Toshimitsu Majima

VEGF, basic-FGF, and TGF-β in Crohn's disease and ulcerative colitis: a novel mechanism of chronic intestinal inflammation

OBJECTIVE:Inflammatory bowel disease (IBD), the precise etiology of which remains unknown, is comprised of two forms of chronic intestinal inflammation; ulcerative colitis (UC) and Crohns disease (CD). Recent evidence increasingly suggests that IBD is the result of dysfunctional immunoregulation manifested by inappropriate production of mucosal cytokines. An abnormal microcirculatory system has also been implicated in its pathogenesis. To elucidate the mechanism of ischemic change in IBD, we assessed serum concentration levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF), and plasma level of endothelin-1 (ET-1). We also investigated the expression of VEGF, b-FGF, and transforming growth factor-β1,2,3 (TGF-β1,2,3) in tissue by immunostaining.METHODS:Blood samples were obtained from 11 patients with UC, 11 patients with CD, and 10 patients as controls. Paraffin-embedded samples were used for an immunohistochemical study.RESULTS:The concentration levels (in picograms per milliliter) were as follows: for ET-1, UC: 127 ± 47.0, CD: 167.3 ± 35.1, and controls (asthma: 38.5 ± 23.8, p < 0.01; diverticulitis: 40.5 ± 25.6, p < 0.01), for b-FGF, UC: 9.2 ± 1.9, CD: 9.1 ± 1.5, and controls (asthma: 5.0 ± 0, p < 0.01; diverticulitis: 5.0 ± 0, p < 0.01), for VEGF, UC: 659.8 ± 181.0, CD: 740.0 ± 182.3, and controls (asthma: 193.7 ± 58.7, p < 0.01; diverticulitis: 199.6 ± 59.7, p < 0.01). The levels of VEGF and b-FGF were significantly higher in active IBD than those in the controls. There was a significant positive correlation among the serum levels of VEGF and b-FGF and the plasma level of ET-1; that is, elevated VEGF, b-FGF, and ET-1 levels correlated well with each other. Immunohistochemical studies showed increased venula in the submucosa and lamina propria. Overexpression of VEGF and b-FGF in endothelial cells was revealed and TGF-β2 and TGF-β3 were found in inflammatory cells of active IBD, but no change was observed around the vessels in the controls.CONCLUSIONS:It is suggested that the reciprocal reaction of these cytokines may contribute to angiogenesis in IBD by inducing intestinal ischemia through vasoconstriction.

Inhibitory effects of β-carotene, palm carotene, and green tea polyphenols on pancreatic carcinogenesis initiated by N-nitrosobis(2-oxopropyl)amine in Syrian golden hamsters

The effects of alpha-carotene, beta-carotene, palm carotene, and green tea polyphenols (GTP) on the progression stage of pancreatic carcinogenesis after rapid production of ductal lesions were studied in Syrian hamsters. Dose threshold inhibitory effects were noted for beta-carotene, 25 ppm, and palm carotene, 40 ppm, which includes 24 ppm beta-carotene reducing the numbers of putative preneoplastic lesions of duct epithelial hyperplasia and atypical hyperplasia, as well as carcinoma in situ and invasive carcinomas. GTP at doses of 500 and 5000 ppm, but not 100 ppm, also significantly decreased the numbers of hyperplasia and total duct lesions. Combined administration of 40 ppm palm carotene, and 50 ppm GTP similarly inhibited the lesion development. Alpha-carotene, however, did not affect pancreatic carcinogenesis. The results suggest that chemopreventive effects are exerted by beta-carotene and GTP above critical doses and that combined administration of palm carotene and GTP might be a candidate chemoprevention strategy for pancreatic cancer in humans.

Chemopreventive efficacy of piroxicam administered alone or in combination with lycopene and β-carotene on the development of rat urinary bladder carcinoma after N-butyl-N-(4-hydroxybutyl)nitrosamine treatment

The effects of the non‐steroidal anti‐inflammatory drug (NSAID) piroxicam and the carotenoids lycopene and β‐carotcnc, alone or in combination, on the development of rat superficial urinary bladder carcinomas induced by N‐butyl‐N‐(4‐hydroxybutyI)nitrosamine (BBN) were studied. Male Fischer 344 rats, 6 weeks old, were given 0.05% BBN in the drinking water for 8 weeks followed by administration of piroxicam (0.0075% in the diet), lycopene (0.0025% in the drinking water) and/or β‐carotene (0.0025% in the drinking water) for 12 weeks, then killed for histological analysis of urinary bladder lesions. Cell proliferation potential was analyzed by immunohistochemical staining of the proliferative cell nuclear antigen (PCNA). Piroxicam alone, piroxicam+lycopene, and piroxicam +lycopene+β‐carotene all significantly decreased the incidences and numbers of transitional cell carcinomas (TCCs), but the combination of piroxicam with carotenoids did not result in a clear improvement in the preventive potential of piroxicam. Piroxicam +β‐carotene also caused a significant reduction and lycopene alone a slight but not significant reduction in the number of TCCs. In contrast, β‐carotene alone and lycopene +β‐carotene were without inhibitory influence on any of the lesion categories examined, and the latter significantly increased the proportion of high‐grade TCCs. Nevertheless, all of the chemopreventive agents, either alone or in combination, significantly decreased the TCC PCNA index, the effect extending to the surrounding epithelium in the piroxicam 4‐lycopene and piroxicam+lycopene+β‐carotene groups. These results indicate that the NSAID piroxicam may he a more effective chemopreventive agent than lycopene and β‐carotene for superficial urinary bladder carcinogenesis.

Increased Telomerase Activities in Human Pancreatic Duct Adenocarcinomas

Telomerase is a key enzyme with regard to immortalization of cancer cells and increased activity has been demonstrated in various human malignant neoplasms. Since little is known of its role in pancreatic cancers, we investigated changes in telomerase activity in human pancreatic duct adenocar‐cinomas and compared the frequency of increased telomerase activity with the presence of K‐ras gene mutations. The samples were obtained from 38 pancreatic duct adenocarcinomas and 7 tumor surrounding tissues at surgical resection. Telomerase activity was examined by telomeric repeat amplification protocol assay and terminal restriction fragment (TRF) length was examined by Southern analysis. K‐ras mutation was examined by means of polymerase chain reaction‐single strand conformation polymorphism analysis. Among 38 pancreatic carcinomas, 32 (84%) exhibited increased telomerase activities with no apparent relation to the histological type of tumor, tumor size, regional lymphnode involvement and distant metastasis or clinical stage. In tissue surrounding the tumor, telomerase activity was not detected. TRF length tended to be reduced in pancreatic carcinomas. Mutations of K‐ras gene were found in 24 out of the 38 (63%) cases. Among the 38 cases, 14 showed increased telomerase activity without K‐ras mutation and 4 cases showed K‐ras mutation without telomerase activity. These results suggest that increased telomerase activity might be a sensitive genetic diagnostic marker and could be a target for future therapy of pancreatic duct carcinomas.

Increased Telomerase Activity in Hyperplastic Nodules and Hepatocellular Carcinomas Induced by a Choline-deficient L-Amino Acid-defined Diet in Rats

Activation of telomerase has been reported in several human cancers, including hepatocellular carcinomas (HCCs). We investigated telomerase activity during hepatocarcinogenesis induced by a choline‐deficient L‐amino acid‐defined (CDAA) diet in rats. Male F344 rats were given a CDAA diet or a choline‐supplemented L‐amino acid‐defined (CSAA) diet from 6 weeks of age for 75 weeks, and subgroups were killed 10 weeks, 50 weeks and 75 weeks after the beginning of the experiment. Hyperplastic nodules and HCCs were noted in rats fed a CDAA diet for 50 weeks and 75 weeks, respectively. Normal control liver specimens were obtained from 6‐week‐old rats. Telomerase activity was assessed by using a telomeric repeat amplification protocol (TRAP). Normal liver and background parenchyma of rats fed either of the diets for 10 weeks or 50 weeks showed weak telomerase activity. In contrast, markedly increased levels were demonstrated in hyperplastic nodules and HCCs. These results suggest that increased telomerase activity may be a biological feature of preneoplastic lesions that evolve to HCCs in rat liver.

Increased telomerase activity in intrahepatic cholangiocellular carcinomas induced by N-nitrosobis(2-oxopropyl)amine in hamsters.

Telomerase activities in intrahepatic cholangiocarcinomas induced by N-nitrosobis(2-oxopropyl)amine (BOP) in female hamsters were determined using a telomeric repeat amplification protocol (TRAP) assay followed by densitometric quantification. Each determination was repeated to confirm the results and telomerase activity was also detected by gel electrophoresis. An increase was evident in all of 10 cholangiocarcinomas examined, with levels ranging from 2.48 to 4.40 times the normal liver value by densitometric quantification. This finding of a consistent increase suggests that telomerase activation is involved in the development of intrahepatic cholangiocarcinomas and immortalization of cancer cells.

Mutations of K-ras but not p53 genes in biliary duct and pancreatic duct carcinomas induced in hamsters by cholecystoduodenostomy with dissection of the common duct followed by N-nitrosobis(2-oxopropyl)amine.

An experimental model for the induction of extrahepatic biliary duct carcinomas in hamsters given cholecystoduodenostomy with dissection of the extrahepatic duct at the distal end of the common duct (CDDB) followed by N-nitrosobis(2-oxopropyl)amine (BOP) has been reported [Tajima et al. (1994) Jpn. J. Cancer Res., 85, 780-788]. The CDDB procedure greatly accelerates cell turnover in the biliary epithelium. In the present experiment, mutations of K-ras and p53 genes in the induced lesions were investigated by the reverse transcriptase-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) method followed by direct sequencing. Mutations of K-ras, involving a G to A transition in second position of codon 12 of K-ras exon 1, were detected in six out of eight (75%) extrahepatic bile duct carcinomas and six out of eleven (54.5%) pancreatic duct carcinomas. However, no mutations of p53 were observed in either tumor type. The results indicate an association between anomalous pancreaticobiliary junction and development of biliary carcinomas that may be pertinent to the human situation, and indicate that conditions of the model predispose to mutations occurring in K-ras but not p53.

Infrequent Ki-ras and an absence of p53 mutations in hepatocellular carcinomas induced by a choline deficient l-amino acid defined diet in rats

Mutations of Ki-ras and p53 genes in hepatocellular carcinomas (HCCs) induced by the choline deficient L-amino acid defined (CDAA) diet in rats were investigated by polymerase chain reaction (PCR), single strand conformation polymorphism (SSCP) analysis followed by direct sequencing. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 70 weeks and then sacrificed. Macroscopically detectable nodules which were histologically confirmed to be well-differentiated HCCs were dissected free from the surrounding tissue and subjected to gene mutation analysis along with samples of non-tumor areas. Conformational change in the Ki-ras gene was detected in 1 out of 7 HCCs, involving a GGC to GTC transversion at codon 13. No p53 mutations were detected in 7 HCCs and also neither Ki-ras nor p53 mutations were found in non-tumor areas. The results suggest that neither Ki-ras nor p53 genes play an important role in hepatocarcinogenesis caused by long term expose to a CDAA diet in rats.

Forskolin-stimulated adenylylcyclase activity: a marker to assess islet cell viability following cold storage in different solutions and to predict islet cell function following transplantation.

For clinical islet cell transplantation, short-term storage of islet cells is likely to be necessary, and it is imperative that the islet cells be kept as viable as possible during the period. However, there are little data on which preservative solutions are most suitable for the storage of islet cells after isolations or before transplantation. To estimate islet cell viability and transplantation success rate in the present study, adenylylcyclase activity was measured with a rapid new fluorometric assay in rat islet cells prior to transplantation, because cAMP plays an essential role in determining islet β-cell viability and responsiveness to various hormonal stimuli. Adenylylcyclase activity was measured in islet cells stored for different periods of time (0, 3, 16, 24, 48, 96 h) and in different preservative solutions. Approximately 1,000 islet cells from each preservation group using University of Wisconsin (UW) solution were transplanted to streptozotocin-induced diabetes mellitus (DM) rats. Transplant success was evaluated by measuring blood glucose levels. Preoperative adenylylcyclase activity was compared with posttransplant islet cell function. The adenylylcyclase activity of UW solution was significantly higher than that of Euro-Collins solution and lactate-Ringers solution through the different preservation time periods. Preoperative adenylylcyclase activity correlated well with posttransplant islet cell function in a rat model of DM. We conclude that adenylylcyclase activity can be used as a marker to assess islet cell viability as well as differences in preservation media and may predict islet cell transplant success.

A pitfall of MRCP for preoperative cholangiography: report of a case

bowel are usually discovered in the late stage. Thus, the 5-yr survival rate for patients with small bowel cancer is poor, averaging 30% (2–4). Among small bowel tumors, 35% of benign tumors and 17% of malignant tumors are located in the duodenum (2). With the development of new endoscopic techniques such as strip biopsy, it has become possible to achieve adequate tumor resection including not only pedunculated or sessile lesions but also flat or erosive lesions. Because strip biopsy permits the resection of large specimens, it is useful not only for therapy but also for assessing the pathological characteristics and degree of invasion of lesions. Although strip biopsy has a number of limitations, patients who are not candidates for surgery because of severe underlying disease or refusal to grant consent are good candidates for this procedure. Because the duodenum mucosa is quite thin, endoscopic resection is associated with a high risk of perforation. However, the risk of this complication can be minimized in strip biopsy procedures by injecting saline solution into the submucosa to lift the lesion away from underlying tissues as well as to prevent excessive spread of the electrocoagulation effect (5). The excellent clinical outcome observed in the case presented here strongly suggests that strip biopsy may prove to be useful for the treatment of early duodenal cancer.