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Tu1613 Evaluation of Mucin and Neuroendocrine Expression in Diffuse Gastric Cancer With Signet Ring Cell Morphology
AGA Abstracts
Tu1613
Tu1614
Evaluation of Mucin and Neuroendocrine Expression in Diffuse Gastric Cancer With Signet Ring Cell Morphology Øystein Sørdal, Gunnar Qvigstad, Ivar S. Nordrum, Helge Waldum
Ghrelin Receptor Modulates CD4 T Cell Function During Intestinal Inflammation Martina Di Giovangiulio, Pedro J. Gomez-Pinilla, Giovanna Farro, Inge Depoortere, Guy E. Boeckxstaens, Gianluca Matteoli
Background and Aims: A proportion of diffuse gastric carcinoma according to the Lauren classification system has signet ring cell morphology. Signet ring cells are characterized by a peripherally placed nucleus and a central amorphous substance, which is reported to be mucin based on Periodic Acid Schiff positivity (PAS). PAS is not a specific staining method for mucin, and it stains several substances including various glycoproteins. Neuroendocrine expression has previously been reported in tumour cells in diffuse type gastric cancer. Methods: PAS, PAS-Diastase, Alcian blue and cytokeratin stainings were undertaken. Relevant gastrointestinal mucins (MUC1, 2, 3, 4, 5AC, 6 and MUC13) were assessed by immunohistochemistry and in situ hybridization (ISH) by the use of a new and improved ISH method. The neuroendocrine properties were evaluated by mRNA and protein expression of the general neuroendocrine markers chromogranin A, synaptophysin as well as Regenerating islet-derived family member 4 (Reg-IV). Results: All cases had cytokeratin positive tumor cells that also were PAS and PAS-Diastase and Alcian blue positive. No mucin mRNA or protein expression was observed in signet ring tumor cells including the amorphous substance in any of the nine cases. Focal or diffuse expression of mucin by immunohistochemistry was observed in non-signet ring tumors cells in five out of nine cases. All cases showed immunoreactivity to synaptophysin, and seven out of nine cases immunoreactivity to chromogranin A in signet ring and non-signet ring tumor cells. Chromogranin A and Reg-4 mRNA expression was observed in tumor cells in all samples with retained mRNA. Conclusions: The lack of MUC protein and mRNA in signet ring tumor cells suggests the amorphous substance is not mucin. The in situ hybridization method utilized in this study has several specificity steps and clear advantages over conventional methods. The discrepancy between mucin mRNA and protein expression suggest that the focal or diffuse positivity in non-signet ring tumor cells by immunohistochemistry could be non-specific. The abundant expression of the general neuroendocrine markers CgA and synaptophysin, and CgA as well as Reg-4 mRNA in tumour cells gives support to a neuroendocrine origin. In conclusion, these findings question exocrine expression in these tumours and do not lend support to the view that these tumours are mixed adenoneuroendocrine carcinomas (MANEC).
Introduction: Recent work suggests that the orexigenic hormone ghrelin have potent antiinflammatory properties in preclinical model of intestinal inflammation. Ghrelin is secreted predominantly from enteroendocrine cells of the stomach and acts as an appetite-regulating hormone increasing food intake and long-term regulation of body weight. The effects of ghrelin are mediated via the G protein-coupled receptor (GPCR) named as growth hormone secretagogue receptor (GHSR). Interestingly, the expression of this receptor has been shown on innate and adaptive immune cells. Taken together, the above suggests that ghrelin may have immunomodulatory properties. However, the mechanism, by which ghrelin suppresses intestinal inflammation, is still unclear. Therefore, we decided to investigate the effect of ghrelin on T cell function during colitis. Methods: T cell transfer chronic colitis was induced in the recombination activating gene 1 knockout mice (Rag1-/-) by adoptive transfer of Naïve CD4 T helper cells (CD4-Ths) from genetically-compatible wild-type (WT) or growth hormone secretagogue receptor knockout mice (GHSR-/-). Development of colitis was evaluated every week by assessing body weight loss, diarrhea score and blood in the feces. Eight weeks after T cell transfer, the degree of intestinal inflammation was assessed by analyzing spleen weight, colon length, histology and cytokine expression in the colon. Results: The lack of the ghrelin receptor on CD4-transferred T cells significantly worsened the course of colitis in Rag1-/- mice. Indeed, Rag1-/- mice reconstituted with GHSR -/- CD4 Ths (GHSR→Rag1) showed colitis symptoms (body weight loss, alteration in the feces) significantly earlier than Rag1-/- mice reconstituted with WT CD4 Ths (WT →Rag1). In line, GHSR→Rag1 mice displayed increased body weight loss (WT →Rag1, 14.5±4.0% vs GHSR→Rag1, 25.5±3.0%) and higher diarrhea score compared to WT →Rag1 (WT→Rag1, 2.1±0.2 vs GHSR→Rag1, 3.5±0.5). Eight weeks after T cells transfer, GHSR →Rag1 mice had significantly increased spleen weight (WT →Rag1, 70.5±21.2mg vs GHSR →Rag1, 147.5±42.1mg) and shortening of the colon (WT →Rag1, 8.9±0.4cm vs GHSR →Rag1, 7.9±0.5) Histological analysis also showed a higher lesion score in GHSR →Rag1 mice compared to WT→Rag1 mice (WT→Rag1, 7.9±4.0 vs GHSR→Rag1, 10.3±4.8). In addition, gene expression of colonic specimen revealed higher levels of IL6 (4.3±1.3 fold), TNF α (4.6±1.6 fold), IFNγ (5.6±2.3 fold) and IL17a (2.8±0.8 fold) in GHSR →Rag1 mice versus WT→Rag1 mice. Conclusions: Our observations strongly suggest that ghrelin may significantly ameliorate experimental chronic colitis by modulating T helper effector cell function in the gut. A better understanding of the underlying regulatory mechanisms of ghrelin will be crucial to further explore the potential therapeutic properties of ghrelin in inflammatory bowel disease. Tu1615 Modeling the Distinct Roles for IL-23 and IL-17 in Inflammatory Bowel Disease Using Mdr1a-/- Mice Joe Maxwell, William Brown, Yu Zhang, James B. Rottman, Alison Budelsky, Jennifer Towne Th17 cells and their signature cytokine IL-17A have been implicated in a number of autoimmune diseases including inflammatory bowel disease (IBD). IL-23 is a closely related cytokine to IL-12 in that it shares both a ligand and a receptor subunit. IL-23, IL-17A and IL-17F expression are elevated in IBD and SNPs in the IL-23R show strong association with Crohn's disease and ulcerative colitis. To examine the role of these cytokines in the gut, we compared inhibition of IL-23p19 and IL-17RA in the Helicobacter bilis-infected mdr1a-/- mouse model of colitis. Inhibition of IL-23 alone provided strong efficacy comparable to that seen with an IL-12/23p40 dual inhibitor. IL-17RA antagonism did not protect mice from disease, and led to acute disease exacerbation. To better understand which IL-17 family cytokines drive inflammation through IL-17RA in the gut, we compared inhibition of IL-17RA with inhibition of IL-17A, IL-17F or IL-25. IL-17A inhibition resulted in disease exacerbation similar to that seen with inhibition of IL-17RA, while inhibition of the other IL-17 family cytokines had no effect. Subsequent studies found that IL-17RA inhibition did not exacerbate disease in uninfected mdr1a-/- mice. The dependence on the presence of H. bilis for the exacerbation suggests either a role for IL-17A in maintenance of barrier integrity or in control of pathogens in the gut. To this point, we observed an increase in circulating markers indicative of barrier breach and bacterial dissemination. In order to further investigate the mechanisms underlying disease exacerbation in anti-IL-17RA treated H. bilis-infected mdr1a-/- mice, we examined gene expression changes in the gut. IL-23 inhibition led to decreased expression of a number of cytokines and chemokines in the gut while IL-17RA or IL-17A inhibition led to increased expression of many genes including IFN γ, TNFα, IL-6 and a number of chemokines. To determine whether an increase in IFN γ was key to the anti-IL-17RA mediated exacerbation, we treated mice with anti-IFNγ along with anti-IL-17RA. Inhibition of IFN γ alone was highly efficacious; however, IFNγ blockade did not affect disease in the presence of anti-IL-17RA. These data suggest that pathways other than IFN γ may be driving anti-IL-17RA mediated disease exacerbation in this mouse model of colitis and the exacerbation is not likely due to a shift from Th17 cells to Th1 cells. The data in this H. bilis-infected mdr1a-/- mouse colitis model are similar to what is emerging from Crohn's disease clinical trials: IL-12/ 23p40 inhibitors are showing promise while inhibition of IL-17A with secukinumab led to exacerbation in a subset of patients. Together the emerging clinical data suggest that the role of Th17 cells and IL-17A in colitis is different from other inflammatory diseases and that IL-23 and IL-17 biology are distinct in the gut.
AGA Abstracts
S-806
Tu1613
Tu1614
Evaluation of Mucin and Neuroendocrine Expression in Diffuse Gastric Cancer With Signet Ring Cell Morphology Øystein Sørdal, Gunnar Qvigstad, Ivar S. Nordrum, Helge Waldum
Ghrelin Receptor Modulates CD4 T Cell Function During Intestinal Inflammation Martina Di Giovangiulio, Pedro J. Gomez-Pinilla, Giovanna Farro, Inge Depoortere, Guy E. Boeckxstaens, Gianluca Matteoli
Background and Aims: A proportion of diffuse gastric carcinoma according to the Lauren classification system has signet ring cell morphology. Signet ring cells are characterized by a peripherally placed nucleus and a central amorphous substance, which is reported to be mucin based on Periodic Acid Schiff positivity (PAS). PAS is not a specific staining method for mucin, and it stains several substances including various glycoproteins. Neuroendocrine expression has previously been reported in tumour cells in diffuse type gastric cancer. Methods: PAS, PAS-Diastase, Alcian blue and cytokeratin stainings were undertaken. Relevant gastrointestinal mucins (MUC1, 2, 3, 4, 5AC, 6 and MUC13) were assessed by immunohistochemistry and in situ hybridization (ISH) by the use of a new and improved ISH method. The neuroendocrine properties were evaluated by mRNA and protein expression of the general neuroendocrine markers chromogranin A, synaptophysin as well as Regenerating islet-derived family member 4 (Reg-IV). Results: All cases had cytokeratin positive tumor cells that also were PAS and PAS-Diastase and Alcian blue positive. No mucin mRNA or protein expression was observed in signet ring tumor cells including the amorphous substance in any of the nine cases. Focal or diffuse expression of mucin by immunohistochemistry was observed in non-signet ring tumors cells in five out of nine cases. All cases showed immunoreactivity to synaptophysin, and seven out of nine cases immunoreactivity to chromogranin A in signet ring and non-signet ring tumor cells. Chromogranin A and Reg-4 mRNA expression was observed in tumor cells in all samples with retained mRNA. Conclusions: The lack of MUC protein and mRNA in signet ring tumor cells suggests the amorphous substance is not mucin. The in situ hybridization method utilized in this study has several specificity steps and clear advantages over conventional methods. The discrepancy between mucin mRNA and protein expression suggest that the focal or diffuse positivity in non-signet ring tumor cells by immunohistochemistry could be non-specific. The abundant expression of the general neuroendocrine markers CgA and synaptophysin, and CgA as well as Reg-4 mRNA in tumour cells gives support to a neuroendocrine origin. In conclusion, these findings question exocrine expression in these tumours and do not lend support to the view that these tumours are mixed adenoneuroendocrine carcinomas (MANEC).
Introduction: Recent work suggests that the orexigenic hormone ghrelin have potent antiinflammatory properties in preclinical model of intestinal inflammation. Ghrelin is secreted predominantly from enteroendocrine cells of the stomach and acts as an appetite-regulating hormone increasing food intake and long-term regulation of body weight. The effects of ghrelin are mediated via the G protein-coupled receptor (GPCR) named as growth hormone secretagogue receptor (GHSR). Interestingly, the expression of this receptor has been shown on innate and adaptive immune cells. Taken together, the above suggests that ghrelin may have immunomodulatory properties. However, the mechanism, by which ghrelin suppresses intestinal inflammation, is still unclear. Therefore, we decided to investigate the effect of ghrelin on T cell function during colitis. Methods: T cell transfer chronic colitis was induced in the recombination activating gene 1 knockout mice (Rag1-/-) by adoptive transfer of Naïve CD4 T helper cells (CD4-Ths) from genetically-compatible wild-type (WT) or growth hormone secretagogue receptor knockout mice (GHSR-/-). Development of colitis was evaluated every week by assessing body weight loss, diarrhea score and blood in the feces. Eight weeks after T cell transfer, the degree of intestinal inflammation was assessed by analyzing spleen weight, colon length, histology and cytokine expression in the colon. Results: The lack of the ghrelin receptor on CD4-transferred T cells significantly worsened the course of colitis in Rag1-/- mice. Indeed, Rag1-/- mice reconstituted with GHSR -/- CD4 Ths (GHSR→Rag1) showed colitis symptoms (body weight loss, alteration in the feces) significantly earlier than Rag1-/- mice reconstituted with WT CD4 Ths (WT →Rag1). In line, GHSR→Rag1 mice displayed increased body weight loss (WT →Rag1, 14.5±4.0% vs GHSR→Rag1, 25.5±3.0%) and higher diarrhea score compared to WT →Rag1 (WT→Rag1, 2.1±0.2 vs GHSR→Rag1, 3.5±0.5). Eight weeks after T cells transfer, GHSR →Rag1 mice had significantly increased spleen weight (WT →Rag1, 70.5±21.2mg vs GHSR →Rag1, 147.5±42.1mg) and shortening of the colon (WT →Rag1, 8.9±0.4cm vs GHSR →Rag1, 7.9±0.5) Histological analysis also showed a higher lesion score in GHSR →Rag1 mice compared to WT→Rag1 mice (WT→Rag1, 7.9±4.0 vs GHSR→Rag1, 10.3±4.8). In addition, gene expression of colonic specimen revealed higher levels of IL6 (4.3±1.3 fold), TNF α (4.6±1.6 fold), IFNγ (5.6±2.3 fold) and IL17a (2.8±0.8 fold) in GHSR →Rag1 mice versus WT→Rag1 mice. Conclusions: Our observations strongly suggest that ghrelin may significantly ameliorate experimental chronic colitis by modulating T helper effector cell function in the gut. A better understanding of the underlying regulatory mechanisms of ghrelin will be crucial to further explore the potential therapeutic properties of ghrelin in inflammatory bowel disease. Tu1615 Modeling the Distinct Roles for IL-23 and IL-17 in Inflammatory Bowel Disease Using Mdr1a-/- Mice Joe Maxwell, William Brown, Yu Zhang, James B. Rottman, Alison Budelsky, Jennifer Towne Th17 cells and their signature cytokine IL-17A have been implicated in a number of autoimmune diseases including inflammatory bowel disease (IBD). IL-23 is a closely related cytokine to IL-12 in that it shares both a ligand and a receptor subunit. IL-23, IL-17A and IL-17F expression are elevated in IBD and SNPs in the IL-23R show strong association with Crohn's disease and ulcerative colitis. To examine the role of these cytokines in the gut, we compared inhibition of IL-23p19 and IL-17RA in the Helicobacter bilis-infected mdr1a-/- mouse model of colitis. Inhibition of IL-23 alone provided strong efficacy comparable to that seen with an IL-12/23p40 dual inhibitor. IL-17RA antagonism did not protect mice from disease, and led to acute disease exacerbation. To better understand which IL-17 family cytokines drive inflammation through IL-17RA in the gut, we compared inhibition of IL-17RA with inhibition of IL-17A, IL-17F or IL-25. IL-17A inhibition resulted in disease exacerbation similar to that seen with inhibition of IL-17RA, while inhibition of the other IL-17 family cytokines had no effect. Subsequent studies found that IL-17RA inhibition did not exacerbate disease in uninfected mdr1a-/- mice. The dependence on the presence of H. bilis for the exacerbation suggests either a role for IL-17A in maintenance of barrier integrity or in control of pathogens in the gut. To this point, we observed an increase in circulating markers indicative of barrier breach and bacterial dissemination. In order to further investigate the mechanisms underlying disease exacerbation in anti-IL-17RA treated H. bilis-infected mdr1a-/- mice, we examined gene expression changes in the gut. IL-23 inhibition led to decreased expression of a number of cytokines and chemokines in the gut while IL-17RA or IL-17A inhibition led to increased expression of many genes including IFN γ, TNFα, IL-6 and a number of chemokines. To determine whether an increase in IFN γ was key to the anti-IL-17RA mediated exacerbation, we treated mice with anti-IFNγ along with anti-IL-17RA. Inhibition of IFN γ alone was highly efficacious; however, IFNγ blockade did not affect disease in the presence of anti-IL-17RA. These data suggest that pathways other than IFN γ may be driving anti-IL-17RA mediated disease exacerbation in this mouse model of colitis and the exacerbation is not likely due to a shift from Th17 cells to Th1 cells. The data in this H. bilis-infected mdr1a-/- mouse colitis model are similar to what is emerging from Crohn's disease clinical trials: IL-12/ 23p40 inhibitors are showing promise while inhibition of IL-17A with secukinumab led to exacerbation in a subset of patients. Together the emerging clinical data suggest that the role of Th17 cells and IL-17A in colitis is different from other inflammatory diseases and that IL-23 and IL-17 biology are distinct in the gut.
AGA Abstracts
S-806