Culture substrates made of elastomeric micro-tripod arrays for long-term expansion of human pluripotent stem cells.

Culturing and expansion of human pluripotent stem cells (hPSCs) depend on the chemical and physical properties of the substrates. Gel-coated substrates providing low stiffness are commonly used for long-term pluripotency maintenance of hPSCs. We previously reported that gelatin nanofibrous substrates also allow long-term culturing of hPSCs, suggesting the importance of three-dimensional (3D) organization of extracellular matrix proteins. To further evaluate the significance of 3D features and material stiffness, we fabricated elastomeric micro-tripod arrays (MTAs) and maintained hPSC propagation over 10 passages without observing the pluripotency loss or the development of chromosomal abnormality. We also found that the hPSC colonies on MTAs were more rounded than those on gel-coated flat substrates. Theoretical analyses suggested that the effective stiffness of elastomeric MTAs is comparable to that of gel-coated substrates and that the effect of structural anisotropy is negligible. Cells on MTAs benefit from enhanced diffusion underneath the hPSC colonies as well as enzyme-free detachment from the substrate during passage.

Adhesion patterning by a novel air-lock technique enables localization and in-situ real-time imaging of reprogramming events in one-to-one electrofused hybrids.

Although fusion of somatic cells with embryonic stem (ES) cells has been shown to induce reprogramming, single-cell level details of the transitory phenotypic changes that occur during fusion-based reprogramming are still lacking. Our group previously reported on the technique of one-to-one electrofusion via micro-slits in a microfluidic platform. In this study, we focused on developing a novel air-lock patterning technique for creating localized adhesion zones around the micro-slits for cell localization and real-time imaging of post fusion events with a single-cell resolution. Mouse embryonic fibroblasts (MEF) were fused individually with mouse ES cells using a polydimethylsiloxane (PDMS) fusion chip consisting of two feeder channels with a separating wall containing an array of micro-slits (slit width ∼3 µm) at a regular spacing. ES cells and MEFs were introduced separately into the channels, juxtaposed on the micro-slits by dielectrophoresis and fused one-to-one by a pulse voltage. To localize fused cells for on-chip culture and time-lapse microscopy, we implemented a two-step approach of air-lock bovine serum albumin patterning and Matrigel coating to create localized adhesion areas around the micro-slits. As a result of time-lapse imaging, we could determine that cell division occurs within 24 h after fusion, much earlier than the 2-3 days reported by earlier studies. Remarkably, Oct4-GFP (Green Fluorescent Protein) was confirmed after 25 h of fusion and thereafter stably expressed by daughter cells of fused cells. Thus, integrated into our high-yield electrofusion platform, the technique of air-lock assisted adhesion patterning enables a single-cell level tracking of fused cells to highlight cell-level dynamics during fusion-based reprogramming.

Development of low-fluorescence thick photoresist for high-aspect-ratio microstructure in bio-application.

In this study, we propose and evaluate a novel low-auto-fluorescence photoresist (SJI photoresist) for bio-application, e.g., in gene analysis and cell assay. The spin-coated SJI photoresist has a wide thickness range of ten to several hundred micrometers, and photoresist microstructures with an aspect ratio of over 7 and micropatterns of less than 2 µm are successfully fabricated. The emission spectrum intensity of the SJI photoresist is found to be over 80% less than that of the widely used SU-8 photoresist. To evaluate the validity of using the proposed photoresist in bio-application for fluorescence observation, we demonstrate a chromosome extension device composed of the SJI photoresist. The normalized contrast ratio of the SJI photoresist exhibits a 50% improvement over that of the SU-8 photoresist; thus, the SJI photoresist is a versatile tool for bio-application.

Interactions among gastric somatostatin, interleukin-8 and mucosal inflammation in Helicobacter pylori-positive peptic ulcer patients.

BACKGROUND: To investigate whether Helicobacter pylori infection, but not drugs, affects gastric somatostatin, interleukin-8 (IL-8), histological inflammation through eradication therapy, and interactions among these parameters. METHODS: Twenty-eight H. pylori-positive patients (21 males; mean age 47.0 years) with either gastric ulcer (GU: n = 11) or duodenal ulcer (n = 17) diagnosed endoscopically were treated with dual therapy. Eradication was defined as negative microbiologic tests and 13C-urea breath test. Levels of antral and gastric juice somatostatin and mucosal IL-8 were measured by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Histology was assessed by the Sydney system. RESULTS: H. pylori was eradicated in 15 patients (10 males, 6 GU) out of 28 (54%). The patients' backgrounds did not affect the eradication of H. pylori. Successes in eradication significantly increased antral and juice somatostatin contents, and dramatically decreased IL-8 levels and histological gastritis. In contrast, persistent H. pylori infection did not affect somatostatin and histological gastritis. An inverse correlation was present between changes in somatostatin levels and histological activity. No relationship was observed in changed values between antral somatostatin and IL-8. CONCLUSIONS: These results indicate that eradication of H. pylori, but not the drugs used, induced an increase in somatostatin levels in the antrum and gastric juice, suggesting a close relationship between H. pylori and gastric somatostatin regulation. A close correlation between an increase in gastric somatostatin levels and the normalization of histological activity was present, suggesting that certain peptide-immune interactions in the gastric mucosa exist in H. pylori infection.

Improvement of soft ground using solidified coal ash and its effects on the marine environment.

This paper presents some research results pertaining to the application of solidified coal ash (SCA) for improving the stability of soft ground. The results pertain to the physical properties of SCA required for use as an alternative to sand in sand compaction piles (SCPs), and the assessment of possible environmental impacts resulting from construction of SCA piles in marine environments. The results of field tests indicate that the physical properties of SCA (permeability, internal friction angle, and grain-size distribution) are favorable for use in soil improvement applications. Also, the results show that SCA is sufficiently suitable as an alternative to sand in SCPs, although SCA piles cannot be compacted to the same extent as sand piles. Finally, test results showed no adverse environmental impacts on natural benthos resulting from placement of SCA piles in marine environments. Thus, the results of this study confirm that SCA is a viable alternative material to sand in SCPs that are used for ground improvement in marine environments, and that large quantities of SCA may be required for such applications resulting in an alternative use for an otherwise waste material.

Effect of intragastric ammonia on gastrin-, somatostatin-and somatostatin receptor subtype 2 positive-cells in rat antral mucosa.

Somatostatin suppresses gastrin and somatostatin secretion via somatostatin receptors (SSTRs). Ammonia produced by Helicobacter pylori has been reported to modify gastric gastrin and somatostatin levels. We investigated the distribution of SSTR-subtype 2 (SSTR-2) in relation to gastrin- and somatostatin-containing cells and the effect of ammonia solution (0.01%-0.1%) administered orally for 2 to 4 weeks on these cells in rat antral mucosa by immunohistochemistry. The majority of SSTR-2 peptide [31-41]-positive cells were located in the basal third of the glands. Double staining experiments revealed that SSTR-2 peptide [31-41]-positive cells are co-localized in 85.0 +/- 2.2% of the gastrin-containing cells and in 34.4 +/- 4.8% of the somatostatin-containing cells. Ammonia solution significantly decreased the number of somatostatin-containing cells and increased the proportion of SSTR-2 peptide [31-41]-labeling in the somatostatin-containing cells in a duration-dependent manner. Maximum changes were observed in rats treated with ammonia solution at the lowest level of 0.01% accompanied by an increase in serum gastrin levels in the portal vein. Sodium hydroxide at the similar pH to 0.01% ammonia solution had no effect. These findings suggest that SSTR-2 are localized in antral endocrine cells and that ammonia solution mainly decreases somatostatin-containing cells without SSTR-2 expression, resulting in an increase in gastrin secretion into the portal vein.

Effects of pharmacokinetic modulating chemotherapy using oral UFT and continuous venous 5FU infusion on the prognosis of irradiated rectal carcinomas with p53 overexpression.

We previously found that patients with irradiated rectal carcinomas with p53 overexpression had poor prognoses after radical resection. In the present study, we attempted to improve the prognosis by the introduction of adjuvant chemotherapy. We administered pharmacokinetic modulating chemotherapy, based on the concept that the benefit of a continuous venous 5-fluorouracil (5FU) infusion can be potentiated by low-dose oral UFT, a combination of 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur) and uracil at a molar ratio of 1:4. Forty-two of 107 patients examined between January 1992 and December 1997 with an irradiated rectal carcinoma (39%) showed positive immunohistochemical staining for p53. Among them, 14 patients received adjuvant chemotherapy (CT group). The percentage of highly malignant tumors in the CT group was higher than that in the no-chemotherapy (NCT) group (n=28). However, the rate of cumulative local recurrence in the CT group was 0%, while that in the NCT group was 28.6% (p=0.0392). The distant recurrence rate in the CT group was also significantly lower than that in the NCT group (7.1% vs. 42.9%, p=0.0376). The cumulative 3-year survival rate was 100% in the CT group and 64.3% in the NCT group (p=0.0245). These results suggested that the antitumor property of 5FU enhanced by pharmacokinetic modulation might have a lethal effect on rectal tumors with a loss of the p53-related apoptosis pathway. These preliminary findings are encouraging for the treatment of rectal cancers with possible poor prognosis.

The regrowth of rectal adenocarcinoma after pre-operative radiotherapy with a report of two cases.

To evaluate the factors that contribute towards the regrowth of rectal carcinoma after pre-operative radiotherapy. Of 120 patients, we pre-operatively treated with intraluminal brachytherapy or external beam radiotherapy since 1986, two showed regrowth of rectal carcinoma. For them, the case reports and findings for the resected specimen obtained using immunohistochemical staining for the P53 tumor suppressor gene (P53) and proliferating cell nuclear antigen (PCNA) were retrospectively evaluated. The regrowth of rectal carcinoma was observed 40 days after intraluminal brachytherapy in one patient, and 33 days after external radiotherapy in the other. Both P53 and PCNA staining was seen in the recurrent tumors or during the examination of the resected specimen. These results suggest that polypoid tumors with regrowth have a malignant potential. The interval between radiotherapy and operation reported in the literature varies widely. The potential for regrowth should be considered in the preoperative radiotherapy for rectal cancer.

Second-look hepatectomy after 5FU arterial infusion in patients with primary unresectable hepatic colorectal metastases.

We treated primary unresectable hepatic colorectal metastases by hepatic arterial chemotherapy (HAC) combined with resection of the tumor. Patients underwent a resection of the primary colorectal tumor and a placement of HAC system, and received a 5-fluorouracil (5FU) administration once a week (320 mg/m(2)/day). Five patients underwent a 'second-look' hepatectomy in this series. Their shrinkage rate of the primary lesion ranged from 80-99%, as seen by computed tomography. The resected liver tumors were characterized as p53-positive and proliferating cell nuclear antigen-positive. The levels of fluorodeoxyuridylate (FdUMP) and thymidylate synthetase inhibition were low in the tumor tissue. These results might reflect a kind of resistance to 5FU therapy. Hepatectomy is one of the possible options to eradicate the residual SFU-resistant component of the malignancy. Our preliminary experience possibly indicates longer survival from combination approach than from HAC alone.

A possible correlation between p53 overexpression and prognosis after radiotherapy combined surgery for rectal carcinoma.

One hundred and twenty patients with rectal carcinoma in the lower two-thirds of the rectum were treated with preoperative radiotherapy and radical surgery between October 1986 and March 1996. The expression of p53 was examined in 58 of these patients, all of whom had undergone surgery more than 5 years previously and whose prognosis had been recorded. Eighteen of 58 (31%) cases showed pS3 overexpression. Clinicopathological variables other than pathological T stage did not correlate with p53 expression. The proportion of residual tumor cells in p53 positive group was significantly higher than that in the negative group. Survival time was significantly shorter in p53 positive than in p53 negative patients (p = 0.0042). The proportion of cumulative local recurrence in the p53 negative group was 2.8%, while that of the p53 positive group was 43.2% (p = 0.01). The cumulative survival rate in the p53 negative group was 76.3%, while that in the p53 positive group was 24.4% (p = 0.04). The amount of p53 positive cells in both local and distant recurrence cases was significantly higher than that without recurrence (p < 0.0001, p = 0.0016) respectively. These results suggest that p53 overexpression might predict a poor outcome at the resection of the irradiated rectal carcinoma.

Nizatidine accelerates gastric emptying of a solid meal in rats.

Nizatidine, a new histamine-2-receptor antagonist, stimulates gastrointestinal motility in dogs and gastric emptying of liquids in rats. Effect of nizatidine on gastric emptying of a solid meal was investigated using a novel gastric emptying model in rats. Male Wistar rats (weighing 200-300 g) were supplied with powdered food containing 30 w/w% barium 14 hr before the beginning of the experiment and x-ray photography of rat stomach was taken under light ether anesthesia. Gastric emptying was assessed by percentage of a decrease in area 30 min after drug was injected intraperitoneally. There was a positive correlation between the area of the gastric outline and the weight of the gastric contents (r = 0.94, P < 0.01). Ether anesthesia itself did not affect gastric emptying. Nizatidine increased gastric emptying dose-dependently (emptied percentage; vehicle: 4.9 +/- 1.5%, 1 mg/kg: 7.2 +/- 0.4%, 3 mg/kg: 10.4 +/- 2.0%, 10 mg/kg: 16.7 +/- 4.9%, 30 mg/kg: 25.7 +/- 7.4%). N-Desmethyl nizatidine (NDM) also stimulated gastric emptying, but nizatidine S-oxide, cimetidine, an famotidine had no significant effects on gastric emptying. Nizatidine and neostigmine, but not NDM, at a subthreshold dose accelerated gastric emptying treated with a low dose of acetylcholine (0.1 mg/kg). Atropine (2 mg/kg, -30 min) did not modulate the gastroprokinetic action of nizatidine, but blocked that of NDM. These findings suggest that this noninvasive method may allow measurement of gastric emptying of solids accurately and that nizatidine and NDM facilitate gastric emptying probably mediated by a direct and/or an indirect (acetylcholinesterase inhibition) cholinergic mechanism.

Effect of plaunotol on hypergastrinemia induced by long-term omeprazole administration in humans.

Omeprazole markedly inhibits basal and stimulated gastric acid secretion and has the ability to produce hypergastrinemia and hyperplasia of enterochromaffin-like cells in humans. On the other hand, plaunotol, an acyclic diterpene alcohol, has been reported to inhibit gastrin release by stimulating endogenous secretion release. We investigated the effect of plaunotol on serum gastrin levels after six to eight weeks of omeprazole (20 mg/day) administration in 22 patients (16 males, 6 females; mean age 52.3, range 36-70 years) with peptic ulcer disease. The patients were randomized to the following two groups: 11 subjects with omerprazole alone (single group) and 11 with omeprazole plus plaunotol (240 mg/day) (combination group) treatment. There were no significant differences between the two groups concerning age, sex, ulcer stage, ulcer history, environmental factors, and Helicobacter pylori (HP) prevalence. After complete drug(s) administration, serum immunoreactive (ir) -gastrin levels increased significantly in the single group (P < 0.001) in contrast to the combination group, and plaunotol significantly inhibited hypergastrinemia induced by omeprazole administration (P < 0.001). Significant increases in serum ir-calcitonin gene-related peptide concentrations were observed in the combination group compared to the single group (P < 0.05). However, there were no significant changes in sereum ir-secretin, somatostatin, and vasoactive intestinal polypeptide levels as well as ulcer healing and HP prevalence between the two groups. These findings suggest that plaunotol may suppress hypergastrinemia induced by long-term omeprazole administration, at least partly, via a certain brain-gut hormone affecting gastrin release.

[The possibility of transendoscopic transmission of Helicobacter pylori and the method of disinfection of endoscopic instruments].

Although Helicobacter pylori (HP) infection has been considered to have an etiologic role in the development of antral gastritis and recurrence of duodenal ulcer, the source and normal route of transmission of HP remains unknown. Ironically, iatrogenic infection was the first route of transmission established. Through many investigations using HP DNA analysis and anti-HP antibody change, the possibility of transendoscopic transmission of HP has been determined. From the fundamental analysis of the method, by which disinfection may be sufficient after an usual endoscopic examination, our results suggested that on several occasions, HP may be transmitted even if disinfection was performed with alcohol. Careful cleaning of the endoscope with alkaline glutaraldehyde appears to be sufficient to avoid transmission of HP from one positive patient to another.

Immunoreactive-somatostatin concentrations of the human stomach and mood state in patients with functional dyspepsia: a preliminary case-control study.

Immunoreactive-somatostatin (ir-SS) concentrations of the gastric mucosa and mood state in patients with functional dyspepsia were examined. The subjects were 12 patients with upper abdominal discomfort, nausea and/or vomiting (motility disorder group) and 14 patients complaining of upper abdominal pain (ulcer-like disorder group) for more than a month without any organic upper-gastrointestinal tract disease proven by endoscopy. These patients were compared with either an age- and sex-matched group of asymptomatic outpatients without any organic disease (control group: n = 26) or to a group of patients with peptic ulcer (n = 19). Somatostatin concentrations of the stomach were measured by radio-immunoassay, and the mood state of each subject was assessed by Manifest Anxiety Scale (MAS) and Self-rating Depression Scale test. Immunoreactive-somatostatin concentrations of the gastric mucosa were significantly higher in the ulcer-like disorder group than in the peptic ulcer, motility disorder or control group, and gastric juice levels were higher in the ulcer-like disorder group. The psychometric tests showed that the motility disorder group was more depressive than the ulcer-like disorder group, but there were no differences between the motility disorder, ulcer-like disorder and peptic ulcer group in MAS scores or environmental factors. These results indicate that there may be two different subgroups in functional dyspepsia influenced by both ir-SS concentration of the stomach and/or mood state.

[Effect of a long-term oral ammonia administration on immunoreactive-somatostatin concentrations of rat stomach].

The effect of a long-term oral ammonia administration on immunoreactive-somatostatin concentrations was investigated in rat stomach. The gastric ir-somatostatin concentrations in the group treated with 0.01% ammonia (pH 9.6) for four weeks were significantly higher than those in both the group treated with 0.1% ammonia (pH 10.4), 0.1 mM-NaOH (pH 9.6), or distilled water (pH 7.0) for four weeks and the group treated with 0.01% ammonia for two weeks. On the contrary, ir-somatostatin levels in the gastric juice and serum tended to decrease with ammonia administration. Further, ammonia administration significantly induced the decrease in mucosal thickness in the pyloric gland area and parietal cell numbers in a dose- and time-dependent manner. From these findings, it was suggested that a long-term oral treatment with 0.01% ammonia, which was clinically estimated as the concentration of the gastric juice in patients with Helicobacter pylori infection, induced not only atrophic changes on gastric mucosa, but the inhibitory effect on somatostatin secretion in rat stomach.