, ALLOGENEIC EXPANDED ADIPOSE-DERIVED MESENCHYMAL STEM CELLS (EASC), FOR COMPLEX PERIANAL FISTULAS IN CROHN'S DISEASE: LONG-TERM RESULTS FROM A PHASE III RANDOMIZED CONTROLLED TRIAL Julian Panes, Damian Garcia-Olmo, Gert A. Van Assche, Jean Frederic Colombel, Walter Reinisch, Daniel C. Baumgart, Maria Nachury, Marc Ferrante, Lili Kazemi-Shirazi, Jeancharles Grimaud, Fernando de la Portilla, Eran Goldin, Marie Paule Richard, Ignacio Tagarro, Silvio Danese
EGFR-ACTIVATED NOTCH SIGNALING CONTRIBUTES TO PATHOGENESIS OF MÉNÉTRIER'S DISEASE Won Jae Huh, Robert J. Coffey Background and Aims: Ménétrier's disease is a rare acquired protein-losing hypertrophic gastropathy characterized by giant gastric rugal folds, decreased acid secretion, increased gastric mucus production, and hypoalbuminemia due to protein loss in the gastric mucosa. Microscopically, it is characterized by massive foveolar hyperplasia, oxyntic gland atrophy, and repatterning of cell specification. We previously reported transgenic mice overexpressing the EGF receptor (EGFR) ligand, TGF-α, in the stomach phenocopy Ménétrier's disease, that TGF-α is overexpressed in the stomach of Ménétrier's disease patients, and that the EGFR neutralizing monoclonal antibody (mAb), cetuximab, is the first effective medical therapy for Ménétrier's disease. In a proteomic analysis of gastric tissue of Ménétrier's disease patients before and after cetuximab treatment, we observed decreased levels of the Notch ligand, Jagged1, after cetuximab treatment. The aim of this study was to examine whether Notch signaling is a downstream target of EGFR signaling and whether it contributes to the pathogenesis of Ménétrier's disease. Methods: We tested whether Notch signaling is activated in the stomach of TGF-α transgenic mice and whether Notch activation is reduced by MM151, a cocktail of EGFR mAbs that blocks mouse EGFR, and/ or dibenzazepine (DBZ), a γ-secretase inhibitor, as determined by Hes-1 immunofluorescent staining. We also examined the level of foveolar hyperplasia and proliferation, and the number of parietal and chief cells in the gastric mucosa of these mice using markers: UEA1 (foveolar pit cell), Ki-67 (proliferation), H+/K+ ATPase (parietal cell), and gastric intrinsic factor (chief cell). Results: Nuclear Hes-1 expression was upregulated in the pit, isthmus, and neck compartments in the stomach of TGF-α transgenic mice compared to wild-type mice. Number of nuclear Hes-1 positive cells were decreased by MM-151 and/ or DBZ treatment in TGF-α transgenic mice. Also, MM-151 and/ or DBZ treatment led to decreased foveolar hyperplasia and proliferation, and increased numbers of parietal and chief cells in the gastric mucosa of TGF-α transgenic mice. Conclusions: These results show that Notch signaling is activated in TGF-α transgenic mice and activated Notch signaling contributes, at least in part, to the pathogenesis of Ménétrier's disease. Moreover, these findings suggest that blockade of Notch signaling may be a novel therapeutic strategy for Ménétrier's disease.
Background Complex perianal fistulas are a common complication of Crohn's disease (CD), which are challenging to treat, as they are associated with a high rate of treatment failure. The objectives of this study were to assess the long-term safety and efficacy of Cx601, added on to current therapies, in CD patients with treatment-refractory complex perianal fistulas. Methods This was a phase III, double-blind, parallel group multicenter study in which patients with inactive or mildly-active luminal CD (CDAI≤220) and complex active perianal fistulas were randomized (1:1) to Cx601 (single injection of 120 million eASC to all tracts + standard of care) or control (placebo + standard of care). Patients underwent fistula curettage and seton placement (if indicated) at least 2 weeks before study treatment, and could continue current treatment (immunosuppressants and/or anti-TNFs) without dose or regimen modification; antibiotics were allowed for up to four weeks. Pre-specified efficacy endpoints evaluated at week 52 in the mITT population (randomized, treated and ≥1 postbaseline efficacy assessment) were combined remission (CR, closure of all treated external openings that were draining at baseline assessed clinically, and absence of collections >2 cm of the treated perianal fistulas confirmed by blinded central MRI reading) and clinical remission (closure of all treated external openings that were draining at baseline assessed clinically). Sustained CR at week 52 after CR at week 24 was also evaluated. Results 107 patients were randomized to Cx601 and 105 to control; 70 patients in the Cx601 and 61 in the control group completed the 52-week follow-up. As shown in the Table, the beneficial effect of Cx601 on CR and clinical remission at week 24 was sustained at week 52. A significantly greater proportion of Cx601- vs control-treated patients achieved CR and clinical remission at week 52. Among patients with CR at week 24, a greater proportion of those treated with Cx601 vs control had no relapse at week 52 (75.0% vs 55.9%). The frequency and type of treatment-related adverse events were similar in the two groups (Cx601: 20.4%; control: 26.5%), the most common of which were anal abscess and proctalgia. Conclusions A single administration of Cx601 was safe and effective for the treatment of complex perianal fistulas in CD patients, with efficacy and tolerability sustained for up to one year.
988 ZONULIN TRANSGENIC MOUSE MODEL SHOWS ALTERED SMALL INTESTINAL PERMEABILITY AND INCREASED MORBIDITY AND MORTALITY IN THE DSS MODEL OF COLITIS Craig Sturgeon, Jinggang Lan, Alessio Fasano Background: Increased intestinal permeability has been proposed to be an integral element, along with genetic makeup and environmental triggers in the pathogenies of chronic inflammatory diseases (CID). We identified zonulin to be a master regular of intercellular tight junctions. It has been linked by our group and others to the development of several CIDs. We identified human zonulin as pre-haptoglobin(HP)-2. HP has two alleles, HP1 and HP2, with HP2 arising from a duplication event which only occurs in humans. Aims: To study the role of zonulin (alias preHP2)-mediated small intestinal permeability in the pathogenesis and development of CIDs. Methods: HP2-2 zonulin transgenic mice, the transgenic zonulin HP2 mouse model created through genetic engineering to be homologous to human HP2, was subjected to dextran-sodium-sulfate (DSS) treatment and compared to C57Bl/6 (WT) HP1-1 mice. Mice were treated with DSS for 7 days and followed for 4-7 days recovery. Intestinal permeability was measured both in vivo, by serum FITC-dextran, and ex vivo by mounting small intestinal tissue in microsnapwells and measuring cascade-blue-dextran passage. To mechanistically link the clinically outcome of DSS treatment in zonulin transgenic HP2 mice, animals were treated with the zonulin inhibitor AT1001 in addition to DSS. Results: We observed a mortality rate of 70% at 14 days post DSS treatment in HP2 mice compared to 0% in WT(p=0.0002). Additionally, DSS-treated HP2 animals showed increased loss of body weight (p<0.0001. In vivo HP2 mice had increased small intestinal permeability at baseline compared to WT (p<0.01) which was exacerbated by DSS treatment (p<0.05). Ex vivo measurement of intestinal permeability showed similar changes with HP2 mice showing increased permeability compared to WT after DSS in both the duodenum (p<0.01) and jejunum (p<0.05). These changes in permeability were accompanied by upregulation of HP2 gene expression by 4-fold in the duodenum (p<0.01) and 8-fold in the jejunum (p<0.05) compared to untreated HP2 mice. Treatment with AT1001 prevented the DSSinduced increased small intestinal permeability both in vivo (p<0.01) and ex vivo in both the duodenum (p<0.01), jejunum (p<0.05), and colon (p<0.05). In addition, the AT1001 completely reverted mortality due to the recovery of weight loss after DSS withdrawn. Conclusions: Combined, these results suggest that compared to C57Bl/6 WT mice, zonulin transgenic HP2 mice treated with DSS showed a more pronounced functional impairment of small intestinal permeability, a more severe decrease in body weight, and ~70% mortality rate (compared to 0% of WT mice). These data show that increased small intestinal permeability can be linked to inflammation in the colon and zonulin dependent gut barrier impairment is an early step leading to the break of tolerance with subsequent development of CIDs.
*97.5% CI; **95.0% CI
986 DEFICITS IN ENTEROCYTE APICAL TRANSPORTERS ASSOCIATED WITH LOSS OF MYOSIN VB Amy C. Engevik, Victoria G. Weis, Byron C. Knowles, Cameron Schlegel, Nadia A. Ameen, Hermann Koepsell, Nicholas C. Zachos, Mark Donowitz, James R. Goldenring Background: Microvillus Inclusion Disease (MVID) is a rare form of congenital diarrhea resulting from inactivating mutations in Myosin Vb (MYO5B). Treatment options for patients with MVID are extremely limited and carry a high risk of complications and mortality. Thus, despite the rarity of MVID new treatment strategies are necessary to improve patient outcome. Currently, the mechanism driving MVID diarrhea is unknown. We hypothesize that loss of MYO5B results in aberrant expression of key apical enterocyte membrane transporters that promote the absorption of nutrients and water. Methods: Our lab has generated a mouse model of MVID with a germline deletion of MYO5B (MYO5B KO) to elucidate the mechanism by which loss of MYO5B results in untreatable diarrhea. Duodenum tissue was collected from neonatal MYO5B KO mice and heterozygous or wildtype littermates (control). Immunofluorescence staining and quantitative real time PCR (qPCR) were performed to determine the localization of apical transporters and the relative gene expression of transporters respectively in intestinal tissue of MYO5B KO and control mice. Results: Phalloidin conjugates identified apical F-actin in the intestinal brush border of both MYO5B KO and control mice. Similar to MVID patients, numerous F-actin positive inclusions were observed in MYO5B KO enterocytes. To assess the localization of key transporters known to regulate water absorption, we performed immunofluorescence for the sodium hydrogen exchanger isoform 3 (NHE3), cystic fibrosis transmembrane regulator (CFTR), sodium glucose co-transporter (SGLT1) and downregulated in adenoma (DRA). Additionally we assessed NHE regulatory factor 1 (NHERF1), a component known to anchor ion transporters in the plasma membrane. Control mice showed apical localization of NHE3, CFTR, SGLT1, DRA and NHERF1. In contrast MYO5B KO mice exhibited NHE3 and CFTR in F-actin positive inclusions, with some basolateral expression of NHE3. Likewise, SGLT1 was present in intracellular inclusions and had decreased apical expression in MYO5B KO mice. MYO5B KO mice also had decreased apical expression of DRA. Alkaline phosphatase 1, an anchored ectoenzyme highly expressed in the brush border membrane of duodenal epithelial cells, was present in control mice, but absent in MYO5B KO mice. qPCR showed decreased expression of NHE3, DRA and GLUT2 and increased expression of SGLT1 in the intestinal tissue of MYO5B KO mice compared to control mice. Enteroids generated from MYO5B KO mice recapitulated in vivo findings with the presence of F-actin positive inclusions and subapical expression of SGLT1 and CFTR. Conclusions: Our data suggest that loss of MYO5B results in altered expression of transporters known to regulate water and nutrient absorption. These data implicate intestinal ion transporter deficits as a likely explanation for the pathogenesis of MVID.
S-187
AGA Abstracts
AGA Abstracts
985