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W1687 Nicotine Enhances Phagocytosis in Macrophages via Recruitment of Dynamin-2 to the Phagocytic Cup
(n=5). Lesion formation by IND was markedly decreased in cats given PDF added with 3% pectin, guar gum, polydextrose or mucin; MLAs were 0.6±0.3 cm2, 0.0±0.0 cm2, 1.3±0.8 cm2 and 1.6±0.5 cm2 (n=4), respectively. The inhibitory effects were significant (p<0.05 vs PDF alone). Viscosities of the SDFs and mucin (0.03-30%) were increased in a concentrationdependent manner, and the viscosities (mPa-S) of pectin, guar gum, polydextrose and mucin (3% concentration) were 414, >1200, 1 and 4, respectively. Pectin (10%, 4 ml/kg, p.o.) given after a 16h-fast did not affect the intestinal absorption of IND (3 mg/kg, p.o.), i.e., Cmax: 4.6±1.2 vs. 3.6±0.1 μg/ml, AUC0-4h: 6.4±0.8 vs. 7.1±1.3 μg x h/ml (n=5). Addition of pectin 3% to PDF did not inhibit intestinal hypermotility induced by IND. CONCLUSIONS: SDFs and exogenous mucin can prevent the formation of small intestinal lesions by NSAIDs, probably by protecting the mucosa similar to endogenous mucin from aggressive factors. Viscosity may relate, at least in part, to the protective effects of SDFs on the small intestine. These results suggest a new therapeutic method for preventing the formation of small intestinal lesions by NSAIDs.
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Introduction: Mucositis results from chemotherapy and radiotherapy and is a debilitating disorder of the alimentary tract. Symptoms include abdominal pain, bloating and diarrhoea. Current treatments for mucositis are often ineffective. In animal models, Emu Oil (EO) has been demonstrated to exert a beneficial effect in some forms of arthritis and other inflammatory disorders. We sought to investigate the effects of EO in a rat model of mucositis. Methods: Female dark agouti rats were gavaged with EO (0.5ml or 1ml) for 5 days prior to intraperitoneal injection of 5-Fluorouracil (5-FU: 150 mg/kg), and this continued up to the day of kill (48, 72 and 96 hours post 5-FU administration). Histological (crypt depth and villus height) and biochemical (sucrase activity and myeloperoxidase [MPO] levels) parameters were examined. Sucrase activity In Vivo was determined by the Sucrose Breath Test [SBT] at -120, 0, 48, 72 and 96 hours. Statistical analysis was by one-way ANOVA, where p<0.05 was considered significant. Results: MPO activity, a marker of neutrophil infiltration, was significantly decreased in the ileum at 96 hours in both the 0.5ml (451±168 U/g) and 1ml (503±213 U/g) EO groups, relative to the 5-FU treated controls (1724±431 U/g, p<0.05). Similar results were also evident in the jejunum (1501±466: 1ml EO; 5FU control: 3196±374, p<0.05) and mid-small intestine (376±93: 1ml EO; 5-FU control: 2268±435, p<0.05). These results were further supported by significant improvements in villus height (230±17μm) and crypt depth (152±8μm) in the ileum at 96 hours following 1ml EO compared to 5-FU treated control villus height (180±18μm, p<0.05) and crypt depth (106±12μm, p<0.05). At 48 hours, 5-FU treatment decreased the SBT (2.1±1.1%CD90) compared to untreated animals (15.0±0.8%CD90), and this was maintained for the remainder of the trial. However, EO was unable to reverse the effects of 5-FU on sucrase activity, as assessed biochemically, nor by the SBT compared to 5-FU treated controls. Conclusion: In this preliminary study, EO decreased some indicators of acute inflammation in the chemotherapy-damaged small intestine, and improved villus and crypt structure in the intestinal mucosa. These promising findings suggest potential for EO to act as a preventative therapy in mucositis. Further studies are required to determine its mode of action and the effects of dose escalation and inter-batch variation.
W1684 Helicobacter Heilmannii Peroral Infection Exacerbates NSAIDs-Induced Small Intestinal Injury in IL-10 (-/-) Mouse Masahiko Nakamura, Tetsufumi Takahashi, Toshifumi Hibi, Kanji Tsuchimoto Recently, capsule endoscopic observation has shown the high prevalence of NSAIDs-induced small intestinal injury, while the interaction of bacterial infection and NSAIDs on the gastrointestinal mucosal damage remains to be clarified. Thus, the present study was undertaken to clarify the interaction of effect of loxoprofen, one of the NSAIDs, and Helicobacter heilmannii (Hh) obtained from cynomolgus monkeys on the small intestinal mucosal injury. Materials and Methods: C57BL/6 mice and IL-10-knockout (KO) mice was used. Half of the mice was infected with Hh by intragastric intubation three months before the experiments. The mice was treated with 30mg/kg b.w. of loxoprofen every twelve hours for three days. The mucosal injury was observed after staining with indigo carmin and graded. The alteration of the microvascular permeabillity was investigated by the intravenous administration of FITC-dextran (MW 50, 000, 5mg/mouse) and CD31 immunoreactivity. The localization of Hh bacilli was investivated with immunohistochemistry using anti-Helicobacter pylori polyclonal antibody which was shown to react with Hh. The apoptosis was detected by the immunohistochemical method using polyclonal antibody against active caspase 3 and 9. In addition, cyclooxygenase-2 and microsomal prostaglandin E synthase-1 immunoreactivity was also investigated. Rebamipide (30mg/kg b.w.), one of the mucoprotective agent, was administered to one fourth of the mice concomitantly with loxoprofen to clarify its preventive effect. Results: In the IL-10-KO mice treated with Hh for three months, accumulation of the Hh bacilli was detected especially in the mucus layer. Electron microscopic cytochemistry clearly showed the multispiral bacilli immnoreactive to Hp polyclonal antibodies. In situ hybridization study identified this bacilli as Hh. As to the mucosal injury, C57BL/6 mice treated with loxoprofen showed mild gastrointestinal mucosal damage, while in the IL10-KO mice treated with loxoprofen the severe erosive lesion was detected in the small intestine, while only mild changes were observed in the stomach and large intestine. Hh-infected mice had more severe lesion in the small intestine. The apoptosis and microvascular permeability showed the similar tendency. Rebamipide treatment almost suppressed the mucosal damages. In conclusion, NSAIDs and Hh showed the addtive effect on the small intestinal mucosal damage which was abolished by the rebamipide treatment.
W1687 Nicotine Enhances Phagocytosis in Macrophages via Recruitment of Dynamin2 to the Phagocytic Cup Esmerij P. van der Zanden, Guy E. Boeckxstaens, Wouter de Jonge INTRODUCTION The cholinergic nervous system attenuates the production of proinflammatory cytokines by macrophages and inhibits inflammatory processes. We previously showed that vagus nerve signaling not only blunts inflammation in macrophages, but also enhances their phagocytic capacity. Here, we study the molecular pathways involved in this process. METHODS The spleen macrophage cell line MF4/4 and primary peritoneal macrophages were pre-treated with nicotine (0-1μM) and challenged with Zymosan(5P/ Cell). After 5 or 10min stimulation, membrane proteins were isolated using the proteome cell compartment kit (Qiagen) and prepared for Western Blot. Whole cell lysates for immunoblotting were isolated after 30min. For Rac1 GTPase activity assays, 96-wells plates were coated with Rho-family effector proteins, to which the active GTP-bound form of Rac from a biological sample can bind. Cells were grown on glass slides (Nuncbrand), pretreated with nicotine, challenged with FITC-labeled Zymosan and phagocytosis was allowed for 5min. Cells were stained with antibodies against Dynamin-2 and F-actin and slides were analyzed using confocal laser scanning microscope. Recruitment to the phagocytic cup was scored by analysis of 80-100 cells in a blinded fashion. RESULTS In peritoneal and MF4/4 cells, nicotine enhanced phagocytic uptake of Zymosan particles (up to 2.5 fold). Exposure to Zymosan stimulated pathways that are known to be involved in the phagocytic process; phosphorylation of PI-3K (10% vs 90% of total PI3K control vs Zymosan), Akt (10% vs 40% of total P38 in control vs Zymosan) and p38 MAP kinase (1% vs 35% of total P38 in control vs Zymosan). However, the phosphorylation of PI3K, Akt, or p38 MAPK was not affected by nicotine. In addition, nicotine did not enhance Rac1 GTPase activity. On the other hand, pretreatment with nicotine augmented the recruitment of the large GTPase Dynamin-2 towards the phagocytic cup and strongly stimulated cup formation (p>0.03, as quantified by confocal microscopy). This observation was corroborated by Western analysis of membrane protein: nicotine treatment led to a significant (p>0.05) increase in membraneassociated Dynamin-2 after 5min (increase to 190% of control) and 10min (increase to 160% of control) of Zymosan treatment. CONCLUSION Our data show that cholinergic agonists induce phagocytosis in splenic and peritoneal macrophages. This mechanism involves nAChR-mediated enhancement of Dynamin-2 recruitment to the phagocytic cup, rather than PI3K, Akt or p38 signaling pathways.
W1685 Intraperitoneal Secretion of Cytokines and Chemokines in Response to the Surgical Stress Rei Kawashima, Yuki I. Kawamura, Vongsavanh Phongsisay, Toshihiko Okada, Noriko Toyama-Sorimachi, Yutaka Kawamura, Fumio Konishi, Taeko Dohi [Background and Aim] Peritoneal adhesions cause significant signs and symptoms including intestinal obstruction, chronic pelvic pain and infertility, and eventually a second more serious surgery is often required. Thus, adhesions in the peritoneal cavity are both life threatening and an enormous cost for patient care. Our previous studies using mice showed that peritoneal macrophages and mesothelial cells secreted chemokine CCL1 by various inflammatory stimuli, and expression of its receptor CCR8 in macrophages was upregulated by CCL1. Blocking CCL1/CCR8 interaction inhibited macrophage aggregation and thus prevented the formation of peritoneal adhesions developed after surgical stress and intestinal inflammation (J. Immunol 2007;178:5296). In this study we aimed to identify cytokines and chemokines secreted into the human peritoneal cavity during laparotomy, which may have potential as a target of prevention of peritoneal adhesion. [Methods] Samples were collected from 7 cases who underwent laparotomy for partial colectomy. To get peritoneal lavage fluid, one liter of saline was applied into the peritoneal cavity and recovered as much as possible, at the beginning and the end of operation. After clearing by centrifuge, supernatant was used to measure levels of 28 kinds of cytokines and chemokines by Multiplex system or ELISA. The precipitated cells were stained with diff-quick and were also subjected to RT-PCR for expression of chemokine receptors. [Results] At the beginning of the operation, mesothelial cells were observed in the peritoneal lavage fluid; at the end of operation, macrophages and eosinophils were frequently detected. Transcripts of CCR8 were detected in the cell pellets in both beginning and end of the operation. Concentration of CCL1 in peritoneal lavage fluid was elevated in the end of operation. A dramatic ~5-40-fold increase of IL-6 and CCL5 (RANTES) was observed in the end of operation in all cases. To a lesser extent, levels of IL-1β, IFN-γ and other macrophage chemokines also elevated in all cases. Of interest, change of TNF-α was not seen in 5 out 7 cases. Considerable amount of IL-17 was detected in the samples obtained at the beginning of operation (200-1200 pg/ml) and the level decreased (3 out of 7 cases) or did not change (3 out of 7) after operation. [Conclusion] In humans, macrophages and eosinophils were recruited in to the peritoneal cavity in the end of the operation. IL-6 and CCL5 were the major cytokine and chemokine secreted into peritoneal cavity during laparotomy. As shown in mice, we think that chemokines are possible target for prevention of postoperative adhesion.
W1689 Latent Infection of the Human Enteric Nervous System By Varicella Zoster Virus (VZV) Jason J. Chen, Michael D. Gershon, Shilin Wan, Robert A. Cowles, Alejandro RuizElizalde, Stephan C. Bischoff, Anne A. Gershon Varicella-zoster virus (VZV) is one of the most infectious of the 8 human herpesviruses. Primary VZV infection causes varicella (chickenpox), during which VZV becomes latent in sensory nerve ganglia and remains for life. In about 30% of individuals, VZV reactivates from latency to cause zoster (shingles), which is associated with considerable morbidity and may be followed by post-hepetic neuralgia. Because VZV lacks an animal reservoir, its perpetuation depends on latency and reactivation. Despite its strong preference for human tissues, we have found that VZV infects guinea pig enteric neurons In Vitro. When fibroblastfree cultures are inoculated with cell-free virus, infection is latent; however, if fibroblasts are present when VZV is introduced, infection is lytic. Latently infected enteric neurons
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AGA Abstracts
AGA Abstracts
The Effects of Orally-Administered Emu Oil in a Rat Model of ChemotherapyInduced Mucositis Ruth J. Lindsay, Mark S. Geier, Roger Yazbeck, Kerry A. Lymn, Ross N. Butler, Gordon S. Howarth
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Introduction: Mucositis results from chemotherapy and radiotherapy and is a debilitating disorder of the alimentary tract. Symptoms include abdominal pain, bloating and diarrhoea. Current treatments for mucositis are often ineffective. In animal models, Emu Oil (EO) has been demonstrated to exert a beneficial effect in some forms of arthritis and other inflammatory disorders. We sought to investigate the effects of EO in a rat model of mucositis. Methods: Female dark agouti rats were gavaged with EO (0.5ml or 1ml) for 5 days prior to intraperitoneal injection of 5-Fluorouracil (5-FU: 150 mg/kg), and this continued up to the day of kill (48, 72 and 96 hours post 5-FU administration). Histological (crypt depth and villus height) and biochemical (sucrase activity and myeloperoxidase [MPO] levels) parameters were examined. Sucrase activity In Vivo was determined by the Sucrose Breath Test [SBT] at -120, 0, 48, 72 and 96 hours. Statistical analysis was by one-way ANOVA, where p<0.05 was considered significant. Results: MPO activity, a marker of neutrophil infiltration, was significantly decreased in the ileum at 96 hours in both the 0.5ml (451±168 U/g) and 1ml (503±213 U/g) EO groups, relative to the 5-FU treated controls (1724±431 U/g, p<0.05). Similar results were also evident in the jejunum (1501±466: 1ml EO; 5FU control: 3196±374, p<0.05) and mid-small intestine (376±93: 1ml EO; 5-FU control: 2268±435, p<0.05). These results were further supported by significant improvements in villus height (230±17μm) and crypt depth (152±8μm) in the ileum at 96 hours following 1ml EO compared to 5-FU treated control villus height (180±18μm, p<0.05) and crypt depth (106±12μm, p<0.05). At 48 hours, 5-FU treatment decreased the SBT (2.1±1.1%CD90) compared to untreated animals (15.0±0.8%CD90), and this was maintained for the remainder of the trial. However, EO was unable to reverse the effects of 5-FU on sucrase activity, as assessed biochemically, nor by the SBT compared to 5-FU treated controls. Conclusion: In this preliminary study, EO decreased some indicators of acute inflammation in the chemotherapy-damaged small intestine, and improved villus and crypt structure in the intestinal mucosa. These promising findings suggest potential for EO to act as a preventative therapy in mucositis. Further studies are required to determine its mode of action and the effects of dose escalation and inter-batch variation.
W1684 Helicobacter Heilmannii Peroral Infection Exacerbates NSAIDs-Induced Small Intestinal Injury in IL-10 (-/-) Mouse Masahiko Nakamura, Tetsufumi Takahashi, Toshifumi Hibi, Kanji Tsuchimoto Recently, capsule endoscopic observation has shown the high prevalence of NSAIDs-induced small intestinal injury, while the interaction of bacterial infection and NSAIDs on the gastrointestinal mucosal damage remains to be clarified. Thus, the present study was undertaken to clarify the interaction of effect of loxoprofen, one of the NSAIDs, and Helicobacter heilmannii (Hh) obtained from cynomolgus monkeys on the small intestinal mucosal injury. Materials and Methods: C57BL/6 mice and IL-10-knockout (KO) mice was used. Half of the mice was infected with Hh by intragastric intubation three months before the experiments. The mice was treated with 30mg/kg b.w. of loxoprofen every twelve hours for three days. The mucosal injury was observed after staining with indigo carmin and graded. The alteration of the microvascular permeabillity was investigated by the intravenous administration of FITC-dextran (MW 50, 000, 5mg/mouse) and CD31 immunoreactivity. The localization of Hh bacilli was investivated with immunohistochemistry using anti-Helicobacter pylori polyclonal antibody which was shown to react with Hh. The apoptosis was detected by the immunohistochemical method using polyclonal antibody against active caspase 3 and 9. In addition, cyclooxygenase-2 and microsomal prostaglandin E synthase-1 immunoreactivity was also investigated. Rebamipide (30mg/kg b.w.), one of the mucoprotective agent, was administered to one fourth of the mice concomitantly with loxoprofen to clarify its preventive effect. Results: In the IL-10-KO mice treated with Hh for three months, accumulation of the Hh bacilli was detected especially in the mucus layer. Electron microscopic cytochemistry clearly showed the multispiral bacilli immnoreactive to Hp polyclonal antibodies. In situ hybridization study identified this bacilli as Hh. As to the mucosal injury, C57BL/6 mice treated with loxoprofen showed mild gastrointestinal mucosal damage, while in the IL10-KO mice treated with loxoprofen the severe erosive lesion was detected in the small intestine, while only mild changes were observed in the stomach and large intestine. Hh-infected mice had more severe lesion in the small intestine. The apoptosis and microvascular permeability showed the similar tendency. Rebamipide treatment almost suppressed the mucosal damages. In conclusion, NSAIDs and Hh showed the addtive effect on the small intestinal mucosal damage which was abolished by the rebamipide treatment.
W1687 Nicotine Enhances Phagocytosis in Macrophages via Recruitment of Dynamin2 to the Phagocytic Cup Esmerij P. van der Zanden, Guy E. Boeckxstaens, Wouter de Jonge INTRODUCTION The cholinergic nervous system attenuates the production of proinflammatory cytokines by macrophages and inhibits inflammatory processes. We previously showed that vagus nerve signaling not only blunts inflammation in macrophages, but also enhances their phagocytic capacity. Here, we study the molecular pathways involved in this process. METHODS The spleen macrophage cell line MF4/4 and primary peritoneal macrophages were pre-treated with nicotine (0-1μM) and challenged with Zymosan(5P/ Cell). After 5 or 10min stimulation, membrane proteins were isolated using the proteome cell compartment kit (Qiagen) and prepared for Western Blot. Whole cell lysates for immunoblotting were isolated after 30min. For Rac1 GTPase activity assays, 96-wells plates were coated with Rho-family effector proteins, to which the active GTP-bound form of Rac from a biological sample can bind. Cells were grown on glass slides (Nuncbrand), pretreated with nicotine, challenged with FITC-labeled Zymosan and phagocytosis was allowed for 5min. Cells were stained with antibodies against Dynamin-2 and F-actin and slides were analyzed using confocal laser scanning microscope. Recruitment to the phagocytic cup was scored by analysis of 80-100 cells in a blinded fashion. RESULTS In peritoneal and MF4/4 cells, nicotine enhanced phagocytic uptake of Zymosan particles (up to 2.5 fold). Exposure to Zymosan stimulated pathways that are known to be involved in the phagocytic process; phosphorylation of PI-3K (10% vs 90% of total PI3K control vs Zymosan), Akt (10% vs 40% of total P38 in control vs Zymosan) and p38 MAP kinase (1% vs 35% of total P38 in control vs Zymosan). However, the phosphorylation of PI3K, Akt, or p38 MAPK was not affected by nicotine. In addition, nicotine did not enhance Rac1 GTPase activity. On the other hand, pretreatment with nicotine augmented the recruitment of the large GTPase Dynamin-2 towards the phagocytic cup and strongly stimulated cup formation (p>0.03, as quantified by confocal microscopy). This observation was corroborated by Western analysis of membrane protein: nicotine treatment led to a significant (p>0.05) increase in membraneassociated Dynamin-2 after 5min (increase to 190% of control) and 10min (increase to 160% of control) of Zymosan treatment. CONCLUSION Our data show that cholinergic agonists induce phagocytosis in splenic and peritoneal macrophages. This mechanism involves nAChR-mediated enhancement of Dynamin-2 recruitment to the phagocytic cup, rather than PI3K, Akt or p38 signaling pathways.
W1685 Intraperitoneal Secretion of Cytokines and Chemokines in Response to the Surgical Stress Rei Kawashima, Yuki I. Kawamura, Vongsavanh Phongsisay, Toshihiko Okada, Noriko Toyama-Sorimachi, Yutaka Kawamura, Fumio Konishi, Taeko Dohi [Background and Aim] Peritoneal adhesions cause significant signs and symptoms including intestinal obstruction, chronic pelvic pain and infertility, and eventually a second more serious surgery is often required. Thus, adhesions in the peritoneal cavity are both life threatening and an enormous cost for patient care. Our previous studies using mice showed that peritoneal macrophages and mesothelial cells secreted chemokine CCL1 by various inflammatory stimuli, and expression of its receptor CCR8 in macrophages was upregulated by CCL1. Blocking CCL1/CCR8 interaction inhibited macrophage aggregation and thus prevented the formation of peritoneal adhesions developed after surgical stress and intestinal inflammation (J. Immunol 2007;178:5296). In this study we aimed to identify cytokines and chemokines secreted into the human peritoneal cavity during laparotomy, which may have potential as a target of prevention of peritoneal adhesion. [Methods] Samples were collected from 7 cases who underwent laparotomy for partial colectomy. To get peritoneal lavage fluid, one liter of saline was applied into the peritoneal cavity and recovered as much as possible, at the beginning and the end of operation. After clearing by centrifuge, supernatant was used to measure levels of 28 kinds of cytokines and chemokines by Multiplex system or ELISA. The precipitated cells were stained with diff-quick and were also subjected to RT-PCR for expression of chemokine receptors. [Results] At the beginning of the operation, mesothelial cells were observed in the peritoneal lavage fluid; at the end of operation, macrophages and eosinophils were frequently detected. Transcripts of CCR8 were detected in the cell pellets in both beginning and end of the operation. Concentration of CCL1 in peritoneal lavage fluid was elevated in the end of operation. A dramatic ~5-40-fold increase of IL-6 and CCL5 (RANTES) was observed in the end of operation in all cases. To a lesser extent, levels of IL-1β, IFN-γ and other macrophage chemokines also elevated in all cases. Of interest, change of TNF-α was not seen in 5 out 7 cases. Considerable amount of IL-17 was detected in the samples obtained at the beginning of operation (200-1200 pg/ml) and the level decreased (3 out of 7 cases) or did not change (3 out of 7) after operation. [Conclusion] In humans, macrophages and eosinophils were recruited in to the peritoneal cavity in the end of the operation. IL-6 and CCL5 were the major cytokine and chemokine secreted into peritoneal cavity during laparotomy. As shown in mice, we think that chemokines are possible target for prevention of postoperative adhesion.
W1689 Latent Infection of the Human Enteric Nervous System By Varicella Zoster Virus (VZV) Jason J. Chen, Michael D. Gershon, Shilin Wan, Robert A. Cowles, Alejandro RuizElizalde, Stephan C. Bischoff, Anne A. Gershon Varicella-zoster virus (VZV) is one of the most infectious of the 8 human herpesviruses. Primary VZV infection causes varicella (chickenpox), during which VZV becomes latent in sensory nerve ganglia and remains for life. In about 30% of individuals, VZV reactivates from latency to cause zoster (shingles), which is associated with considerable morbidity and may be followed by post-hepetic neuralgia. Because VZV lacks an animal reservoir, its perpetuation depends on latency and reactivation. Despite its strong preference for human tissues, we have found that VZV infects guinea pig enteric neurons In Vitro. When fibroblastfree cultures are inoculated with cell-free virus, infection is latent; however, if fibroblasts are present when VZV is introduced, infection is lytic. Latently infected enteric neurons
A-717
AGA Abstracts
AGA Abstracts
The Effects of Orally-Administered Emu Oil in a Rat Model of ChemotherapyInduced Mucositis Ruth J. Lindsay, Mark S. Geier, Roger Yazbeck, Kerry A. Lymn, Ross N. Butler, Gordon S. Howarth