Rakkyo fructan as a cryoprotectant for serum-free cryopreservation of mammalian cells.

Cryopreservation refers to the long-term storage of mammalian cells. Mammalian serum is generally used as a cryoprotectant, but is associated with problems including the risk of contamination by pathogens and quality control issues. Therefore, a serum-free cryopreservation method needs to be established. In this study, we focused on rakkyo fructan, a fructose polymer, derived from the Japanese shallot as an alternative factor to serum. Fructan contributes to tolerance to frost and dehydration in plants by stabilizing the plant membrane. However, whether fructan protects mammalian cells against freezing stress remains unknown. The ability of rakkyo fructan to be an alternative cryoprotectant to fetal bovine serum (FBS) was examined in the present study. 2E3-O, a mouse hybridoma, was preserved in rakkyo fructan, was highly viable after being defrosted, and then proliferated rapidly. When rakkyo fructan was combined with dimethylsulfoxide (DMSO), its ability to protect the hybridoma against freezing stress was improved. The rakkyo fructan and DMSO mixture was used in the cryopreservation of the mammalian cell lines CHO-DP12, a producer of recombinant antibodies, and HepG2, human hepatoma cells frequently tested in bio-artificial livers. Following the freezing and thawing processes, CHO-DP12 cells retained their ability to produce recombinant antibodies and as did HepG2 cells for albumin and mRNA expression of cytochrome P450 enzymes. These results indicate that rakkyo fructan is a promising cryoprotectant that prevents mammalian cells from freezing stress similar to FBS.

Gastric emptying of liquid and solid meals at various temperatures: effect of meal temperature for gastric emptying.

BACKGROUND: Patients with functional dyspepsia frequently show delayed gastric emptying, and dietary advice is frequently given for its improvement. If meal temperature influences gastric emptying, advice regarding the meal temperature may become a possible component of dietary therapy. However, little information exists concerning the thermal effect of meals on gastric emptying. The aim of this study was to determine the thermal effect of liquid and solid meals on gastric emptying. METHODS: The gastric emptying of liquid and solid test meals was examined in healthy volunteers (liquid, n = 25, mean age = 35.7 +/- 9.6 years, male-to-female ratio = 22:3; solid, n = 25, mean age = 35.2 +/- 8.8 years, male-to-female ratio = 20:5). Gastric emptying after the ingestion of liquid or solid meals at three different temperatures (4, 37, and 60 degrees C) was investigated with the [(13)C]-labeled acetate breath test. The lag phase time (T (max-calc)) and the half-emptying time (T (1/2)) were calculated from the (13)CO(2) breath excretion curve as indices of gastric emptying. RESULTS: The values of T (max-calc) at 60 degrees C with both the liquid and solid meals were significantly smaller than those at 37 degrees C (P < 0.05). However, there was no difference in the T (1/2) values. In the analysis of the percent excretion of (13)CO(2) in 1 h (% dose/h) data with the liquid meal test in the earlier phase within 30 min, significantly larger values were found at 60 degrees C than at the other temperatures. These findings suggest that a hot meal significantly accelerates gastric emptying. CONCLUSIONS: Meal temperature may be considered as a component of dietary therapy for patients with functional dyspepsia.

Barrett's esophagus in Japanese patients: its prevalence, form, and elongation.

BACKGROUND: Barrett's esophagus is a well-known acquired condition resulting from gastroesophageal reflux disease (GERD). However, it is still unknown whether Barrett's esophagus develops gradually over time in patients with GERD. To address this issue, we investigated the change in the prevalence and length of short-segment Barrett's esophagus (SSBE) over time. METHODS: From January 2005 to March 2007, we enrolled 5338 patients who received upper gastrointestinal endoscopy. Prevalence and length of endoscopically identified SSBE were evaluated within groups divided on the basis of 10-year age intervals. The factors possibly influencing SSBE length such as symptoms, antacid use, and endoscopic findings were also evaluated. Additionally, the length change in 236 patients with histologically confirmed Barrett's esophagus was evaluated over a 2-year follow-up. RESULTS: Of the 5338 enrolled patients, 1997 had SSBE. The prevalence of endoscopically identified SSBE was significantly higher and its length was significantly longer in elderly patients. Multiple regression analysis showed that age, presence of reflux esophagitis, reflux symptoms, and hiatal hernia were positively correlated with SSBE length. Analysis of the 2-year follow-up study of histologically confirmed SSBE revealed significant extension of Barrett's length in 28.0% of 236 patients. Presence of reflux symptoms and hiatal hernia were identified as positive predictors and proton pump inhibitor administration as a negative predictor of SSBE elongation. CONCLUSIONS: Positive predictors for the extension of SSBE were presence of hiatal hernia and reflux symptoms, but not age.

Efficacy of prokinetic agents in improving bowel preparation for colonoscopy.

BACKGROUND AND AIM: Colonoscopy plays an important role in the diagnosis and treatment of gastrointestinal illness in both Western countries and Japan. However, preparative bowel cleansing for colonoscopy is frequently troublesome for elderly and/or constipated patients, since they must drink larger volumes of lavage solution for adequate cleansing. We investigated the use of prokinetic agents for improving the efficacy and tolerability of bowel cleansing prior to colonoscopy. METHODS: 613 patients were divided into two groups according to oral lavage solution used (polyethylene glycol or magnesium citrate), and were further randomized to receive either vehicle (100 ml water) alone, vehicle with 5 mg mosapride citrate, or vehicle with 50 mg itopride hydrochloride 30 min before administration of lavage solution. Experimental parameters included bowel cleansing quality, times to first defecation and completion of bowel cleansing, and incidence of uncomfortable abdominal symptoms during colonoscopy preparation. RESULTS: Administration of mosapride citrate or itopride hydrochloride prior to oral lavage solution did not significantly improve bowel cleansing quality. However, statistically significantly fewer uncomfortable abdominal symptoms were found in patients who received mosapride citrate or itopride hydrochloride versus vehicle alone. CONCLUSION: Prokinetic agents effectively decreased the incidence of uncomfortable abdominal symptoms experienced during colonoscopy preparation.

REG Ialpha protein expression in Barrett's esophagus.

BACKGROUND AND AIM: Accelerated cellular proliferation in Barrett's esophagus has been implicated in Barrett's elongation and malignant transformation. Therefore, growth factors may play important roles in the pathophysiology of Barrett's esophagus. Regenerating gene (REG), an epithelial growth factor, has been reported to link mucosal inflammation and subsequent carcinogenesis in the gastrointestinal tract. The aim of this study was to investigate whether REG is expressed in Barrett's esophagus and to elucidate the relationship between REG protein expression and clinicopathological factors of Barrett's esophagus. METHODS: Between July 2003 and June 2004, 266 patients with endoscopically and histologically proven Barrett's esophagus were enrolled in this study. Before endoscopic examination, all participants were requested to answer structured questionnaires on gastroesophageal reflux symptoms and drugs usage. Mucin phenotype, cyclooxygenase-2 expression, cellular proliferation, apoptosis and REG Ialpha protein expression were investigated in the biopsy samples taken from Barrett's esophagus. Clinicopathological factors that correlated with REG Ialpha protein expression in patients with Barrett's esophagus were evaluated using multivariate logistic regression analysis. RESULTS: REG Ialpha protein expression was observed in 48 (18.0%) of 266 patients with Barrett's esophagus by immunohistochemistry. Newly developed squamous re-epithelialization of Barrett's esophagus at biopsy sites, presence of hiatal hernia and aging were shown to correlate with REG Ialpha protein expression. CONCLUSIONS: The present study is the first to show REG expression in Barrett's esophagus. Expression of REG Ialpha was more frequently observed in patients who showed squamous re-epithelialization of Barrett's esophagus at biopsy sites.

What is the clinical significance of stromal angiogenesis in Barrett's esophagus?

BACKGROUND: Enriched blood vessels occur in the stroma of Barrett's esophagus and are related to the ease of tumor invasion in the early stage of the cancer. Hypoxia-inducible factor (HIF) is closely related with angiogenesis through expression of vascular endothelial growth factor. The aim of the present study was to determine the predictors for angiogenesis in Barrett's esophagus and evaluate their clinical significance. METHODS: Between December 2003 and May 2004, 209 patients with endoscopically and histologically proven Barrett's esophagus were enrolled. Before endoscopic examination, all participants answered structured questionnaires for gastroesophageal reflux symptoms and drug usage. HIF-1alpha and COX-2 protein expressions, cellular proliferation, and apoptosis were investigated immunohistochemically in biopsy samples taken from the esophagus of each patient. The degree of angiogenesis was determined by CD34 immunostaining analysis. Predictors for angiogenesis were evaluated with multivariate logistic regression analysis. RESULTS: Sixty (28.7%) of the 209 enrolled patients with Barrett's esophagus had a high CD34 score. Factors proven as positive predictors for a high CD34 score were presence of gastroesophageal reflux symptoms, reflux esophagitis, COX-2 protein expression, and the proliferating cell nuclear antigen (PCNA) index. Administration of proton-pump inhibitors and HIF-1alpha protein expression were not predictors. CONCLUSIONS: Reflux esophagitis and gastroesophageal reflux symptoms were positive predictors for enriched angiogenesis in the stromal portion of Barrett's esophagus, which has malignant potential because the epithelial cells express COX-2 and have accelerated cellular proliferation.

[Diagnosis and treatment of NSAIDs induced colonic mucosal lesions].

NSAIDs induced colonic mucosal lesions have been reported to be found in about 3% of chronic NSAIDs users in Japan, suggesting that a particular attention should be paid for the clinical management of NSAIDs users. Differential diagnosis of these lesions is considered to be difficult only by the morphological examination since endoscopic images of the lesions show very various types such as ulcerative and colitis type. Therefore, the diagnostic criteria proposed by Iida et al. play an important role for the confirmation of the clinical diagnosis. Treatment for the lesions is principally the discontinuation of NSAIDs administration since many possibly agents have not been fully evidenced the effect by clinical cohort study. The establishment of the guideline for clinical management of patients with NSAIDs induced colonic mucosal lesions is sincerely required.

Increased expression of midkine in the rat colon during healing of experimental colitis.

Midkine (MK) is a unique growth and differentiation factor that modulates the proliferation and migration of various cells; however, little is known regarding its relationship to intestinal diseases. The aim of this study was to investigate MK expression and its role in dextran sulfate sodium (DSS)-induced colitis in rats. The expressions of MK, receptor-like protein-tyrosine phosphatase (RPTP)-beta, and proinflammatory cytokines were examined in rat colonic tissues after the development of DSS-induced colitis using Northern blotting, immunohistochemistry, and laser-capture microdissection (LCM) coupled with RT-PCR. The effects of MK on the migration of intestinal epithelial cells (IEC-6) were also evaluated in vitro using an intestinal wound repair model. MK expression was significantly increased in damaged colonic mucosa, mainly from day 3 to day 5 after the end of DSS administration, with abundant MK immunoreactive signals detected in submucosal fibroblasts. Expressions of proinflammatory cytokines were most strongly induced on day 1, which preceded the augmentation of MK expression. Results of LCM coupled with RT-PCR clearly indicated RPTP-beta expression in colonic epithelial cells. The migration assay showed that wound repair in the MK-treated groups was accelerated dose dependently. The present results showed for the first time that intestinal inflammation upregulates the MK-RPTP-beta system, which may stimulate mucosal regeneration during the process of healing of colitis. Additional investigations regarding the role of MK may contribute to the development of new options for the treatment of inflammatory bowel diseases.

Ulinastatin shows preventive effect on post-endoscopic retrograde cholangiopancreatography pancreatitis in a multicenter prospective randomized study.

BACKGROUND AND AIM: Endoscopic retrograde cholangiopancreatography (ERCP) is a useful diagnostic and therapeutic procedure; however, ERCP occasionally causes post-ERCP pancreatitis. The administration of gabexate mesilate has been reported to be effective for the prevention for post-ERCP pancreatitis when given during and after the procedure. The aim of the present study was to investigate the preventive effect of the novel protease inhibitor ulinastatin on post-ERCP pancreatitis. METHODS: One hundred and thirty-nine patients who underwent the ERCP procedure were studied. These patients were randomly divided into three groups based on the agent and dose given during and following the ERCP procedure: gabexate mesilate (900 mg), high-dose ulinastatin (450 000 units) and low-dose ulinastatin (150 000 units). Serum amylase, interleukin (IL)-6 and IL-8 levels and plasma polymorphonuclear leukocyte elastase (PMN-E) activity were measured after ERCP. In addition, post-ERCP hyperamylasemia and post-ERCP pancreatitis were recorded. RESULTS: There were no significant differences in serum amylase, IL-6 and IL-8 levels and PMN-E activity after ERCP procedure between the three groups. Post-ERCP pancreatitis was observed in two (4.3%), three (6.5%) and four (8.5%) cases in the gabexate mesilate, high-dose ulinastatin and low-dose ulinastatin groups, respectively. Multiple logistic regression analysis showed that the addition of endoscopic sphincterotomy during the ERCP procedure was the only significant risk factor for the development of post-ERCP hyperamylasemia and post-ERCP pancreatitis (P = 0.03 and P = 0.04, respectively), but there was no significant difference in the occurrence of post-ERCP hyperamylasemia and post-ERCP pancreatitis between the three groups receiving different preventative treatments. CONCLUSION: The administration of low- and high-dose ulinastatin has similar effects to high-dose gabexate in the prevention of post-ERCP pancreatitis.

Localization of calcitonin gene-related peptide receptors in rat gastric mucosa.

The location of calcitonin gene-related peptide (CGRP) receptors in the rat stomach has not been elucidated. It was recently reported that the CGRP receptor is formed when a calcitonin-receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1 are co-expressed on the cell membrane. The aim of this study was to determine the location and the role of CGRP receptors in the rat gastric mucosa. Gene expressions of CRLR and RAMP1 were investigated by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization. Immunohistochemical stainings for CGRP, somatostatin, gastrin, and chromogranin A were performed. Gastric endocrine cells were collected by counterflow-elutriation and their responses to CGRP were studied. CRLR and RAMP1 mRNA was expressed mainly in small gastric epithelial cells in the pyloric glands. The mRNA expression had a similar distribution to that of D cells. In cultured gastric endocrine cells, CGRP enhanced somatostatin production, while it inhibited the secretion of histamine and gastrin. Our results suggest that CGRP receptors are expressed in D cells in the rat gastric mucosa and control production and secretion of somatostatin.