Endoscopic submucosal dissection as excisional biopsy for anorectal malignant melanoma: A case report.

BACKGROUND: Anorectal malignant melanoma (AMM) is a rare disorder with an extremely poor prognosis. Although there is currently no consensus on the treatment methods for AMM, surgical procedures have been the most common treatment methods used until now. We recently encountered a case of AMM that we diagnosed using endoscopic submucosal dissection (ESD). To our knowledge, this is the first case of ESD for AMM, suggesting that ESD can potentially be a diagnostic and treatment method for AMM. CASE SUMMARY: A 77-year-old woman visited our hospital with a chief complaint of anal bleeding and a palpable rectal mass. Colonoscopy revealed a 20-mm protruded lesion in the lower rectum. After obtaining biopsy specimens from the lesion, although a malignant rectal tumor was suspected, a definitive diagnosis was not made. Endoscopic ultrasonography revealed tumor invasion into the submucosal layer but not the muscular layer. Therefore, we performed an excisional biopsy using ESD. Immunohistochemical examination of the ESD-resected specimen revealed tumor cells positive for Human Melanin Black-45, Melan-A, and S-100. Moreover, the tumor cells lacked melanin pigment; thus, a diagnosis of amelanotic AMM was made. Although the AMM had massively invaded the submucosal layer and both lymphatic and venous invasion were present, we closely monitored the patient without any additional therapy on the basis of her request. Six months after ESD, local recurrence was detected, and the patient consented to wide local excision. CONCLUSION: It is suggested that ESD is a potential diagnostic and treatment method for AMM.

Gastric adenocarcinoma of fundic gland type spreading to heterotopic gastric glands.

Herein, we present a case of gastric adenocarcinoma of fundic gland type (GA-FG) spreading to heterotopic gastric glands (HGG) in the submucosa. A 58-year-old man with epigastric pain was referred to our hospital and underwent an esophagogastroduodenoscopy. A Borrmann type II gastric cancer at the antrum and a 10 mm submucosal tumor-like lesion in the lesser curvature of the upper third of the stomach were detected. Histological examination of the biopsy specimens obtained from the submucosal tumor-like lesion suggested a GA-FG. Therefore, endoscopic submucosal dissection was performed as excisional biopsy, and histopathological examination of the resected specimen confirmed a GA-FG and HGG proximal to the GA-FG. Although the GA-FG invaded the submucosal layer slightly, the submucosal lesion of the GA-FG had a poor stromal reaction and was located just above the HGG in the submucosa. Therefore, we finally diagnosed the lesion as a GA-FG invading the submucosal layer by spreading to HGG.

A case of small intestinal intussusception caused by anisakiasis diagnosed and treated by upper gastrointestinal endoscopy.

A 35-year-old man attended our hospital with complaining of epigastric pain and vomiting. He was diagnosed with bowel intussusception based on the target sign revealed in the upper jejunum by abdominal computed tomography. However, the cause of the intussusception was not clear. Insertion of an endoscope into the jejunum revealed prominent edema in the upper part of the jejunum, and Anisakis simplex was identified at the site and removed. Symptoms rapidly improved after endoscopic treatment. We report this case because surgery was avoidable and because we are aware of no previous reports of small intestinal intussusception caused by anisakiasis that has been diagnosed and treated endoscopically.

A case of liver abscess caused by appendicitis which was considered to be drained from hepatobronchial fistula.

A 52-year-old man was referred to our hospital complaining of right lower abdominal pain. He was diagnosed with appendicitis complicated with a liver abscess and underwent an appendectomy. After antibiotic treatment following surgery, the liver abscess penetrated the right lung, which was considered to be drained from a hepatobronchial fistula. Due to the effect of drainage, the liver abscess immediately improved and the patient was subsequently discharged.

Carbon monoxide promotes gastric wound healing in mice via the protein kinase C pathway.

Previous studies have shown that carbon monoxide (CO) is involved in a variety of physiological and pathophysiological processes including anti-inflammatory, anti-apoptotic and anti-oxidant responses. However, it remains unclear whether CO promotes gastric ulcer healing. In the present study, we evaluated the efficacy of CO-saturated saline in the treatment of gastric ulcers and its underlying mechanism. Acute gastric ulcers were induced in C57BL/6 male mice using acetic acid. A CO-saturated solution was prepared by bubbling 50% CO gas into saline. To investigate the effect of CO on gastric mucosal healing, CO solution was orally administrated twice a day beginning on day 3 after the induction of gastric ulcer. Mice were sacrificed on day 7 after ulcer induction. The stomach was removed, and the ulcerated lesions were measured. In vitro wound healing assays were used to determine the mechanism of action of CO in the restoration of murine gastric epithelial cells. The oral administration of CO solution accelerated the gastric ulcer healing by promoting re-epithelialization. Furthermore, the wound healing assay performed using the murine gastric epithelial cells revealed that the CO-saturated medium enhanced cell migration through the activation of protein kinase C (PKC). Based on these data, CO may represent a novel therapeutic approach for the treatment of gastric mucosal injuries.

Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic acid-induced small intestinal apoptosis, a hallmark of acetylsalicylic acid-induced enteropathy.

Daikenchuto, a Kampo medicine, regulates intestinal fibrosis associated with decreasing expression of heat shock protein 47 and collagen content in a rat colitis model.

Heat shock protein (HSP) 47 may play an important role in the pathogenesis of intestinal fibrosis. Daikenchuto (DKT), a traditional Japanese herbal (Kampo) medicine, has been reported to ameliorate intestinal inflammation. The aims of this study were to determine time-course profiles of several parameters of fibrosis in a rat model, to confirm the HSP47-expressing cells in the colon, and finally to evaluate DKT's effects on intestinal fibrosis. Colitis was induced in male Wistar rats weighing 200 g using an enema of trinitrobenzene sulfonic acid (TNBS). HSP47 localization was determined by immunohistochemistry. Colonic inflammation and fibrosis were assessed by macroscopic, histological, morphometric, and immunohistochemical analyses. Colonic mRNA expression of transforming growth factor ß1 (TGF-ß1), HSP47, and collagen type I were assessed by real time-polymerase chain reaction (PCR). DKT was administered orally once a day from 8 to 14 d after TNBS administration. The colon was removed on the 15th day. HSP47 immunoreactivity was coexpressed with α-smooth muscle actin-positive cells located in the subepithelial space. Intracolonic administration of TNBS resulted in grossly visible ulcers. Colonic inflammation persisted for 6 weeks, and fibrosis persisted for 4 weeks after cessation of TNBS treatment. The expression levels of mRNA and proteins for TGF-ß1, HSP47, and collagen I were elevated in colonic mucosa treated with TNBS. These fibrosis markers indicated that DKT treatment significantly inhibited TNBS-induced fibrosis. These findings suggest that DKT reduces intestinal fibrosis associated with decreasing expression of HSP47 and collagen content in the intestine.

Role of tumor necrosis factor-α in the pathogenesis of indomethacin-induced small intestinal injury in mice.

The pathogenesis of small intestinal damage caused by non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is still unclear. For this reason, there is currently no therapeutic strategy for ameliorating such damage. On the other hand, molecular treatment strategies targeting tumor necrosis factor (TNF)-α exert beneficial effects on intestinal lesions in patients with inflammatory bowel disease (IBD). To clarify the participation of TNF-α in NSAID-induced small intestinal damage, we investigated the effects of indomethacin administration in mice with targeted deletion of the TNF-α gene. Indomethacin (10 mg/kg) was administered subcutaneously to male C57BL/6 (wild-type: WT) mice and TNF-α-deficient (TNF-α-/-) mice to induce small intestinal damage. The ulcer score, the tissue-associated myeloperoxidase (MPO) activity as an index of neutrophil infiltration, and the expression of keratinocyte chemoattractant (KC) mRNA in the small intestinal mucosa were measured. In addition, we performed a TUNEL assay to evaluate indomethacin-induced apoptosis of intestinal epithelial cells and measured the expression of caspase-3 protein and Bcl-2 mRNA. The ulcer score, MPO activity, and expression of KC mRNA were significantly increased after indomethacin administration. These increases were significantly inhibited in TNF-α-/- mice compared with WT mice. Apoptotic cells were observed by the TUNEL assay in the area of the ulcerative lesion, and they were significantly fewer in TNF-α-/- mice compared with WT mice. The expression of cleaved caspase-3 protein was induced by indomethacin administration, and significantly inhibited in TNF-α-/- mice compared with that of WT mice. The expression level of Bcl-2 mRNA in indomethacin-treated TNF-α-/- mice was significantly higher than that in WT mice. TNF-α plays an important role in the pathogenesis of indomethacin-induced small intestinal damage. These results suggest that TNF-α could become a new therapeutic target for NSAID-induced small intestinal damage.

Carbon monoxide liberated from carbon monoxide-releasing molecule exerts an anti-inflammatory effect on dextran sulfate sodium-induced colitis in mice.

BACKGROUND: Endogenous carbon monoxide (CO) is one of the three products of heme degradation by heme oxygenase-1 (HO-1) and exerts novel anti-inflammatory and anti-apoptotic effects as a gaseous second messenger. The purpose of this investigation was to determine whether exogenous CO could modulate intestinal inflammation. METHODS: Acute colitis was induced with 2% DSS in male C57BL/6 mice. CO-releasing molecule-2 (CORM-2; tricarbonyldichlororuthenium(II) dimer) was intraperitoneally administered twice daily and the disease activity index (DAI) was determined. We measured tissue-associated myeloperoxidase (MPO) activity as an index of neutrophil infiltration, and the production of keratinocyte chemoattractant (KC) and tumor necrosis factor-α (TNF-α) protein in the intestinal mucosa. In an in-vitro study, young adult mouse colonic epithelial (YAMC) cells were incubated with TNF-α, and KC mRNA/protein expression and nuclear translocation of nuclear factor-kappa B (NF-κB) were measured with or without CORM-2 treatment. RESULTS: After DSS administration, DAI score increased in a time-dependent manner, and this increase was ameliorated by CORM-2 treatment. Increases in MPO activity and in the production of KC and TNF-α after DSS administration were significantly inhibited by CORM-2. TNF-α-induced KC production in YAMC cells was also inhibited by CORM-2 treatment. Further, nuclear translocation of NF-κB in YAMC cells was inhibited by CORM-2. CONCLUSION: CORM-liberated CO significantly inhibited inflammatory response in murine colitis by inhibition of cytokine production in the colonic epithelium. These results suggest that CO could become a new therapeutic molecule for inflammatory bowel disease.

Endogenous hydrogen sulfide is an anti-inflammatory molecule in dextran sodium sulfate-induced colitis in mice.

BACKGROUND: Endogenous hydrogen sulfide (H(2)S) is increasingly being recognized as an important gaseous physiological mediator. Accumulating evidence shows the functions of H(2)S in various models of disease, but rarely in colitis. In this study, we investigated the role of endogenous H(2)S in a dextran sodium sulfate (DSS)-induced colitis model. METHODS: Acute colitis was induced using 8% DSS in male BALB/c mice. The mRNA expression of cystathionine γ-lyase (CSE), the primary synthetase of H(2)S in the gastrointestinal tract, and cystathionine-ß-synthetase (CBS) was measured by real-time RT-PCR. The amount of H(2)S in the colonic mucosa was measured by gas chromatography. Colitis severity was evaluated clinically, histologically, and biochemically under the condition of co-treatment with DL-propargylglycine (PAG), an irreversible CSE inhibitor, and sodium sulfide (Na(2)S), an H(2)S donor. RESULTS: The mRNA expression levels of CSE and CBS, and the H(2)S content in the colonic mucosa were increased with time after DSS administration. The disease activity index, which was determined by weight loss, stool consistency, and intestinal bleeding, increased after DSS administration. PAG significantly enhanced the increase in the disease activity index scores. PAG also significantly increased tissue-associated myeloperoxidase activity and thiobarbituric acid-reactive substances in the inflamed mucosa. Moreover, Na(2)S counteracted these effects of PAG. CONCLUSIONS: Taken together, the results indicated that the inhibition of endogenous H(2)S generation caused the deterioration of DSS-induced colitis. We conclude that physiological H(2)S might act as an anti-inflammatory molecule in colitis.

Increased expression of microRNA in the inflamed colonic mucosa of patients with active ulcerative colitis.

BACKGROUND AND AIMS: MicroRNA (miRNA) are endogenous, approximately 22-nucleotide non-coding RNA that suppress gene expression at post-transcriptional levels by binding to the 3'-untranslated region of specific mRNA targets through base-pairing. It has been recently reported that miRNA have critical functions in key biological processes such as cell proliferation and cell death in various cancer cells. However, the relationship between intestinal inflammation and miRNA expression remains unclear. In the present study, we used microarray technology to identify miRNA induced in the colonic mucosa of patients with active ulcerative colitis (UC). METHODS: Two colonic biopsy specimens from patients with active stage (>Matts grade 2) of UC under colonoscopy and two colonic biopsy specimens from healthy volunteers were obtained for gene expression profiles. Total RNA was extracted, and miRNA expression profiles were investigated using miRNA Microarray. Subsequently, to confirm the result of the Microarray investigation, we checked the expression of several selected miRNA using real-time polymerase chain reaction (PCR) in 12 colonic biopsy specimens from patients with active UC under colonoscopy and 12 specimens from the healthy volunteers. RESULTS: In the microarray study, the expression of several miRNA was upregulated in the colonic mucosa of patients with active UC. Furthermore, two miRNA (miR-21, miR-155) were selected in the study using real-time PCR. CONCLUSION: Upregulated miRNA may be responsible for the development of intestinal inflammation in UC.

Reactive oxygen species-quenching and anti-apoptotic effect of polaprezinc on indomethacin-induced small intestinal epithelial cell injury.

BACKGROUND: To protect the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs is one of the critical issues in the field of gastroenterology. Polaprezinc (PZ), a gastric muco-protecting agent, has been widely used for the treatment of gastric ulcer and gastritis for its unique effects, such as its strong reactive oxygen species (ROS)-quenching effect. The aim of this study was to clarify the mechanism by which indomethacin-induced small intestinal mucosal injury occurs, by using a rat intestinal epithelial cell line (RIE-1). In addition, the protective role of PZ and the possible mechanism of its effect on indomethacin-induced small intestinal injury were investigated. METHODS: Cell death was evaluated by methyl thiazolyl tetrazolium (MTT) assay and a double-staining method with Hoechst33342 dye and propidium iodide. Indomethacin-induced ROS production was evaluated by detecting the oxidation of a redox-sensitive fluorogenic probe, RedoxSensor, and the oxidation of cysteine residues of proteins (protein S oxidation). The activation of cytochrome c, smac/DIABLO, and caspase-3 was assessed by western blotting. In some experiments, PZ or its components, L: -carnosine and zinc, were used. RESULTS: We found that indomethacin caused apoptosis in RIE-1 cells in a dose- and time-dependent manner. Indomethacin also induced ROS production and an increase in the protein S oxidation of RIE-1. Pretreatment of RIE-1 with PZ or zinc sulfate, but not L: -carnosine, significantly reduced the indomethacin-induced apoptosis. PZ prevented ROS production and the increase in protein S-oxidation. PZ inhibited indomethacin-induced cytochrome c and smac/DIABLO release and subsequent caspase-3 activation. CONCLUSIONS: The protective effect of PZ on indomethacin-induced small intestinal injury may be dependent on its ROS-quenching effect.

Inhalation of carbon monoxide ameliorates TNBS-induced colitis in mice through the inhibition of TNF-α expression.

BACKGROUND: Carbon monoxide (CO), long considered a toxic gas, has recently been shown to mediate anti-inflammatory effects in various animal models. The aim of this study was to investigate whether the inhalation of CO ameliorated 2,4,6-trinitrobenzine sulfonic acid (TNBS)-induced colitis in mice. METHODS: The CO treatment group was exposed to CO gas at a concentration of 200 ppm in a closed cage starting on the day when TNBS was administered and throughout the remaining study period. The distal colon was removed, and ulcerative lesions were subsequently evaluated with macroscopic damage scores. Furthermore, thiobarbituric acid (TBA)-reactive substances and tissue-associated myeloperoxidase (MPO) activity in colonic mucosa were measured as indices of lipid peroxidation and neutrophil infiltration. The expressions of TNF-α in colonic mucosa were also measured by enzyme-linked immunosorbent assay. In additional experiments in vitro, CD4(+) T cells isolated from the spleen were stimulated with anti-CD3/CD28 Ab, and the cells and supernatants were collected and evaluated for TNF-α expression. RESULTS: The increased colonic damage after TNBS administration was significantly inhibited by the treatment with CO. Furthermore, CO significantly inhibited the increases in TBA-reactive substances, MPO activity and TNF-α production in colonic mucosa after the induction of TNBS colitis. In CD4(+) T cells isolated from mice treated with CO inhalation, the production of TNF-α was significantly inhibited. CONCLUSIONS: The inhalation of CO protected mice from developing intestinal inflammation. Based on these data, the beneficial effects of CO in a murine colitis model may be attributed to its anti-inflammatory properties.

Carbon monoxide enhance colonic epithelial restitution via FGF15 derived from colonic myofibroblasts.

Carbon monoxide (CO) has been reported to ameliorate colonic inflammation and improve experimental colitis. It is well known that mucosal restitution is important to improve colitis as well as reduction of mucosal inflammation. However, it has not been clear whether CO effects to colonic mucosal restitution or not. In general, colonic myofibroblast (MF) has been reported to play an important role of colonic epithelial cell restitution via constitutive secretion of TGF-beta. In this study, we showed CO (supplied by CO-releasing molecule; CORM) treated MF conditioned medium enhanced colonic epithelial cell (YAMC) restitution and we determined gene expression in colonic MF treated with CO using microRNA. The microRNA array suggested that miR-710 was significantly reduced in MF by CO treatment and the target gene of miR-710 is determined to fibroblast growth factor (FGF)15. The CO treated MF conditioned medium which FGF15 expression was silenced extinguished the enhancement effect of epithelial cell restitution. Our findings demonstrate that CO treatment to MF increased FGF15 expression via inhibition of miR-710 and FGF15 enhanced colonic epithelial cell restitution.

Heat-shock protein 70-overexpressing gastric epithelial cells are resistant to indomethacin-induced apoptosis.

BACKGROUND/AIMS: Protecting intestinal mucosa from nonsteroidal anti-inflammatory drugs is still an unsolved problem. It has been revealed that apoptosis in epithelial cells as a result of mitochondrial injury is an important pathogenesis in indomethacin-induced gastric mucosal injury. In this study, we revealed the effect of overexpressed heat-shock protein 70 (HSP70) in indomethacin-induced apoptosis and oxidative stress. METHODS: HSP70-overexpressing rat gastric mucosal cells (7018-RGM-1 cells) and control cells (pBK-CMV-12 cells) were used and treated with 0-500 microM of indomethacin for 24 h. Cell viability and cytotoxity were measured by a WST-8 assay and a lactate dehydrogenase release assay, respectively. Apoptosis was observed by fluorescence microscopy staining with Hoechst 33342 and propidium iodide. The expression of Bcl-2 family proteins, activation of caspase-3, and 4-hydroxy-2-nonenal (4-HNE)-modified proteins were assessed by Western blot analysis. RESULTS: Indomethacin caused apoptosis of gastric epithelial cells. The 7018-RGM-1 cells survived significantly after indomethacin treatment compared to the control cells. The increase in pro-apoptotic Bad proteins, the decrease in anti-apoptotic Bcl-2 proteins, and caspase activation were all suppressed in the 7018-RGM-1 cells. A lower level of indomethacin-induced 4-HNE-modification was detected in the 7018-RGM-1 cells than in the control cells. CONCLUSION: Overexpressed HSP70 may potentiate resistance to apoptosis and oxidative stress in indomethacin-induced gastric epithelial cell injury.

Involvement of reactive oxygen species in indomethacin-induced apoptosis of small intestinal epithelial cells.

BACKGROUND: The precise pathogenic mechanism of nonsteroidal antiinflammatory drug-induced small intestinal injury is still unknown. In the present study, we investigated the mechanism by which indomethacin induced mucosal injury by using an in vitro model of small intestine. METHODS: The colon cancer cell line Caco-2, exhibiting a small intestinal phenotype starting as a crypt cell and differentiating to a villous phenotype, and RIE, a rat intestinal epithelial cell line, were employed. Indomethacin was added to differentiated the Caco-2 and RIE monolayer, and cell death was quantified by MTT assay and LDH release in the cell culture supernatant. Indomethacin-induced cell death was also qualified by fluorescent probes under the fluorescent microscope. As a functional study, the permeability of the Caco-2 monolayer was assessed by measuring transepithelial electrical resistance (TEER) and the flux of FITC-conjugated dextran across the monolayer. Indomethacin-induced reactive oxygen species production in Caco-2 and RIE was evaluated by redoxsensitive fluorogenic probes using a fluorometer. In some experiments, antioxidants were used to clarify the role of reactive oxygen species on indomethacin-induced Caco-2 cell death. RESULTS: Indomethacin caused cell death (mainly apoptosis) of Caco-2 and RIE in a dose-and time-dependent manner that was correlated with increased permeability of the Caco-2 monolayer. Exposure of Caco-2 and RIE with indomethacin also resulted in a significant reactive oxygen species production that was inhibited by the pretreatment of these cells with antioxidants. CONCLUSIONS: Taken together, reactive oxygen species production is one of the mechanisms by which indomethacin induced small intestinal injury.

Increased intestinal expression of heme oxygenase-1 and its localization in patients with ulcerative colitis.

BACKGROUND: Heme oxygenase-1 (HO-1) is regarded as a sensitive and reliable indicator of cellular oxidative stress. Two end products of heme degradation, carbon monoxide (CO) and bilirubin, are involved in the protective role of HO-1 against oxidative injury. We have demonstrated enhanced expression of this enzyme and increased concentration of CO in experimental models of colitis, but the role of HO-1 in patients with ulcerative colitis (UC) has not been extensively investigated. The aim of the present study was to determine the intestinal levels and localization of ho-1 mRNA and HO-1 protein in patients with UC. METHODS: Eighteen patients with UC and 13 patients with colon cancer were prospectively selected from subjects who underwent colonoscopy. Biopsy specimens were obtained from the inflamed mucosa of UC patients and from the normal mucosa at least 5 cm from the margin of carcinoma. The expression of ho-1 mRNA was assayed by real-time polymerase chain reaction (PCR). The colonic expression of HO-1 was determined by immunohistochemistry and western blotting using a monoclonal antibody against HO-1. RESULTS: The expression of ho-1 mRNA and HO-1 protein was significantly increased in the colonic mucosa of patients with active UC compared with normal mucosa. In the patients with active UC, mononuclear cells in the submucosa of the colon were positive for HO-1, and there was negligible staining in the epithelial cells. CONCLUSION: The present findings are evidence of the induction of HO-1 in the colon of UC patients.