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M1242 Pharmacological Characterization of Murine Lower Esophageal Sphincter (LES) Relaxation
AGA Abstracts
PYY3-36 in a restraint cage. PYY peptide effects on food intake and FPO were monitored simultaneously in fasted or non-fasted mice. Gastric emptying was determined 2 h after PYY peptides injected IP in fasted/re-fed mice. In the distal colon, Y2 receptor expression was assessed by RT-PCR in the mucosa or layers of submucosa and muscle, and location in whole mounts of longitudinal muscle/myenteric plexus by immunohistochemistry. Results: PYY3-36, PYY, and NPY (8 nmol/kg) inhibited the stimulated 1-h FPO in response to novelty stress by 89%, 58% and 52% respectively compared with saline; lower doses (0.8, 2.5 nmol/ kg) had no effect. Inhibition induced by PYY3-36, PYY and NPY reached 94%, 92% and 57% respectively within 15 min. PYY and PYY3-36, but not Y1 agonists, prevented the 1h restraint-induced FPO and colonic contractions, and abolished 5-HTP (10 mg/kg)-induced FPO and diarrhea. BIIE-0246 (5 mg/kg) blocked PYY and PYY3-36 inhibitory actions on the colonic responses to novelty stress and 5-HTP. PYY3-36 (25 nmol/kg) suppressed bethanechol (5 mg/kg, IP)-stimulated FPO and diarrhea. PYY3-36 inhibitory action on FPO stimulated by novel environment was still observed in VIP knockout mice. PYY and PYY336, but not Y1 agonist, reduced the 2-h gastric emptying by 56% and 48% respectively. PYY3-36 inhibitory effects on food intake and FPO were correlated both in fasted mice in light phase and non-fasted mice in dark phase. Y2 mRNA was expressed in both mucosa and muscle layers and Y2-like immunoreactivity was detected in the colonic myenteric plexus. Conclusions: PYY and PYY3-36 potently blocked colonic contractions, fecal output and diarrhea stimulated by stress, 5-HT or cholinergic agonist. PYY inhibitory action is mediated through Y2 receptors present in the colon. These data suggest that Y2 receptors may play a role in the development of constipation.
indicate that an increase in 5-HT2C receptor in hypothalamus and an increase in 5-HT3 receptor in stomach are involved in anorexic effect of cisplatin. M1241 Contractile and Secretory Responses of Luminal Short-Chain Fatty Acids and the Expression of These Receptors, GPR41 and GPR43, in the Human Small and Large Intestines Shin-ichiro Karaki, Hideaki Tazoe, Izumi Kaji, Yasuko Otomo, Takaji Yajima, Atsukazu Kuwahara Background and aim: Short-chain fatty acids (SCFAs) are luminal fermented products from some kinds of dietary fibers especially in large intestine. In animal experiments, luminal SCFAs are reported to induce a variety of physiological effects on intestinal functions including smooth muscle contraction and epithelial secretion. However, there is little evidence of the effects of SCFAs in the human intestine. Therefore, the aim of present study tried to show the physiological effects of SCFAs in the human small and large intestines, and examined the expression of the SCFA receptors, GPR41 and GPR43, possibly mediating the responses. Methods: Human terminal ileum and colon were used in nonpathological region of surgical specimens obtained from the patients of large intestinal cancer following informed consent. Smooth muscle strips with mucosa and without mucosa were tied to an isometric force transducer and suspended in a tissue bath in order to record the smooth muscle contractile activity. To record transepithelial ion transport, mucosa-submucosal preparations were mounted on Ussing flux chamber and short-circuit current (Isc) were measured. For RT-PCR and Western blot analysis for GPR41 and GPR43, total RNA and protein were extracted from the tissues, respectively. Moreover, tissues were fixed in Zamboni's fixative, and 10 µm-thin cryostat sections were made for immunohistochemistry of GPR41 and GPR43. Results: In terminal ileum, propionate-induced transient contractions were observed in circular muscle strip with mucosa, but not without mucosa. This is the first to show the SCFA-induced contractile response in the human small intestine. In Ussing chamber experiments, propionate evoked negative Isc probably by K+ secretion in large intestine, but rarely evoked positive Isc by Cl- secretion as previously reported in rat distal colon. Moreover, mRNA and proteins of both SCFA receptors, GPR41 and GPR43, were detected in the human intestinal mucosa. In immunohistochemistry, these receptors were observed to localize on epithelial cells especially enteroendocrine cells containing peptide YY. Conclusion: The present study first shows the physiological effects of SCFAs and the SCFA receptor expressions in the human intestine. However, there is no direct evidence that GPR41 and/or GPR43 mediate(s) these physiological effects of SCFAs yet. Therefore, further study is necessary to perform in order to reveal whether these receptors really involve with the effects of SCFAs in the human intestine.
M1239 Alterations in Synaptic Transmission in the Enteric Nervous System and Correlation to Motility Defects in CB1 Receptor-Deficient Mice Ian M. Hons, Catherine M. Keenan, Martin Storr, Beat Lutz, Keith A. Sharkey The endocannabinoid (EC) system has been shown to be important in regulating the plasticity of central nervous system synapses. However, the role of ECs on peripheral neurons has not been extensively investigated. The enteric nervous system (ENS) is a self-contained integrative network possessing intrinsic reflexes and rhythmic network activity located in the wall of the gut. The ENS possesses extensive synaptic connectivity, and additionally, these synapses have been shown to be highly plastic in pathological situations. The CB1 receptor is localized in the ENS. Using CB1 receptor-deficient mice, we sought to study the role of ECs in the ENS, and investigated the functional consequences of inactivation of the CB1 receptor gene. Female CB1-/- mice on a C57BL/6N background were bred in Calgary. Comparisons were made between CB1-/- mice (KO) and littermate controls (CB1+/+, WT). The distal ileum was removed and maintained in oxygenated Krebs solution containing nicardipine and scopolamine. The myenteric plexus was dissected from a segment of ileum, and S and AH type neurons were examined by intracellular voltage recordings. Passive and active electrical properties were evaluated as well as synaptic events evoked by focal stimulation of adjacent ganglia. Motility was assessed in WT and KO mice gavaged with Evans blue (5% in 5% gum arabic). 15 min after the gavage, animals were sacrificed and the distance traveled by the dye front was measured and expressed as a percentage of the length of the entire small intestine. Neurons where assigned to 2 groups using electrophysiological and morphological criteria: Dogiel type I S neurons which receive fast excitatory post-synaptic potentials (EPSPs), and AH neurons which posses a characteristic action potential shape, prolonged after-hyperpolarization, and Dogiel type II morphology. Recordings of the basal electrical activity from neurons of KO animals displayed a higher number of neurons receiving spontaneous EPSPs in S type neurons when compared to WT controls (1/6 WT, 6/6 KO, P < 0.05). Measurements of upper GI transit indicated an increase in transit in KO animals (70.3 ± 3.27% WT, 92.1 ± 2.5% KO, P < 0.01). These results are consistent with ECs playing a role in dampening synaptic activity in the ENS. In addition, they indicate that normal gut function requires a basal level of EC production to control activity in enteric neural networks. When disrupted, the ENS becomes spontaneously active, leading to abnormal gut function (i.e. rapid transit). Our data indicate that CB1 receptors and ECs are involved in synaptic communication in the ENS and that this system contributes to normal gut function.
M1242 Pharmacological Characterization of Murine Lower Esophageal Sphincter (LES) Relaxation Scott Carmichael, John Sauvé, David V. Miller, Yong Zhang, William G. Paterson AIM: Because of the availability of genetic knockouts, the mouse has been increasingly utilized as a model for studying GI motility. To date there is limited data available on the physiology of the LES motor function in normal or mutant mice. METHODS: We characterized pharmacologically the putative cholinergic, nitrergic and purinergic innervation of the LES circular smooth muscle from CD-1 mice using isometric tension recording from isolated LES rings. The magnitude of LES relaxation (LESR) was expressed as a percent of maximal LESR induced by 1 µM isoproterenol. Preliminary experiments were also conducted in W/ Wv mutant mice, which lack intramuscular interstitial cells of Cajal (ICCs). RESULTS: Rings of LES muscle from CD-1 mice developed spontaneous tone shortly after suspension in the tissue bath. Electrical field stimulation of intrinsic nerves (100V, 10 Hz, 0.5 ms pulse duration, 5 sec) evoked a reproducible LESR (45.4 ± 6.8 %; n=30) followed by a postrelaxation contraction. This relaxation was abolished by application of the nitric oxide synthase antagonist N(G)-nitro-L-arginine methyl ester (L-NAME; 100 µM). In the presence of L-NAME, cumulative application of atropine (3 µM) restored LESR to a level not significantly different than control (55.8 ± 8.4%). Furthermore, atropine virtually abolished the postrelaxation contraction. Subsequent application of apamin (300 nM), a small conductance Ca2+-activated K+ channel blocker, significantly attenuated the LESR to 20.4 ± 4.2% (p<0.05) suggesting a purinergic component to LESR. Application of the neurokin-2 receptor antagonist MEN-10376 (3 µM) or Substance P tachyphylaxis had no consistent effects on the postrelaxation contraction. LES rings from W/Wv mutant mice developed significantly less spontaneous tone and weaker KCl-induced contraction than strips from control mice. When tone was restored by pre-application of carbachol in LES rings from W/Wv mutant mice, electrical field stimulation induced relaxation that was abolished by L-NAME. CONCLUSIONS: These experiments indicate that in the mouse LES both nitrergic and purinergic inhibitory neurotransmission is present. Acetylcholine appears to be the major excitatory neurotransmitter. In ICC deficient mice, LES muscle contractility is impaired, but nitrergic relaxation is intact, suggesting that ICCs are not required for nitrergic neurotransmission. (Supported by CIHR)
M1240 The Roles of Serotonin 5-HT2C and 3 Receptors in Anorexia and Delayed Gastric Emptying in Cisplatin-Treated Rats Koji Yakabi, Susumu Kurosawa, Tomohisa Hattori, Yuzurihara Mitsutoshi, Shino Ohno Background/Aim: It has been indicated that the acute gastrointestinal tract disorders caused by anticancer drugs involve serotonin secreted from the enterochromaffin cells. Moreover, cerebral serotonin contributes to regulate feeding behavior and energy balance. Mutated serotonin 5-HT2C receptor display leptin-independent hyperphagia that leads to a late onset of obesity (Nature 374, 1995). In order to clarify the mechanisms of cisplatin-induced anorexia, we aimed to investigate the regulation of several 5-HT receptors expression between fasting condition and anorexia induced by anticancer drugs. Methods: Exp.1: Anorexia was induced in S.D. rats by consecutive (1, 2, 4 mg/kg, i.p.) injection of cisplatin for 4 days. The stomach and hypothalamus were collected to determine 5-HT receptor gene expression by RT-PCR. Exp.2: In order to investigate the function of 5-HT receptor in cisplatin-treated rats, 5-HT2C receptor antagonist (SB242084HCl at 10, 30 nmol/rat icv) or 5-HT3 receptor antagonists (granisetoron at doses of 0.1 and 0.5 mg/kg, s.c. or ondansetron at dose of 1 mg/kg, s.c.) was administered to rats 6hr after cisplatin treatment, and the 24hrs food intake and solid gastric emptying were determined. Results: The food intake in cicplatin-treated rats was markedly decreased, compared with saline-treated rats. Moreover, serotonin 5HT2C receptor gene expression in cisplatin-treated rats was increased in hypothalamus, and it was unchanged in stomach. Serotonin 5-HT3 receptor gene expression in cisplatin-treated rats was significantly increased in stomach, while it was not increased in hypothalamus. Administration of SB242084HCl was significantly inhibited the decrease in 16hr food intake in cisplatin-treated rats, whereas it in 5-HT3 receptor antagonist-treated group was unchanged. Granisetron at dose of 0.1 mg/kg and 0.5 mg/kg, and ondansetron inhibited the decrease in solid gastric emptying in cisplatin-treated rats. Conclusion: The results
AGA Abstracts
M1248 Bacterial and Fungal Colonization of Endoscopic Intragastric Balloons Inserted in Patients with Morbid Obesity John A. Karagiannis, Nicoletta G. Mathou, Konstantia D. Paraskeva, Georgios K. Anagnostopoulos, Konstantinos Zografos, Nausika Kontostergiou, Stamatina Konstantinou, Zoi Roussou INTRODUCTION Endoscopic intragastric balloon insertion consists an acceptable method for, at least short-term, weight loss in morbid obese patients. Upon removal of the balloon it was noticed in several patients that it looked macroscopically abnormal with a pattern reminiscent of fungal colonization. AIM OF THE STUDY Aim of the study was to visually
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PYY3-36 in a restraint cage. PYY peptide effects on food intake and FPO were monitored simultaneously in fasted or non-fasted mice. Gastric emptying was determined 2 h after PYY peptides injected IP in fasted/re-fed mice. In the distal colon, Y2 receptor expression was assessed by RT-PCR in the mucosa or layers of submucosa and muscle, and location in whole mounts of longitudinal muscle/myenteric plexus by immunohistochemistry. Results: PYY3-36, PYY, and NPY (8 nmol/kg) inhibited the stimulated 1-h FPO in response to novelty stress by 89%, 58% and 52% respectively compared with saline; lower doses (0.8, 2.5 nmol/ kg) had no effect. Inhibition induced by PYY3-36, PYY and NPY reached 94%, 92% and 57% respectively within 15 min. PYY and PYY3-36, but not Y1 agonists, prevented the 1h restraint-induced FPO and colonic contractions, and abolished 5-HTP (10 mg/kg)-induced FPO and diarrhea. BIIE-0246 (5 mg/kg) blocked PYY and PYY3-36 inhibitory actions on the colonic responses to novelty stress and 5-HTP. PYY3-36 (25 nmol/kg) suppressed bethanechol (5 mg/kg, IP)-stimulated FPO and diarrhea. PYY3-36 inhibitory action on FPO stimulated by novel environment was still observed in VIP knockout mice. PYY and PYY336, but not Y1 agonist, reduced the 2-h gastric emptying by 56% and 48% respectively. PYY3-36 inhibitory effects on food intake and FPO were correlated both in fasted mice in light phase and non-fasted mice in dark phase. Y2 mRNA was expressed in both mucosa and muscle layers and Y2-like immunoreactivity was detected in the colonic myenteric plexus. Conclusions: PYY and PYY3-36 potently blocked colonic contractions, fecal output and diarrhea stimulated by stress, 5-HT or cholinergic agonist. PYY inhibitory action is mediated through Y2 receptors present in the colon. These data suggest that Y2 receptors may play a role in the development of constipation.
indicate that an increase in 5-HT2C receptor in hypothalamus and an increase in 5-HT3 receptor in stomach are involved in anorexic effect of cisplatin. M1241 Contractile and Secretory Responses of Luminal Short-Chain Fatty Acids and the Expression of These Receptors, GPR41 and GPR43, in the Human Small and Large Intestines Shin-ichiro Karaki, Hideaki Tazoe, Izumi Kaji, Yasuko Otomo, Takaji Yajima, Atsukazu Kuwahara Background and aim: Short-chain fatty acids (SCFAs) are luminal fermented products from some kinds of dietary fibers especially in large intestine. In animal experiments, luminal SCFAs are reported to induce a variety of physiological effects on intestinal functions including smooth muscle contraction and epithelial secretion. However, there is little evidence of the effects of SCFAs in the human intestine. Therefore, the aim of present study tried to show the physiological effects of SCFAs in the human small and large intestines, and examined the expression of the SCFA receptors, GPR41 and GPR43, possibly mediating the responses. Methods: Human terminal ileum and colon were used in nonpathological region of surgical specimens obtained from the patients of large intestinal cancer following informed consent. Smooth muscle strips with mucosa and without mucosa were tied to an isometric force transducer and suspended in a tissue bath in order to record the smooth muscle contractile activity. To record transepithelial ion transport, mucosa-submucosal preparations were mounted on Ussing flux chamber and short-circuit current (Isc) were measured. For RT-PCR and Western blot analysis for GPR41 and GPR43, total RNA and protein were extracted from the tissues, respectively. Moreover, tissues were fixed in Zamboni's fixative, and 10 µm-thin cryostat sections were made for immunohistochemistry of GPR41 and GPR43. Results: In terminal ileum, propionate-induced transient contractions were observed in circular muscle strip with mucosa, but not without mucosa. This is the first to show the SCFA-induced contractile response in the human small intestine. In Ussing chamber experiments, propionate evoked negative Isc probably by K+ secretion in large intestine, but rarely evoked positive Isc by Cl- secretion as previously reported in rat distal colon. Moreover, mRNA and proteins of both SCFA receptors, GPR41 and GPR43, were detected in the human intestinal mucosa. In immunohistochemistry, these receptors were observed to localize on epithelial cells especially enteroendocrine cells containing peptide YY. Conclusion: The present study first shows the physiological effects of SCFAs and the SCFA receptor expressions in the human intestine. However, there is no direct evidence that GPR41 and/or GPR43 mediate(s) these physiological effects of SCFAs yet. Therefore, further study is necessary to perform in order to reveal whether these receptors really involve with the effects of SCFAs in the human intestine.
M1239 Alterations in Synaptic Transmission in the Enteric Nervous System and Correlation to Motility Defects in CB1 Receptor-Deficient Mice Ian M. Hons, Catherine M. Keenan, Martin Storr, Beat Lutz, Keith A. Sharkey The endocannabinoid (EC) system has been shown to be important in regulating the plasticity of central nervous system synapses. However, the role of ECs on peripheral neurons has not been extensively investigated. The enteric nervous system (ENS) is a self-contained integrative network possessing intrinsic reflexes and rhythmic network activity located in the wall of the gut. The ENS possesses extensive synaptic connectivity, and additionally, these synapses have been shown to be highly plastic in pathological situations. The CB1 receptor is localized in the ENS. Using CB1 receptor-deficient mice, we sought to study the role of ECs in the ENS, and investigated the functional consequences of inactivation of the CB1 receptor gene. Female CB1-/- mice on a C57BL/6N background were bred in Calgary. Comparisons were made between CB1-/- mice (KO) and littermate controls (CB1+/+, WT). The distal ileum was removed and maintained in oxygenated Krebs solution containing nicardipine and scopolamine. The myenteric plexus was dissected from a segment of ileum, and S and AH type neurons were examined by intracellular voltage recordings. Passive and active electrical properties were evaluated as well as synaptic events evoked by focal stimulation of adjacent ganglia. Motility was assessed in WT and KO mice gavaged with Evans blue (5% in 5% gum arabic). 15 min after the gavage, animals were sacrificed and the distance traveled by the dye front was measured and expressed as a percentage of the length of the entire small intestine. Neurons where assigned to 2 groups using electrophysiological and morphological criteria: Dogiel type I S neurons which receive fast excitatory post-synaptic potentials (EPSPs), and AH neurons which posses a characteristic action potential shape, prolonged after-hyperpolarization, and Dogiel type II morphology. Recordings of the basal electrical activity from neurons of KO animals displayed a higher number of neurons receiving spontaneous EPSPs in S type neurons when compared to WT controls (1/6 WT, 6/6 KO, P < 0.05). Measurements of upper GI transit indicated an increase in transit in KO animals (70.3 ± 3.27% WT, 92.1 ± 2.5% KO, P < 0.01). These results are consistent with ECs playing a role in dampening synaptic activity in the ENS. In addition, they indicate that normal gut function requires a basal level of EC production to control activity in enteric neural networks. When disrupted, the ENS becomes spontaneously active, leading to abnormal gut function (i.e. rapid transit). Our data indicate that CB1 receptors and ECs are involved in synaptic communication in the ENS and that this system contributes to normal gut function.
M1242 Pharmacological Characterization of Murine Lower Esophageal Sphincter (LES) Relaxation Scott Carmichael, John Sauvé, David V. Miller, Yong Zhang, William G. Paterson AIM: Because of the availability of genetic knockouts, the mouse has been increasingly utilized as a model for studying GI motility. To date there is limited data available on the physiology of the LES motor function in normal or mutant mice. METHODS: We characterized pharmacologically the putative cholinergic, nitrergic and purinergic innervation of the LES circular smooth muscle from CD-1 mice using isometric tension recording from isolated LES rings. The magnitude of LES relaxation (LESR) was expressed as a percent of maximal LESR induced by 1 µM isoproterenol. Preliminary experiments were also conducted in W/ Wv mutant mice, which lack intramuscular interstitial cells of Cajal (ICCs). RESULTS: Rings of LES muscle from CD-1 mice developed spontaneous tone shortly after suspension in the tissue bath. Electrical field stimulation of intrinsic nerves (100V, 10 Hz, 0.5 ms pulse duration, 5 sec) evoked a reproducible LESR (45.4 ± 6.8 %; n=30) followed by a postrelaxation contraction. This relaxation was abolished by application of the nitric oxide synthase antagonist N(G)-nitro-L-arginine methyl ester (L-NAME; 100 µM). In the presence of L-NAME, cumulative application of atropine (3 µM) restored LESR to a level not significantly different than control (55.8 ± 8.4%). Furthermore, atropine virtually abolished the postrelaxation contraction. Subsequent application of apamin (300 nM), a small conductance Ca2+-activated K+ channel blocker, significantly attenuated the LESR to 20.4 ± 4.2% (p<0.05) suggesting a purinergic component to LESR. Application of the neurokin-2 receptor antagonist MEN-10376 (3 µM) or Substance P tachyphylaxis had no consistent effects on the postrelaxation contraction. LES rings from W/Wv mutant mice developed significantly less spontaneous tone and weaker KCl-induced contraction than strips from control mice. When tone was restored by pre-application of carbachol in LES rings from W/Wv mutant mice, electrical field stimulation induced relaxation that was abolished by L-NAME. CONCLUSIONS: These experiments indicate that in the mouse LES both nitrergic and purinergic inhibitory neurotransmission is present. Acetylcholine appears to be the major excitatory neurotransmitter. In ICC deficient mice, LES muscle contractility is impaired, but nitrergic relaxation is intact, suggesting that ICCs are not required for nitrergic neurotransmission. (Supported by CIHR)
M1240 The Roles of Serotonin 5-HT2C and 3 Receptors in Anorexia and Delayed Gastric Emptying in Cisplatin-Treated Rats Koji Yakabi, Susumu Kurosawa, Tomohisa Hattori, Yuzurihara Mitsutoshi, Shino Ohno Background/Aim: It has been indicated that the acute gastrointestinal tract disorders caused by anticancer drugs involve serotonin secreted from the enterochromaffin cells. Moreover, cerebral serotonin contributes to regulate feeding behavior and energy balance. Mutated serotonin 5-HT2C receptor display leptin-independent hyperphagia that leads to a late onset of obesity (Nature 374, 1995). In order to clarify the mechanisms of cisplatin-induced anorexia, we aimed to investigate the regulation of several 5-HT receptors expression between fasting condition and anorexia induced by anticancer drugs. Methods: Exp.1: Anorexia was induced in S.D. rats by consecutive (1, 2, 4 mg/kg, i.p.) injection of cisplatin for 4 days. The stomach and hypothalamus were collected to determine 5-HT receptor gene expression by RT-PCR. Exp.2: In order to investigate the function of 5-HT receptor in cisplatin-treated rats, 5-HT2C receptor antagonist (SB242084HCl at 10, 30 nmol/rat icv) or 5-HT3 receptor antagonists (granisetoron at doses of 0.1 and 0.5 mg/kg, s.c. or ondansetron at dose of 1 mg/kg, s.c.) was administered to rats 6hr after cisplatin treatment, and the 24hrs food intake and solid gastric emptying were determined. Results: The food intake in cicplatin-treated rats was markedly decreased, compared with saline-treated rats. Moreover, serotonin 5HT2C receptor gene expression in cisplatin-treated rats was increased in hypothalamus, and it was unchanged in stomach. Serotonin 5-HT3 receptor gene expression in cisplatin-treated rats was significantly increased in stomach, while it was not increased in hypothalamus. Administration of SB242084HCl was significantly inhibited the decrease in 16hr food intake in cisplatin-treated rats, whereas it in 5-HT3 receptor antagonist-treated group was unchanged. Granisetron at dose of 0.1 mg/kg and 0.5 mg/kg, and ondansetron inhibited the decrease in solid gastric emptying in cisplatin-treated rats. Conclusion: The results
AGA Abstracts
M1248 Bacterial and Fungal Colonization of Endoscopic Intragastric Balloons Inserted in Patients with Morbid Obesity John A. Karagiannis, Nicoletta G. Mathou, Konstantia D. Paraskeva, Georgios K. Anagnostopoulos, Konstantinos Zografos, Nausika Kontostergiou, Stamatina Konstantinou, Zoi Roussou INTRODUCTION Endoscopic intragastric balloon insertion consists an acceptable method for, at least short-term, weight loss in morbid obese patients. Upon removal of the balloon it was noticed in several patients that it looked macroscopically abnormal with a pattern reminiscent of fungal colonization. AIM OF THE STUDY Aim of the study was to visually
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