Toshiya Horibe

Targetability and intracellular delivery of anti-BCG antibody-modified, pH-sensitive fusogenic immunoliposomes to tumor cells.

We prepared tumor-specific immunoliposomes by coupling anti-BCG monoclonal antibodies to pH-sensitive fusogenic liposomes modified with succinylated polyglycidol (sucPG), in order to obtain efficient binding to, and endocytotic internalization into, the tumor cells. Mouse colon carcinoma 26 cells, which are known to share a common antigen with BCG, were used in in vitro experiments. BCG-sucPG immunoliposomes showed fusion ability under acidic conditions. Fluorescence microscopic observation indicated that BCG-sucPG immunoliposomes bound to colon 26 tumor cells and induced receptor-mediated endocytosis at 37 degrees C. Fusion assay by resonance energy transfer using N-(7-nitro-2-1,3-benzoxadiazol-4-yl) diacyl phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl) diacyl phosphatidylethanolamine suggested that fusion between BCG-sucPG immunoliposomes and endosomal and/or lysozomal membrane did occur. These results imply that the BCG-sucPG immunoliposomes transfer their content into the cytoplasm by fusing with the endosomal and/or lysozomal membrane after recognition of target cells and subsequent internalization into the cells by endocytosis.

Nuclear cyclin D1 overexpression is a critical event associated with cell proliferation and invasive growth in gallbladder carcinogenesis.

Abstract: Cyclin D1 overexpression is remarkably frequent in several human carcinomas and is believed to be a critical event in oncogenesis. We examined cyclin D1 expression, p53 expression, and the Ki-67 labeling index by immunostaining in human gallbladder mucosa in conditions varying from normal to malignant tissue. We also examined K-ras codon 12 mutations in these tissues with a two-step polymerase chain reaction. Nuclear cyclin D1 overexpression was observed in 48% of carcinomas occurring independently of adenoma, but not in adenomas, carcinomas arising in adenomas, or nonneoplastic lesions. Cytoplasmic cyclin D1 overexpression was observed in about 15% of abnormal specimens, irrespective of the type of epithelial abnormality. Carcinomas showing nuclear cyclin D1 overexpression had significantly higher Ki-67 labeling indexes than those with no overexpression. Moderately to poorly differentiated adenocarcinomas showed a higher incidence of nuclear cyclin D1 overexpression than papillary to well differentiated carcinomas. Specimens with cyclin D1 overexpression showed a high incidence of lymph permeation, venous permeation, and lymph node metastasis. We conclude that nuclear cyclin D1 overexpression is a critical event importantly associated with cell proliferation and invasive growth in gallbladder carcinogenesis, and that cyclin D1 immunostaining may become a useful marker for evaluating gallbladder carcinomas.

A case of hemangioma accompanied by inflammatory pseudotumor of the spleen.

Both hemangioma and inflammatory pseudotumor (IPT) of the spleen are rare benign mass lesions. Moreover, a splenic hemangioma accompanied by IPT is extremely rare. A 61-year-old woman who suffered from liver cirrhosis had a splenic cavernous hemangioma surrounded by granuloma. The literature on IPT of the spleen has described several possibilities of its causes; however, it is still unknown. This case was accompanied by portal hypertension due to liver cirrhosis, which may cause microrupture of hemangioma resulting in an IPT.

Visualization of the drainage veins with contrast-enhanced sonography was useful in diagnosis of small focal nodular hyperplasia.

ocal nodular hyperplasia (FNH) is a benign localized lesion in the liver first reported by Edmondson1 in 1956. Histologically, FNH is defined as a type of tumorlike lesion. Histopathologically, it is sometimes difficult to differentiate FNH from adenoma. Macroscopically, FNH is a nodule without a capsule showing a central stellate fibrous scar spreading from the center of the nodule radially toward the periphery.2 These features have been regarded as the determining factors for imaging diagnosis. In dynamic computed tomography (CT) studies, the FNH is isoattenuating before contrast, showing homogenous hyperattenuation in the early arterial phase and isoenhancement during the portal venous phase. Diagnosis is facilitated by the presence of a central scar, but this feature can only be observed in approximately 50% of cases.3 Although the vascular structure of FNH is said to be characterized by a typical spoke-wheel pattern and a stellate fibrous scar,4 these features are not readily visualized in small FNH lesions. In such cases, it is necessary to differentiate from other hypervascular malignant tumors such as hepatocellular carcinoma (HCC) and metastatic lesions.5 The recent advent of multi–detector row CT, 3-dimensional CT, and single-level dynamic CT during hepatic arteriography has allowed detailed visualization of the hemodynamics of FNH. According to these evaluations, the drainage veins of FNH are mainly the hepatic veins, although they sometimes drain directly into the sinusoids surrounding the tumor.6 However, angiography is invasive, and 3-dimensional volume imaging does not allow real-time presentation. In this case, contrast-enhanced sonography using Levovist (SH U 508A; Schering AG, Berlin, Germany) enhanced the FNH in real time in the early arterial phase and clearly depicted subsequent drainage into the hepatic veins. Received November 23, 2005, from the Department of Diagnostic Ultrasound, Medical Imaging Center, Hyogo College of Medicine, Hyogo, Japan (H.I.); and Departments of Gastroenterology (S.S., F.M., K.Y., M.Ya., T.M., M.Yo., T.H.) and Radiology (N.I., D.K., K.A.), Tokyo Medical University, Tokyo, Japan. Revision requested December 28, 2005. Revised manuscript accepted for publication February 21, 2006. We thank J. Patrick Barron, BA (International Medical Communications Center, Tokyo Medical University), for reviewing the manuscript and Masamichi Kojiro, MD, PhD, (Department of Pathology, Kurume University School of Medicine, Kurume, Japan), for providing advice on histopathologic assessment. Address correspondence to Fuminori Moriyasu, MD, PhD, Department of Gastroenterology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan. E-mail: moriyasu@tokyo-med.ac.jp Abbreviations CT, computed tomography; FNH, focal nodular hyperplasia; HCC, hepatocellular carcinoma; MRI, magnetic resonance imaging

Hemosuccus pancreaticus diagnosed by contrast-enhanced endoscopic ultrasonography (with video)

Hemosuccus pancreaticus, a condition in which blood is expelled into the duodenum via the main pancreatic duct, is a rare cause of acute gastrointestinal bleeding. The common causes of hemosuccus pancreaticus are a pancreatic pseudoaneurysm and pseudocyst due to chronic pancreatitis. We describe a case of hemosuccus pancreaticus caused by a pancreatic pseudoaneurysm due to chronic pancreatitis, which was diagnosed by contrast-enhanced endoscopic ultrasonography (CE-EUS). A 71-year-old man with a long history of alcohol abuse was referred to our hospital with severe anemia and tarry stools. Laboratory data showed a very low hemoglobin level of 4.8 mg/dL. The patient thus received a blood transfusion. Upper GI endoscopy demonstrated no obvious lesion. Computed tomography (CT) revealed a 4.2-cm cystic mass in the pancreatic head with slight contrast-enhancement in the early phase and strong contrast-enhancement in the late phase (Fig. 1). CT also revealed dilatation of the main pancreatic duct and atrophy in the pancreatic body-tail with some small calcifications. Transabdominal ultrasonography showed a 20-mm anechoic area in the pancreatic head and a color Doppler signal was identified in the area. Since a pseudoaneurysm was suspected, angiography was performed. Angiography of the superior mesenteric artery revealed a slight pooling of a contrast medium corresponding to the anechoic area (Fig. 2). However, this was not a typical finding of a pseudoaneurysm. Two days later, since the patient had obstructive jaundice caused by the compression of the enlarged cyst, endoscopic retrograde cholangiopancreatography was performed for biliary decompression. Notably, a duodenoscope showed hemorrhage from the papilla of Vater (Fig. 2). Thus, after biliary stenting, we performed CE-EUS using Sonazoid to identify the origin of the hemorrhage. A fundamental EUS image showed an anechoic lesion similar to that seen on transabdominal ultrasonography (Video S1). Interestingly, 22 seconds after the contrast injection, microbubbles were clearly shown to go into the small feeding artery flowing into the pancreatic head cavity (Fig. 3, Video S1). As previous angiography failed to detect the small feeding artery flowing into the cavity, we speculated that it might be difficult to perform coil embolization on angiography. In addition, the patient had obstructive jaundice due to cyst compression and already received a 16-unit blood cell transfusion. Thus, the patient underwent emergent pylorus-preserving pancreatoduodenectomy as essential therapy. The patient showed good recovery without any adverse events postoperatively. Macroscopic findings showed excessive blood clot in the pancreatic head cavity (Fig. 4). Microscopic specimens showed hemorrhage originating from the ruptured small artery, which flowed into the pancreatic head cavity of which a small portion was covered with an epithelium (Fig. 4). The final diagnosis was hemosuccus pancreaticus derived from a pseudoaneurysm. To the best of our knowledge, this is first report of hemosuccus pancreaticus due to a ruptured artery diagnosed by CE-EUS. CE-EUS may also have other potential applications such as for the close examination of vascular K. Yamamoto · T. Itoi (*) · T. Horibe Department of Gastroenterology and Hepatology, Tokyo Medical University Hospital, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan e-mail: itoi@tokyo-med.ac.jp

Experience with Percutaneous Electronic Choledochoscopy

Experience with percutaneous choledochoscopy using a prototype electronic choledochoscope (Pentax ECN‐1530) is presented herein. This electronic endoscope is 5.3 mm in outside diameter at the tip and has a forceps channel 2.0 mm in diameter. The outside diameter is 0.4 mm larger, while the forceps channel diameter is 0.2 mm smaller, than that of the conventional fiberoptic choledochoscope (FCN‐15X) produced by the same company. Although the new electronic choledochoscope could be inserted through a 16 Fr in size fistula, we considered an 18 Fr fistula to be preferable for insertion without resistance. Various types of accessory equipment for endoscopic treatment, such as an electrohydraulic Shockwave lithotriptor (EHL) and an Nd‐YAG laser, could be used without difficulty. The electronic choledochoscope was useful for examining bile duct carcinoma invasion to the hepatic side and evaluating the efficacy of various multi‐modal treatments, as it provided observation of the bile duct mucosa in great detail due to a very clear dynamic image. Moreover, endoscopic treatment was also greatly facilitated because it provided a clear view on a large, bright monitor screen for the surgeons. We therefore believe that this new electronic choledochoscope is very useful for the accurate diagnosis and treatment of biliary diseases.

Overexpression of cyclin A and p53 in hepatocellular carcinomas. Wild P53 downregurate cyclin a promoter activity

Backround: The p16 INK4a tumor suppressor gene induces a cell cycle G1 arrest. It is the second most commonly inactivated gene identified in human cancers after p53. The p16 gene has been shown to be inactivated in nearly all human colon cancer cell lines and half of colon cancers and adenomas. While the p16 gene has been demonstrated to be inactivated primarily by promoter DNA methylation in hepatocellular carcinomas, there have been no studies examining p16 expression in premalignant liver lesions . Design: 17 macroregenerative and dysplastic nodules and 2 hepatocellular carcinomas from 15 hepatectomy specimens in patients undergoing transplantation for hepatitis C cirrhosis were examined by immunohistochemistry and methylation sensitive polymerase chain reaction (PeR). The nodules were less than 2 cm in diameter and clinically undetected . Sections were stained with anti-human pl6 monoclonal antibody . Human sporadic colon adenocarcinomas served as positive controls . Staining for p16 was considered positive if nuclear staining was greater than cytoplasmic staining. Methylation sensitive PCR was performed on bisulfite-treated DNA extracted from microdissected paraffin sections. Results: No nuclear staining was detected in the macroregenerative or dysplastic nodules. Two nodules demonstrated weak cytoplasmic staining . The surrounding cirrhotic liver showed cytoplasmic and no nuclear staining . Bile ductules at the margins of the nodules showed positive staining and served as internal positive controls. The 2 hepatocellular carcinomas showed a distinct lack of nuclear or cytoplasmic staining. Negative controls lacked cytoplasmic and nuclear staining. One hepatocellular carcinoma and two macroregenerative nodules had methylated p16 gene promoters , while two of the surrounding cirrhotic liver samples had unmethylated pl6 gene promoters. One macroregenerative nodule also showed evidence of both methylated and unmethylated pl6 forms. Conclusions: In patients with hepatitis C, clinically undetected macroregenerative and dysplastic nodules show an absence of pl6 staining. It is likely that pl6 gene methylation is responsible for suppression ofpl6 expression in these nodules. This supports previous observations that a high percentage of hepatocellular carcinomas have inactive p16 genes and suggests that p16 inactivation occurs early in liver tumor progression.

A Case of Chronic Pancreatitis Associated with a Pancreato-gastric Fistula

Abstract: A 51‐year‐old man presented complaining of frequent diarrhea. An ultrasonography and abdominal CT scan revealed a tumor in the tail of the pancreas. An endoscopic retrograde pancreatography revealed contrast medium flowing over the pancreas through the main pancreatic duct. A balloon cathether was then passed into the pancreatic duct, and the scope alone was retracted to the stomach. Maintaining the stomach under endoscopic observation, ICG was injected through the balloon catheter, whereupon it was seen to flow out from two small depressions in the center of a small elevated lesion in the posterior wall of the upper gastric corpus. Based on these endoscopic findings, a diagnosis of chronic pancreatitis with an associated pancreato‐gastric fistula was made.