Ryuzaburo Shineha

Probiotic bacteria stimulate gut epithelial cell proliferation in rat.

Probiotics are used for various intestinaldiseases. However, their effects on gut epithelial cellproliferation have not been investigated. Weadministered 107 colony-forming units ofLactobacillus casei or Clostridium butyricum , or no probiotics(control) by gastric intubation once a day for sevendays to rats fed an elemental diet. We estimated thecrypt cell production rate of the jejunum, ileum, cecum, and distal colon. We also quantified cecalbacteria. Both probiotics increased the crypt cellproduction rate of the jejunum and ileum by 25-40%, ofthe cecum by 70%, and of the distal colon by more than 200% compared with control. Only minor variancein the cecal bacterial composition existed among thethree groups. Probiotics enhanced gut epithelial cellproliferation in rats fed an elemental diet.

Gene Therapy for Pancreatic Cancer Using an Adenovirus Vector Encoding Soluble flt-1 Vascular Endothelial Growth Factor Receptor

Introduction Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. The soluble form of flt-1 VEGF receptor inhibits VEGF activity in a dominant-negative manner. Aim This study demonstrated the regional tumor suppression effect of adenovirus-mediated soluble flt-1 in human pancreatic cancer cells. Methodology The VEGF expression level was examined in nine cell lines. Panc-1 and PK-8 were used as lower- and higher-VEGF-producing cell lines, respectively. The in vitro proliferation of cancer cells infected with adenovirus vectors encoding soluble flt-1 (Adsflt) and control vectors (AdLacZ) was not different. To assess the in vivo tumor growth suppression, cancer cells were inoculated subcutaneously in SCID mice. Adsflt, AdLacZ, or vehicle was injected directly into the tumors. The early process of tumor angiogenesis in a dorsal skinfold chamber was monitored by intravital microscopy. Results In both Panc-1 cells and PK-8 cells, the tumor growth of the Adsflt-treated group was significantly suppressed. The microvessel density, revealed by CD31 immunostaining, was also significantly lower in the Adsflt-treated group. Apoptosis index was higher in the Adsflt group. Immunofluorescence staining revealed the expression of VEGF not only in cancer cells but also in tumor stromal cells. Wild-type cells and AdLacZ-infected cells prompted strong tumor angiogenesis, whereas Adsflt-infected cells failed to exert such an effect. Conclusion These results indicate that antiangiogenic gene therapy using soluble flt-1 might be an effective approach for pancreatic cancer treatment.

Reduced sialidase expression in highly metastatic variants of mouse colon adenocarcinoma 26 and retardation of their metastatic ability by sialidase overexpression

Sialidase expression levels are inversely correlated with the metastatic potential of mouse colon adenocarcinoma 26 sublines, as assessed by activity assays and RT‐PCR, irrespective of total and cell surface sialic acid contents. Compared with low metastatic NL4 and NL44 cell lines, the highly metastatic NL17 and NL22 cells exhibit low expression of sialidases, accompanied with higher levels of sialylLex and GM3. To investigate whether these properties of NL17 cells can be altered by sialidase overexpression, we transfected a cytosolic sialidase gene into NL17 cells. The result was markedly inhibited lung metastasis, invasion and cell motility with a concomitant decrease in sialylLex and GM3 levels, in line with the case of spontaneously low metastatic sublines having relatively high endogenous sialidase levels, implying that sialidase level is a determining factor affecting metastatic ability. Treatment of the cells with antibodies against sialylLex and GM3 affected cell adhesion and/or cell motility, providing evidence that desialylation of these molecules, as targets of sialidase, is involved in the suppression of metastasis.

High incidence of reflux esophagitis observed by routine endoscopic examination after gastric pull-up esophagectomy

A gastric tube has been widely used for reconstruction of the esophagus after esophagectomy for esophageal cancer. Reflux esophagitis after esophagectomy is frequently observed. Therefore we retrospectively investigated the risk factors for reflux esophagitis after gastric pull-up esophagectomy in 74 outpatients with thoracic esophageal cancer. Reflux esophagitis was diagnosed endoscopically. Esophagitis was classified according to the Los Angeles classification. Reflux symptoms, medications, and the surgical procedure were reviewed. The relation between reflux symptoms and reflux esophagitis and the influence of the anastomotic site were evaluated. Reflux esophagitis was observed in 53 patients. Severe esophagitis (grade C or D) was found in 75.6% of these patients. Although all patients with esophagitis took antacid agents, histamine receptor-2 blocker was effective in only 35% of them. The correlation between reflux symptoms and reflux esophagitis was not significant. Reflux esophagitis was present in 56.4% of patients with neck anastomosis and in 88.6% of patients with intrathoracic anastomosis (p = 0.0039). We concluded that routine endoscopic examination is necessary after gastric pull-up esophagectomy because reflux esophagitis is not diagnosed based on reflux symptoms. When a gastric tube is used for reconstruction after esophagectomy, neck anastomosis is recommended to lower the risk of reflux esophagitis.

Overexpression of lysosomal‐type sialidase leads to suppression of metastasis associated with reversion of malignant phenotype in murine B16 melanoma cells

Increased sialylation in cell surface glycoproteins is one characteristic feature of cancer cells, particularly related to their metastatic potential and invasiveness. Expression of lysosomal‐type sialidase, which plays a major role in hydrolysis of such sialo‐glycoproteins, is therefore considered to have a great influence on malignant properties of cancer cells. To investigate whether the sialidase expression level is linked to the malignant phenotype, we transfected B16‐BL6 murine melanoma cells, a highly invasive and metastatic line, with an expression vector harboring a rat lysosomal sialidase cDNA; then clones were isolated and examined for changes in biological character. Sialidase‐overexpressing cells showed suppression of experimental pulmonary metastasis and tumor progression. The transfectants exhibited diminished cell growth, anchorage‐independent growth and increased sensitivity to apoptosis induced by suspension culture or serum depletion in vitro, but no significant alterations in invasiveness, cell motility and cell attachment to fibronectin, collagen IV and laminin. Flow cytometric analysis with either peanut agglutinin (PNA) or Ricinus communis agglutinin (RCA) lectin revealed that desialylated forms of glycoproteins on the cell surfaces were increased. In particular, a desialylated form of a cell surface glycoprotein of 83 kDa was prominent in the transfectants, as determined by galactose oxidase labeling. These observations indicate that sialidase expression is inversely associated with metastatic potential and tumor growth in cancer cells, probably through a regulation mechanism that suppresses cell growth and anchorage‐independent growth and promotes apoptosis with deprivation of cell anchorage.

Angiogenesis inhibition by transdominant mutant Ets-1.

The expression of transcription factor Ets‐1 is induced in endothelial cells (ECs) by angiogenic factor; and in turn Ets‐1 converts ECs to angiogenic invasive phenotype. In order to control angiogenesis, we constructed a transdominant mutant Ets‐1 (TMEts‐1) which acts as a dominant negative molecule. This molecule inhibited the DNA binding and the transactivation activity of the wild‐type Ets‐1. Stable transfection of murine endothelial cell line MSS31 cells with the TMets‐1 gene impaired angiogenic activities including proliferation, migration, invasion, and tube formation in type‐1 collagen gel. Finally, we incorporated the TMets‐1 gene into a non‐proliferative adenovirus vector, designated as AdTMets‐1. AdTMets‐1 significantly inhibited angiogenesis in the Matrigel plugs injected into the subcutaneous tissue of C57BL mice. These results indicate that TMets‐1 would be a tool for angiogenic inhibition. J. Cell. Physiol. 184:255–262, 2000.

Gastric or rectal instillation of short-chain fatty acids stimulates epithelial cell proliferation of small and large intestine in rats.

Short-chain fatty acids stimulate gut epithelial cell proliferation in vivo, although the difference between oral and rectal routes is unknown. Accordingly, we examined the effect of oral or rectal administration of these acids. We instilled a mixture of acetic acid, propionic acid, and n-butyric acid (150, 60, and 60 mmol/liter, respectively; pH 6.5) or saline (270 mM, pH 6.5) into the stomach (2 ml) or rectum (1 ml) three times daily for five days in rats fed an elemental diet. We measured crypt cell production rate of the jejunum, ileum, and distal colon of these rats. The crypt cell production rate of these segments was higher in rats with gastric or rectal instillation of short-chain fatty acids than in saline controls. The rectal route was slightly more effective than the gastric route. The above results indicated that the instillation of short-chain fatty acids orally or rectally stimulated gut epithelial cell proliferation.

Adenocarcinoma at the esophageal gastric junction arising in an 11-year-old girl

Gastric adenocarcinoma is one of the most common gastrointestinal (GI) malignancies, especially among Japanese adults, but represents only 0.05% of all malignant pediatric GI tumors. We report a case of gastric adenocarcinoma arising at the esophageal gastric junction of an 11‐year‐old girl. The tumor was polypoid, measuring 3.0 × 3.0 × 1.2 cm and was light gray and partially red in color with a stalk. Light microscopic examination of the lesion demonstrated adenocarcinoma of variable degrees of both architectural and nuclear atypia with invasion into the submucosa. Immunohistochemical findings of cytokeratin subtypes revealed positive immunoreactivity for cytokeratin subtypes 8, 19 and 20 and negative for 5/6/18, 7, 13 and 14, which is consistent with those of gastric adenocarcinoma. The patient was alive and well 12 months postoperatively.

Association between elevated plasma granulocyte colony-stimulating factor and the degree of surgical stress in patients undergoing gastrointestinal surgery

To characterize the changes in perioperative plasma granulocyte colony-stimulating factor (G-CSF) and analyze the effect of surgical stress on its kinetics, 41 patients undergoing gastrointestinal surgery with varying degrees of surgical stress were examined. The plasma levels of G-CSF significantly increased immediately after the operation, probably in response to surgical injury. This elevation was much higher in the 15 esophagectomy patients, at 883±300 pg/ml on postoperative day (POD) O, than in the 14 gastrectomy patients, with a value of 233±151 on POD O, (P<0.01) or in the 12 cholecystectomy patients, with a value of 64±41 on POD 1 (P<0.01). These findings led us to conclude that G-CSF levels increase significantly in the immediate postoperative period and are most likely associated with the degree of surgical stress. In addition, we studied the priming effect of G-CSF on polymorphonuclear leukocytes (PMNs). G-CSF enhanced PMN superoxide anion (O−2) production and luminol-dependent chemiluminescence (CL) induced by opsonized zymosan in a dose-dependent manner. A significant enhancement was seen in the G-CSF level (1 ng/ml) which was almost the same as the maximum G-CSF level in the esophagectomy patients. Furthermore, postoperative PMN activation occurred after the elevation of plasma G-CSF. Thus, we propose that elevated G-CSF may act as one of the mediators which activate PMN function postoperatively.

Lymph Flow and Lymph Node Metastasis in Esophageal Cancer

This paper delineates which lymph nodes should be dissected due to the high frequency of metastasis associated with different types of primary lesions of the thoracic esophagus. In cancer involving the upper third of the esophagus (Iu), lymph flow was found to be primarily from the superior mediastinal area to the cervical area; in that involving the middle third (Im), it was broadly distributed from the superior, middle, and inferior mediastinal region to the cervical and abdominal regions; and in that involving the lower third (Ei), it tended to extend from the inferior mediastinal region to the abdominal region, with single primary metastatic nodes also being noted in this area. The significance of the “top” nodes, namely, the nodes located along the right recurrent laryngeal nerve in the upper portion of the thorax, was also investigated, and it was confirmed that the prognosis for patients with metastases to both the top nodes and other nodes was unfavorable. An immunohistochemical study on mediastinal lymph flow using the anti-Su-Ps antibody demonstrated interactions between top nodes and cervical and/or thoracic nodes.