Search

Esophageal squamous cell cancer (ESCC) is one of the most common lethal tumors in the world, and development of new diagnostic and therapeutic methods is needed. In this study, cancer‐testis antigen, BORIS, was isolated by functional cDNA expression cloning using screening technique with serum IgG Abs from ESCC patients. BORIS was previously reported to show cancer‐testis antigen like expression, but its immunogenicity has remained unclear in cancer patients. BORIS was considered to be an immunogenic antigen capable of inducing IgG Abs in patients with various cancers, including four of 11 ESCC patients. Immunohistochemical study showed that the BORIS protein was expressed in 28 of 50 (56%) ESCC tissues. The BORIS expression was significantly associated with lymph node metastasis in ESCC patients with pT1 disease (P = 0.036). Furthermore, the patients with BORIS‐positive tumors had a poor overall survival (5‐year survival rate: BORIS‐negative 70.0% vs BORIS‐positive 29.9%, log‐rank P = 0.028) in Kaplan–Meier survival analysis and log‐rank test. Multivariate Cox proportional hazard model demonstrated that BORIS expression was an independent poor prognostic factor (hazard ratio = 4.158 [95% confidence interval 1.494–11.57], P = 0.006). Downregulation of BORIS with specific siRNAs resulted in decreased cell proliferation and invasion ability of ESCC cell lines. BORIS may be a useful biomarker for prognostic diagnosis of ESCC patients and a potential target for treatment including by BORIS‐specific immunotherapy and molecular target therapy.

Photodynamic therapy (PDT) is a noninvasive optical treatment method in which the topical or systemic delivery of photosensitizing drugs is followed by irradiation with broadband red light. Coupling photosensitizers with a specific antibody may allow this approach to target specific cancers. This study determines the antitumor efficacy of coupling verteporfin (Visudyne(®)), a hydrophobic polyporphryin oligomer, with an antiepidermal growth factor receptor (anti-EGFR) antibody. Poly[2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenylcarbonyloxyethyl methacrylate] (PMBN) was conjugated with an anti-EGFR antibody and mixed with verteporfin (verteporfin-PMBN-antibody complex). Tumor-bearing mice were intravenously injected with the verteporfin-PMBN-antibody complex or verteporfin plus PMBN without the antibody. Irradiation was conducted at 640 nm with a dose of 75 J/cm(2). The fluorescence intensity in A431 cells in vitro was threefold higher after exposure to verteporfin-PMBN-antibody complex than after exposure to verteporfin-PMBN. In A431 tumor-bearing mice, the intratumor concentration of verteporfin was 9.4 times higher than that of the skin, following administration of the verteporfin-PMBN-antibody complex. Tumor size significantly decreased within 8 days in mice treated with verteporfin-PMBN-antibody complex compared with those treated with verteporfin-PMBN. PDT using a PMBN-verteporfin-antibody complex offers a promising anticancer therapy.

Esophageal squamous cell carcinoma (ESCC) is more sensitive to radiation and chemotherapy than other cancers of the digestive system, and combined modality therapy may represent a promising treatment method. The radiation-sensitizing effect of docetaxel on ESCC cell lines was investigated. A colony formation assay was performed in which ESCC cell lines (TE2, TE3) and A431 were exposed to docetaxel (from 1.0×10(-11) to 10(-7) M) for 3 h to determine the concentration of docetaxel that was not able to kill individual cells (i.e., the non-cytocidal concentration). Individual cell lines were then exposed to the non-cytocidal concentration of docetaxel prior to, during, and after irradiation to determine whether the timing of docetaxel administration affected cell survival. In addition, flow-cytometry was performed, and the cell cycle was examined prior to and after docetaxel exposure to assess the mechanism of docetaxel as a radiation sensitizer. Docetaxel exhibited a concentration-dependent cytocidal effect, with a different IC(50) for each cell type. Almost no cytocidal effect was observed at the following docetaxel concentrations: A431, ≤1.0×10(-10) M; TE-2 and TE-3, ≤1.0×10(-9) M. Concurrent treatment with docetaxel and radiation tended to decrease cell survival in all the cell lines compared with docetaxel or radiation alone. Cell survival was lowest when the cells were treated using X-ray irradiation after docetaxel exposure (p

Hepatocellular carcinoma (HCC) occurs mainly in the liver associated with chronic hepatitis and hepatic cirrhosis as a result of prolonged viral infection. Transforming growth factor-beta (TGF-beta) induces the fibrosis in hepatic cirrhosis, although it is also an inhibitor of hepatocyte proliferation. To understand the role of TGF-beta signaling in HCC progression, we analyzed gene expression in HCC cells in relation to TGF-beta signaling using a two-way clustering algorithm. By the analysis, five HCC cell lines were classified into two groups according to their metastatic capacity. TGF-beta receptor II (TGFBR2) was downregulated in metastatic cells, which did not show a response to TGF-beta. Immunohistochemistry demonstrated clear membrane distribution of TGFBR2 in noncancerous hepatocytes, whereas reduced TGFBR2 expression was observed in 34 of 136 HCCs. In clinical cases, reduced TGFBR2 expression correlated with larger tumor size (P

Aims: We retrospectively assessed the benefits of 5-fluorouracil (5-FU)-and heparin-based portal infusion chemotherapy combined with systemic administration of mitomycin C (MMC) and cisplatin (CDDP) for 4 weeks following surgery (PI4W). The goal was to determine if this treatment prevented liver metastasis and improved survival for patients with potentially curative resection of pancreatic cancer. Methods: 68 patients who underwent pancreatectomy from January 1995 to August 2007 were treated. Of these cases, 22 patients received portal infusion with 5-FU (250 mg/day) for 2 weeks (PI2W) following surgery, while 25 patients received PI4W therapy (250 mg/day of 5-FU with 2,000 IU/day of heparin everyday for 4 weeks, 4 mg MMC on days 6, 13, 20, 27, and 10 mg CDDP on days 7, 14, 21, 28). The remaining 21 patients were treated without adjuvant therapy during the perioperative period. Results: All patients except one completed the portal infusion chemotherapy without toxicity. The cumulative liver metastasis-free survival rate in the PI4W group was significantly higher than those in the other two groups. Furthermore, in the PI4W group, 3-year survival was 91.6% and 5-year survival was 70.5%, rates which were significantly better than those observed in the other two groups. Conclusion: PI4W therapy after surgery is feasible and could become a promising adjuvant therapy in patients with potentially curative resection of pancreatic cancer.

We report a patient with Castleman's disease arising from the gallbladder neck, which caused difficulty in making the differential diagnosis against gallbladder malignancies. A 50-year-old woman presented to our institution with epigastric pain. An abdominal computed tomography scan (CT) and magnetic resonance cholangiopancreatography (MRCP) study showed a 20-mm tumor located in the gallbladder neck for which malignancy could not be completely ruled out. For the definitive diagnosis and treatment, cholecystectomy was performed. In the operation, the main tumor and resection margins of the cystic duct were submitted for frozen section. The tumor was composed of a proliferation of lymphoid tissue with no signs of dysplasia. The ductal margin was free of tumor. The final histopathological diagnosis was unicentric Castleman's disease, a hyaline vascular variant that developed in the gallbladder. The patient is currently in good condition without any signs of recurrence 28 months after the operation. This is the first detailed report of Castleman's disease of the gallbladder. Making a correct diagnosis was very difficult before the operation, and only a surgical approach enabled confirmation of the diagnosis for this patient.

Using dithioester-capped 2-methacryloyloxyethyl phosphorylcholine (MPC) as a macro chain transfer agent, a diblock copolymer was synthesized with n-butyl methacrylate (BMA) as hydrophobic core-forming blocks. The MPC–BMA unit was copolymerized with an immobilizable unit, p-nitrophenylcarbonyloxyethyl methacrylate (NPMA). The NPMA moiety then was modified by the addition of preS1 domain of hepatitis B surface antigen (HBsAg). This micelle-forming nanoparticle, the poly (MPC-co-BMA-co-NPMA) (PMBN) conjugated with preS1 enables solubilization of paclitaxel (PTX) with increased hepatotropism. The 50% inhibitory concentration (IC50) values of PTX and PTX/PMBN-preS1 against the human hepatocellular carcinoma cell line, HepG2, were 1,008 and 131 nM, respectively (p < 0.05). Conjugation of preS1 to PMBN enhanced strongly the synergistic inhibitory effect of paclitaxel on HepG2 cells in vitro, whereas such a change in IC50 was not detected against the human squamous cell carcinoma cell line, A431. Tumor growth rates of a HepG2 xenograft in Balb/c nude mice after intraperitoneal injection of PTX, PTX/PMBN and PTX/PMBN-preS1 were +97.9%, −74.3% and −96.2%*, respectively (*p < 0.05 versus PTX). The local paclitaxel levels after administration of the PMBN-preS1 conjugate were determined in the xenografts by high-performance liquid chromatography and were 8 times higher than that after administration of paclitaxel alone. No side effects attributable to PMBN-preS1 were observed histologically in vital organs, and body weight loss was significantly less in the PTX/PMBN-preS1 group. These studies demonstrate that PMBN-preS1 may be used as a human hepatocyte-specific drug delivery carrier without serious adverse effects. © 2008 Wiley-Liss, Inc.

Background Surgical site infection (SSI) is an important clinical indicator of quality of patient care and infection control; therefore, we aimed to assess risk factors SSI in colon and gastric surgeries. Methods SSI was assessed according to the National Nosocomial Infection Surveillance (NNIS) system (1999). Risk factors examined included operative approach, operative procedure, duration of operation, diabetes mellitus (DM), body mass index (BMI), age, and sex. Results Among 3152 operated patients, 1675 patients were included in the study. The univariate analysis showed that male sex, high BMI, and long duration of operation were significant risk factors for colon surgery and that advanced age, presence of DM and long duration of operation were significant risk factors for gastric surgery. The multivariate analysis indicated that significant risk factors for SSI were BMI of 25 or above, open surgery, and long duration of operation for colon surgery and open surgery for gastric surgery. The SSI rate of laparoscopic colon surgery was 40%, less than that of open colon surgery, and that of laparoscopic gastric surgery was 75%, less than that of open gastric surgery. Conclusion The risk factors for SSI depend on whether the operation is laparoscopic or open and duration of operation. In addition, BMI (25 or above) and age (70 years or above) are risk factors for colon and gastric surgery, respectively.

Endocrine tumors of the pancreas are uncommon. They occur in approximately 1 in 100,000 of the population and represent 1–2% of all pancreatic neoplasms. The tumors show no significant gender predilection and occur at all ages, with a peak incidence between 30 and 60 years. Some may be part of the multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau (VHL) disease. Microadenomatosis represents the presence of multiple small endocrine tumors in the pancreas [1]. Microadenomatosis of the endocrine pancreas is a hallmark of MEN1 syndrome [2, 3]. Interestingly, this condition is rarely described in a non-MEN1 setting [4, 5]. We herein report a unique case of pancreatic microadenomatosis occurring in a nonfamilial VHL disease patient with renal cell carcinoma (RCC). Moreover, genetic analysis for the VHL gene showed a missense mutation at the codon 167 (Arg167Trp, heterozygous) in this case. The type of VHL mutation has been reported to correlate with the clinical manifestations of the disease [6–9], and we also discuss this issue.

1974年4月から2001年12月までに当教室で切除した浸潤性膵管癌199例のうち，24例が5年以上生存し，そのうち再発死亡した剖検4例について，初回手術時病理組織所見とあわせ術後再発形式について検討した．総合的進行度はStage IIIが2例，IVbが2例，根治度はR0が3例，R1が1例で，組織型は管状腺癌3例，粘液癌1例であった．粘液癌の1例は再発が疑われてから約3年間生存しており，粘液癌は浸潤性膵管癌のなかでも比較的ゆっくりと進行する可能性が示唆された．剖検所見から局所再発と肺転移は全例に認め，肝転移3例（75%），腹膜播種3例（75%），その他遠隔転移3例（75%）に認めた．再発時期に関わらず膵床部局所と肝は膵癌再発の好発部位と考えられたが，長期生存後の再発では肺転移の頻度も増える可能性が示された．膵癌の場合，術後5年以上経過しても再発してくる可能性は否定できず，胸部X線画像を含めた定期検査を継続することが重要と考えられた．

The immunopharmacological profile of novel biocompatible water-soluble interleukin-2 (IL-2)-conjugated 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer immunosuppressive agents was established. MPC-co-n- butyl methacrylate (BMA)-co-p-nitrophenylcarbonyloxyethyl methacrylate (NPMA) (PMBN) was prepared as a backbone for these novel agents. PMBN contained MPC as a biocompatible unit, BMA as a hydrophobic domain in water, and NPMA as an immobilizable unit with IL-2. This research showed that proliferation of cell lines with high-affinity IL-2 receptors derived from T cell malignancies were suppressed by the PMBN conjugated with IL-2 (PMBN-IL2 conjugate) incorporating paclitaxel (PTX) and cyclosporin A at lower concentrations than used conventionally. PMBN-IL2 conjugates incorporating PTX also inhibited the proliferation of responder cells in a human mixed lymphocyte culture at a lower concentration than unconjugated drug. However, PMBN-IL2 conjugates incorporating FK506 inhibited proliferation no more than FK506 alone. The PMBN-IL2 conjugate with PTX may therefore be useful for selectively eliminating activated lymphocytes that hyperproduce high-affinity IL-2 receptors. As an entirely human ‘immunotoxin analogue’ it may not be associated with the dose-limiting toxicity and immunogenicity of conventional immunotoxins.

It is thought that the export of angiogenic fibroblast growth factors (FGF) from tumors may be involved in the onset of tumor angiogenesis. To create a new active targeting drug that inhibits the tumor angiogenic process without toxicities to normal cells, human basic FGF (h-bFGF) was inserted genetically into the Gly89 position of cross-linked RNase1 (the ribonuclease inhibitor protein [RI] binding site of cross-linked human pancreatic RNase) to prevent stereospecific binding to RI. The resultant insertional-fusion protein (CL-RFN89) was active both as h-bFGF and as RNase1. Furthermore, it acquired an additional ability of evading RI through steric blockade of RI binding caused by the fused h-bFGF domain. In the present study, the effect of the resultant protein, CL-RFN89, on the antitumor response though its antiangiogenic properties was investigated in an in vivo model. Continuous systemic treatment with CL-RFN89 significantly inhibited the growth of human A431 squamous cell carcinomas in vivo. Seven days of treatment with CL-RFN89 resulted in a 58.2% inhibition of tumor growth compared with control mice (P

Purpose: Hepatocellular carcinoma (HCC) associated with chronic liver disease is known to show an obvious multistage process of tumor progression. We previously identified heat shock protein 70 as a molecular marker of early HCC during investigation of expression profiling in multistage hepatocarcinogenesis. In this report, we examined cyclase-associated protein 2 (CAP2), which is also listed as an up-regulated gene in early HCC. Experimental Design: We measured the level of CAP2 mRNA by real-time quantitative PCR. We raised a polyclonal antibody against CAP2 and we confirmed the expression of CAP2 by immunoblotting and immunohistochemistry in HCC cell lines and HCC tissues. Results: According to real-time quantitative PCR, the level of CAP2 mRNA was up-regulated in early HCC when compared with noncancerous liver tissue, and it was further up-regulated in progressed HCC. We raised a polyclonal antibody against CAP2, which showed a single 53-kDa band of strong intensity in the human HCC cell lines and HCC tissues but only a weak band in the noncancerous liver tissues in Western blot analysis. Immunohistochemical examination of CAP2 revealed its significant overexpression in early HCC when compared with noncancerous and precancerous lesions and in progressed HCC when compared with early HCC. Conclusion: Our findings show that CAP2 is up-regulated in HCC when compared with noncancerous and precancerous lesions. This is the first report that proves that CAP2 is up-regulated in human cancers and that this is possibly related to multistage hepatocarcinogenesis.

Bionanocapsules (BNCs) are hollow nanoscale particles composed of L protein of the hepatitis B virus surface antigen that represent specific affinity for human hepatocytes. BNCs can transfer genes and drugs into human hepatocytes efficiently and specifically. BNC can be expressed in yeast cells. In this study, we developed a new L particle production system using a stably transfected insect cell line. For this purpose, we established a host-vector system using the Trichoplusia ni insect cell line. L particles were efficiently secreted by the overexpression of the L protein, which was fused to the secretion signal peptide. The concentration of L particles was reached approximately 1.7 microg/ml in 5 days during cultivation in a serum-free medium without antibiotic selective pressure. The production of L particles was maintained for at least 75 days. The secretory production of L particles facilitated their easy purification by chromatography. Furthermore, it was demonstrated that purified L particles can transfect only human hepatocytes. Therefore, an insect cell expression system is an attractive tool for the production of BNC.

Background Removal of the primary lesion with a clear operative margin is the standard treatment for gastrointestinal stromal tumor (GIST) of the stomach. However, there are few reports on the operative indications for relatively small GIST. Methods Clinicopathologic features and survival data of all 60 patients with GIST of the stomach treated at Keio University Hospital from 1993 to 2004 were analyzed. Laparoscopic wedge resection was used as the primary procedure for tumors between 2 to 5 cm. Tumors larger than 5 cm were resected by laparotomy or laparoscopy-assisted operation. Results Thirty-five lesions (58.3%) were resected by laparoscopic wedge resection, 3 by laparoscopic operation with a small skin incision and 22 by conventional open procedures. The mean size of the tumors was 42.5 mm, with a range of 18 to 150 mm and a median value of 35.5 mm. All operative margins were clear, but 1 patient had liver metastases at the time of resection of the primary lesion. The median follow-up period was 53 months and the 5-year disease-free survival rate (DFS) was 96.1%. No local recurrence or distant metastasis was encountered in patients with tumors smaller than 4 cm. A statistically significant correlation was observed between tumor size and mitotic count in this cohort ( P = .010). Tumors from the intermediate- (n = 14) and high-risk (n = 10) groups as classified by the Risk Assessment Classification showed significantly worse DFS than the low-risk and very low risk group (n = 35) (89.9% vs 100% in 5-year DFS, P = .045). Even among tumors smaller than 3 cm, 2 of 14 cases (14.3%) were classified into the intermediate-risk group. Conclusions Although a prospective randomized trial remains to be performed, this study provides additional evidence suggesting that the early removal of GIST, at 5 cm or less in size, provides better DFS than later removal of the tumor at a larger size.

Extraskeletal tumors containing multinucleated, osteoclast-like giant cells (OGCs) are uncommon. These neoplasms are most frequently reported in the breast and pancreas. Recently, some authors have suggested that carcinomas containing OGCs may represent a distinct clinicopathological entity with a more favorable prognosis. Occurrence in the gallbladder is extremely rare, with only one previous case. We report here on an additional case of gallbladder carcinoma with an infiltrate of OGCs. A 72-year-old woman presented with postprandial abdominal pain and was found to have a mass in the body of the gallbladder with direct liver invasion. Histological examination showed an adenosquamous carcinoma with an infiltrate of benign OGCs. Immunohistochemical analysis demonstrated that the giant cells were of histiocytic origin. The patient survived for 6 years without evidence of recurrence. This case adds to a small body of literature on gallbladder carcinoma with OGCs. Further studies are required to clearly define the prognostic significance of these giant cells in gallbladder cancer and the differences between adenosquamous carcinoma with OGCs and other gallbladder carcinomas (such as adenocarcinoma and squamous cell carcinoma) with those cells.

筆者らは, B型肝炎ウイルス (HBV) の外殻L蛋白質から形成され約80nmの平均径を持つL粒子を酵母で量産することに成功した. この粒子はウイルスゲノムを含まない中空のバイオナノ粒子であることから, 薬剤や遺伝子などを肝細胞特異的に送達 (ピンポイントDDS) する安全かつ高効率なナノキャリアとし利用できる. さらに, L蛋白質の肝細胞認識部位 (pre-S領域) を各種のターゲティングペプチドやリガンドに置き換えることで, 任意の組織・臓器に再標的化された粒子も構築できるため, 広範なピンポイントDDSへの応用が期待されている.

The bio-nanocapsule (BNC) is our concept of artificial hollow nanoparticles that have been designed and produced through biotechnological procedures. We proposed an empty virus-like particle, which consists of a recombinant L envelope protein of hepatitis B virus (HBV) and a lipid derived from the host cell, as an engineered BNC. Although this BNC was first developed as an immunogen of hepatitis B vaccine, the pre-S1 region in N-terminus of L envelope protein confers hepatocyte specific infectivity of HBV on the BNC. This recombinant BNC is now being developed as a novel platform of drug delivery system (DDS) vector for selective delivery.

A bio-nanocapsule (BNC), composed of the surface antigen (sAg) of the hepatitis B virus, is an efficient nanomachine with which to accomplish the liver-specific delivery of genes and drugs. Approximately 110 molecules of sAg are associated to form a BNC particle with an average diameter of 130 nm. The L protein is an sAg peptide composed mainly of preS and S regions. The preS region, with specific affinity for human hepatocytes, is localized in the N-terminus. The S region following the preS has two transmembrane regions responsible for the formation of particles. In this study, the fusion of emerald green fluorescent protein (EGFP) at the C-terminus of the S region was designed to deliver proteins to human hepatocytes. Truncation of the C-terminus of the S region was required to obtain sufficient expression levels in Cos7 cells. The nanoparticles that were produced delivered EGFP to human hepatoma cells, displaying the EGFP moiety outside, or enclosing it inside. However, only a single orientation characterizes the particle, so that either type of L fusion particle could be effectively and independently separated by an antibody affinity column. The dual C-terminal topologies of the L fusion particles designed in this study could be applied to various proteins for the C-terminal moiety of the L fusion proteins, depending on the character of the proteins, such as cytoplasmic proteins, as well as cytokines or ligands to cell surface receptors. We suggest that this fusion design is the most efficient way to prepare a BNC that delivers proteins to specific cells or tissues.

Human pancreatic ribonuclease-1 (RNase1) does not exhibit its cytotoxicity unless it is artificially internalized into the cytosol. Furthermore, once it encounters the cytosolic RNase inhibitor (RI), the activity of RNase1 is seriously reduced. To achieve the cellular targeting of RNase1 and the blocking of RI binding simultaneously, the basic fibroblast growth factor (bFGF) sequence was inserted into RNase1 at the RI binding site using a gene fusion technique. The effect of this fusion protein, CL-RFN89, on the angiogenesis, which was accelerated by FGF-FGF receptor interaction, was investigated. It was shown by using fluorescein-labeled CL-RFN89, that the binding to human umbilical vein endothelial cells (HUVECs) was dependent on the existence of the FGF receptors. In addition, CL-RFN89 inhibited the cellular growth of HUVECs in vitro and also inhibited the tube formation, using a three-dimensional tube formation assay. Furthermore, this fusion protein was shown to prevent in vivo tumor cell-induced angiogenesis, using the mouse dorsal air sac assay. These results demonstrated that CL-RFN89 inhibits angiogenesis in vitro and in vivo and that it can be expected to be a potent antiangiogenic agent.

Few studies have reported the incidence and clinical outcomes of gallstone disease after extended (D2) lymph node dissection for gastric cancer. The present study was designed to retrospectively compare limited (D1) and D2 dissections in terms of gallstone formation, presentation of gallstones, and surgery for gallstone disease. A total of 805 Japanese gastric cancer patients (595 male, 210 female) who underwent curative resection with D1 (n = 490) or D2 (n = 315) dissection were retrospectively reviewed. Of those subjects followed for 70.5 +/- 44.3 months (range: 2-196 months), 102 (12.7%) developed gallstones. The incidence of gallstone formation was higher in the D2 group than in the D1 group (17.8% vs. 9.4%, p = 0.001). The interval between gastrectomy and detection of gallstones was shorter in the D2 group than in the Dl group (18.8 +/- 11.4 months vs. 29.4 +/- 18.3 months, p = 0.002). Of those with gallstones followed for 48.0 +/- 28.6 months (range: 1-158 months), 74 (72.5%) remained asymptomatic, and 15 (14.7%) experienced mild biliary pain. Thirteen patients (12.7%) developed recurrent biliary pain (n = 3) or biliary complications (n = 10; 6 acute cholecystitis, 3 obstructive jaundice, and 1 cholangitis), and required surgical treatment. Surgery was more frequently sought in the D2 group than in the D1 group (19.5% vs. 4.3%, p = 0.033). In conclusion, patients with D2 dissection developed gallstones more frequently and earlier than patients with D1 dissection. Of those with gallstones, patients with D2 dissection required surgery more often than patients with D1 dissection. A closer follow-up should be mandatory for gallstone disease after D2 dissection, but further studies are needed before generalizations can be made.

Surgical site infection (SSI) surveillance was examined in gastric cancer patients who had undergone an open gastrectomy between 1997 and 2003 at Keio University Hospital in Tokyo, Japan. National Nosocomial Infections Surveillance (NNIS) reports and several studies have discussed SSI risk factors, but only open gastrectomy was analyzed by regression analysis. The purpose of this study was to examine these issues by performing a regression analysis for the prediction of SSI. SSI was defined by the surgical patient component according to the NNIS system (1999) produced by the Centers for Disease Control and Prevention. Patients undergoing an open gastrectomy were followed up and monitored for SSIs. Risk factors for SSI, after all factors were considered, were studied using single and multivariate analysis. The study enrolled 984 patients who had undergoing an open gastrectomy. Using multivariate and logistic regression analysis, the duration of the operation was identified as a [corrected] risk factor for SSI at open gastrectomy. Although numerous potential risk factors in surgical patients were examined, the duration of the operation was the only significant risk factor for SSIs after open gastrectomy.

Establishing a method to control peritoneal dissemination is one of the most pressing issues in the postsurgical treatment of pancreatic cancer. In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on peritoneal disseminations of hamster pancreatic cancer cells, PGHAM-1. After the orthotopically inoculation of 2×10 6 PGHAM-1 cells, DC pulsed with PGHAM-1-derived tumor lysates, DC alone or PBS as a vehicle was injected intraperitoneally (i.p.) three times at weekly intervals. The group treated with DC or DC+lysate was found to have smaller disseminated tumors than the vehicle-treated. In addition, mean survival time in the DC+lysate groups was significantly longer than the PBS group. These findings suggested that DC-based immunotherapy might be efficient for the treatment of peritoneal disseminations of the pancreatic cancer.

Objective Using a chemoinvasion assay, we show that platelets promote invasiveness of 5 pancreatic adenocarcinoma cell lines. Methods Gelatin zymography and Western blot analysis were performed to detect metalloproteinase-9 (MMP-9) secreted from tumor cells in the presence or absence of platelets. The effects of antiplatelet agents on the invasiveness of tumor cells and the secretion level of MMP-9 were evaluated. Results The number of traversed tumor cells significantly increased when incubated with platelets compared without platelets in all cell lines. The MMP-9 band was detected in all tumor cell lines, and the intensity was obviously greater in conditions of incubation with platelets than without. In the experiment of antiplatelet agents effects, it was confirmed that invasiveness of tumor cells significantly decreased following incubation with cilostazol depending on the concentration in spite of the presence of platelets. The level of MMP-9 also significantly decreased in the ELISA analysis. Conclusions These data mean platelets activate invasiveness of tumor cells because of enhanced MMP-9 secretion. Furthermore, anti-platelet drugs may inhibit invasiveness of tumor cells due to decreased MMP-9 secretion, and this inhibition may lead to the suppression of tumor cell invasion. We propose that antiplatelet agents are applicable in clinical treatment to inhibit metastasis of malignant tumor cells.

Intraductal papillary-mucinous tumors (IPMT) consist of cells with varying histologic degrees of severity and exhibit multiple tumor loci; however, whether or not these lesions exhibit the same genetic changes has not been clarified. To investigate this point, we analyzed K-ras mutations in multiple IPMT lesions from each patient enrolled in our study and compared our findings to those for patients with ductal adenocarcinoma of the pancreas (DC). Twenty IPMT specimens and 7 DC specimens were resected, microdissected and analyzed for the presence of K-ras mutations. The mutated genes were then sequenced using a genetic analyzer. K-ras mutations were observed in 80% of IPMT and 100% of DC patients. More than 2 types of K-ras mutation were observed in the main tumors of 43.8% of IPMT and 0% of DC patients. K-ras mutations in peritumoral and separated lesions were observed in 66.7% and 62.5% of IPMT patients, respectively. At least one identical mutation between the main tumor and the peritumoral or separated lesions was recognized in all of the IPMT patients with those lesions. Different mutations from those in the main tumor were observed in 40% of IPMT patients with separated lesions. The survival curve of IPMT-carcinoma patients with more than 2 types of K-ras mutation in the main tumor was better than that with one type of K-ras mutation. IPMT patients exhibit a remarkably genetic heterogeneity in main tumor and have good prognosis. © 2004 Wiley-Liss, Inc.

B型肝炎ウイルス (hepatitis B virus; HBV) はヒト肝細胞に対し強い感染力を有する. その感染機構を担うHBV表面抗原 (HBV surface antigen; HBsAg) は, 組換え酵母を用いて大量生産が可能であり, 酵母由来の膜成分を取り込んで, 平均直径220nmのHBsAg粒子を形成することが知られている. 最近, HBsAg粒子内部に遺伝子, タンパク質および薬剤を封入して, 静脈注射のみでヒト肝臓特異的送達が可能なキャリヤーとして非常に有効であることを示した. また, HBsAg粒子の表面に存在するヒト肝細胞特異的な認識部位を, 他の細胞を認識する分子へ置換することにより, 粒子表面の提示分子に応じた標的細胞を生体内で標的化する「中空バイオナノ粒子」の創製に成功した. これらの結果は, 生体内ピンポイントデリバリーシステムの新しいキャリヤーとして, 特に遺伝子治療分野で中空バイオナノ粒子が非常に有望であることを示している.

Objective: To investigate the involvement of vasoactive agents, endothelin (ET)-1, and atrial natriuretic peptide (ANP), and the responses of cytokines in patients undergoing total gastrectomy.Design: Prospective study.Setting: University hospital, Japan.Subjects: 20 patients with advanced gastric cancer who had undergone total gastrectomy with lymph node dissection.Interventions: Serum or plasma samples collected on the day before the operation, at the time of skin closure, and on postoperative days 1, 3, 5, and 7.Main outcome measures: Concentrations of acute phase reactants, cytokines (interleukin (IL)-1, tumour necrosis factor (TNF) and interleukin (IL)-6), and vasoactive agents (ET-1 and ANP).Results: There were significant increases in concentrations of IL-6 and acute phase reactants postoperatively. ET-1 and ANP concentrations did not change significantly.Conclusion: There was no correlation between concentrations of the vasoactive agents ET-1 and ANP, and those of acute phase reactants or cytokine...

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.

We investigated whether aberrant p53 and p16 expression, the Ki-67 labeling index (LI), and int-2/cyclin D1 gene amplification predict the response to chemoradiotherapy (CRT) in patients with locally advanced esophageal squamous cell carcinoma (ESCC).p53 and p16 expression status, the Ki-67 LI, and int-2/cyclin D1 amplification were assessed by immunohistochemical staining and slot blot analysis in pretreatment endoscopic biopsy specimens of 41 patients with T4 or M1 Lym (distant lymph node metastasis) ESCC. All patients received a course of chemotherapy (5-fluorouracil and cisplatin) with radiotherapy.The CRT therapeutic response rate was 71%, and resection after CRT was successful in 15 of the cases in which the CRT effect was significant. The cumulative survival rate after CRT in the p53-negative patients was significantly higher than in the p53-positive patients (P =.037). The mean Ki-67 LI in the CRT response cases was significantly higher than in the CRT no-response cases (P =.023). Multivariate regression analysis revealed high Ki-67 LI to be an independent variable linked to a pathologic complete response to CRT (P =.033). The cumulative survival rate after CRT in the group that was p53-negative and int-2/cyclin D1 amplification-positive was significantly higher than in the other groups (P =.008).Evaluating predictive factors in pretreatment endoscopic biopsy specimens may allow selection of more suitable multimodal treatment for ESCC patients and improve their quality of life.

We carried out molecular cytogenetic characterization of 11 cell lines derived from hepatocellular carcinomas (HCCs) and 51 primary HCCs. Comparative genomic hybridization (CGH) revealed frequent amplification at 13q34, where we had detected amplification in several other types of tumor, including esophageal squamous cell carcinomas (ESC). Previously, we suggested possible involvement of TFDP1, encoding a transcription factor DP-1, in the 13q34 amplification observed in a primary ESC. Therefore, we investigated amplifications and expression levels of 5 genes mapped on the amplified region, including TFDP1, for exploring amplification targets at 13q34 in HCCs. 3 of those genes, TFDP1, CUL4A (cullin 4A), and CDC16 (cell division cycle 16), showed distinct amplification and consequent over-expression in some cell lines. Moreover, each was amplified in 3 or 4 of the 51 primary HCCs, and all 3 were amplified in 2 tumors, in which their expression patterns correlated with amplification patterns. To elucidate the functional role of TFDP1 in HCC, we examined expression levels of genes downstream of TFDP1 with real-time quantitative polymerase chain reaction (PCR). Expression of cyclin E gene (CCNE1) correlated closely with that of TFDP1 in not only cell lines, but also primary tumors. Treatment of HCC cells with the antisense oligonucleotide targeting TFDP1 resulted in down-regulation of CCNE1, suggesting that TFDP1 overexpression led to up-regulation of CCNE1 that encoded a positive regulator for cell cycle G1/S transition. In conclusion, our findings suggest that TFDP1, CUL4A, and CDC16 are probable targets of an amplification mechanism and therefore may be involved, together or separately, in development and/or progression of some HCCs.

Background . The presence of tumor cells in the blood stream is considered evidence of a high risk of distant organ metastasis. We examined the usefulness of telomerase activity in peripheral blood polymorphonuclear cells as an indicator of distant metastasis in patients with esophageal squamous cell carcinoma. Methods . Telomerase activity was measured in the peripheral blood mononuclear cell and polymorphonuclear cell fractions obtained from blood samples of healthy volunteers mixed with squamous cell carcinoma cell lines, and cell distribution was analyzed by flow cytometry. Then telomerase activity of forty-two polymorphonuclear cell fractions obtained from esophageal squamous cell carcinoma patients was measured. Results . Telomerase activity was detected in polymorphonuclear cell fractions and cell distribution analysis revealed the presence of esophageal squamous cell carcinoma cells. Organ metastasis was detected in 7 (78%) of the 9 patients with telomerase-positive polymorphonuclear cell fractions as opposed to only five (15%) of the 33 with telomerase-negative cases, and there was a significant positive correlation between telomerase activity and organ metastasis ( p Conclusions . Measurement of telomerase activity in the polymorphonuclear cell fractions is useful for identifying a high risk group for distant organ metastasis in patients with esophageal squamous cell carcinoma.

Eosinophil cationic protein (ECP), one of the major components of basic granules of eosinophils, is cytotoxic to tracheal epithelium. However, the extent of this effect on other cell types has not been evaluated in vitro. In this study, we evaluated the effect of ECP on 13 mammalian cell lines. ECP inhibited the growth of several cell lines including those derived from carcinoma and leukemia in a dose-dependent manner. The IC50 values on A431 cells, MDA-MB-453 cells, HL-60 cells and K562 cells were␣estimated to be ≈ 1–5 µm. ECP significantly suppressed the size of colonies of A431 cells, and decreased K562 cells in G1/G0 phase. However, there was little evidence that ECP killed cells in either cell line. These effects of ECP were not enhanced by extending its N-terminus. Rhodamine B isothiocyanate-labeled ECP started to bind to A431 cells after 0.5 h and accumulated for up to 24 h, indicating that specific affinity for the cell surface may be important. The affinity of ECP for heparin was assessed and found to be reduced when tryptophan residues, one of which is located at a position in the catalytic subsite of ribonuclease in ECP, were modified. The growth-inhibitory effect was also attenuated by this modification. These results suggest that growth inhibition by ECP is dependent on cell type and is cytostatic.

Pulmonary injury with leukocyte infiltration is a frequent occurrence in obstructive cholangitis patients. We wished to evaluate the roles of reactive oxygen species and vascular cell adhesion molecule-1 (VCAM-1) in this distant organ failure. Wild type (WT) and transgenic (SODtg) mice overexpressing superoxide dismutase underwent bile duct ligation and transection (BDL). VCAM-1 expression was quantified, and histopathology was assessed for the liver and lung. BDL resulted in increased leukocyte infiltration to the lung at five days in WT mice. VCAM-1 expression significantly increased in WT mouse liver at three days and WT mouse lung at five days. When these same experiments were performed in SODtg mice, these increases in leukocyte infiltration and VCAM-1 expression in lung were significantly attenuated. These data suggest that reactive oxygen species produced in response to BDL may up-regulate VCAM-1 expression in the lung and play an important role in the pathophysiology of this pulmonary injury.

Molecular genetic analyses have clarified that accumulation of genomic changes provides important steps in carcinogenesis and have identified a number of valuable genetic markers for certain cancers. To date, however, no prognostic markers have been identified for hepatocellular carcinoma (HCC). In this study, we used restriction landmark genomic scanning (RLGS), a new high-speed screening method for multiple genomic changes, to detect unknown genetic alterations in HCC. Thirty-one HCC samples and their normal counterparts were examined by RLGS. Eight spot changes were common in several cases, and all were seen only on the HCC profile. Five of these spots were detected in more than 12 of 31 cases (38.7%). Viral infection had no influence on changes in the RLGS spots. The disease-free survival rate for patients with > or =16 changed RLGS spots was significantly lower than that for patients with fewer changed RLGS spots (< or =15 spots) (P

Background The aim of this study was to examine telomerase activity in affected and adjacent tissue in patients with oesophageal squamous cell carcinoma (SCC). Methods Telomerase activity was measured in oesophageal SCC cells, normal oesophageal culture cells, primary cancer tissues and adjacent normal tissues from patients with oesophageal SCC using a polymerase chain reaction-based assay. Results All oesophageal SCC cells had telomerase activity, whereas normal cultured cells showed no activity. All 57 cancer tissues showed telomerase activity compared with only five (10 per cent) of 50 normal tissues. Cancer cells infiltrating the vessels of mucosal or submucosal tissues in non-cancerous regions were detected in four of five telomerase-positive normal tissues, whereas such infiltration was detected in only three of 45 telomerase-negative normal tissues. Conclusion In patients with oesophageal SCC, measurement of telomerase activity in normal epithelium is a highly sensitive method of detecting the microinvasion of cancer cells.

The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with a gene encoding human epidermal growth factor (hEGF). The hybrid human protein was isolated from Escherichia coli inclusion bodies, refolded and purified to homogeneity. The fusion protein competed with 125I-hEGF for binding to hEGF receptors (EGFR) and had ribonucleolytic activities approaching those of hpRNase1. Several conformations having different enzymatic activities could be detected after reversed-phase high-performance liquid chromatographic analysis, the less hydrophobic molecules being the most active. The hybrid protein was specifically cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line, with an IC50 of approximately 10(-7) M. In contrast, recombinant hpRNase1 had an IC50 higher than 10(-4) M. A mixture of free hEGF and free hpRNasel was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected in EGFR-deficient control cells. Taken together, these data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast cancers overexpressing EGFR, which correlate with a poor prognosis and cannot be cured by surgery alone. Engineering hybrid molecules with endogenous human proteins for targeted therapy may alleviate the dose-limiting immunogenicity and toxicity of conventional immunotoxins.

Missile therapy, which destroys cancer cells specifically, has been advocated as an effective modality for the treatment of carcinoma. We have developed an immunoconjugate consisting of the monoclonal antibody MSN-1 (IgM), which reacts strongly with endometrial adenocarcinomas, combined with a plant hemitoxin named gelonin via a disulfide bond using N-succinimidyl-3-(2-pyridyldithio) propionate and 2-iminothiolane, and examined its selective cytotoxicity in athymic mice. The reductions in resected weights of target tumor cells, at the local site of MSN-1-gelonin immunoconjugate treatment, were 96% with local administration and 75% with caudal vein administration, as compared with the untreated group. There was no weight loss in treated mice. Our results suggest that this MSN-1-gelonin immunoconjugate has potential clinical applications in the treatment of endometrial adenocarcinomas.

A highly specific and effective magnetic resonance imaging (MRI) contrast agent was prepared by coating super-paramagnetite particles with monoclonal antibodies (MAbs) directed against epidermal growth factor receptors (EGFRs), which are over-expressed in esophageal squamous cell carcinoma. The preparation maintained both the immunoreactivity of the MAbs and the full relaxing capability of the magnetite particles. The particles of this EGFR-specific contrast agent are 13.2 +/- 1.9 nm in size, and thus, it is assumed that they are smaller than capillary pores and, hence, able to escape scavenging by reticulo-endothelial system cells. The EGFR-specific T2-relaxing ability of this contrast agent was ascertained first in vitro, using the EGFR-expressing cell line TE8 and the EGFR-deficient cell line H69. The results in athymic rats bearing TE8 or H69 tumors revealed that the agent has EGFR-specific MRI contrast capacity in vivo. The electron-microscopic findings in TE8 tumor-bearing rats revealed that the magnetite particles had been taken up by their lysosomes. In conclusion, immuno-specific MRI using magnetite particles coated with MAbs against EGFR appears to be useful in the diagnosis of squamous cell carcinoma of the esophagus.

A field image acquisition system on the World Wide Web (WWW) was developed. The system contains two sub-systems. One is a real-time field image system to deliver real-time still images to WWW clients at their requests. This sub-system is run by a camera server that is composed of a Windows95 personal computer with a videocapture card and a video camera that can be controlled via a serial connection to the computer. By this sub-system, a WWW client can obtain real-time quality images by remotely controlling the camera angle and zooming rate. The other sub-system is the automated field image database system. This sub-system is run under the cooperation of the camera sever and the database server of a Windows NT-base personal computer. The field image database system automatically manages the WWW image database for the images acquired periodically by the camera server and serves the image retrieval system to WWW clients. Both of the sub-systems have been working well and were found to be useful in several agricultural scenes such as remote diagnoses of crop growth by experts and observation of remotely located experimental fields and etc. A mobile camera server with a wireless LAN system was also developed.

The inflammatory cytokines interleukin (IL) 1 and tumor necrosis factor (TNF) may play an important role in hepatic ischemia-reperfusion (I/R) injury. To study the role of IL-1 in hepatic I-R injury, we investigated the effect of pretreatment with IL-1 receptor antagonist (IL-1ra) on the production of IL-1, TNF, histological findings in the liver, and the survival rate for 7 days. Rats were subjected to 90 min of partial liver warm ischemia by clamping the vessels of the left and middle lobes. In the IL-1ra-treated group, IL-1ra was given 5 min before liver ischemia was induced. IL-1alpha and TNF levels were determined in blood and liver at 0, 30, 90, and 180 min after reperfusion. In a second experiment to determine the effect of IL-1ra pretreatment on survival rate, after 90 min of partial liver ischemia, the right lateral and caudate lobes were excised, leaving only the ischemic lobes. In both groups, IL-1alpha was undetectable in blood, but increased in liver tissue. TNF increased in both blood and liver tissue as reperfusion time increased. Histological evidence of tissue injury was minimal in the IL-1ra-treated group. Furthermore, in the IL-1ra-treated group, the production of TNF decreased in both blood and liver tissue compared with the nontreated group. Survival rates in the IL-1ra-treated and nontreated group were 80% and 30%, respectively. The data demonstrated that the production of IL-1 and TNF increases in hepatic I-R injury and that pretreatment with IL-1ra protects the liver from ischemic insult, indicating an important role for IL-1 in I-R injury.

Mammalian pancreatic ribonuclease (RNase) was conjugated chemically via a disulfide bond to human or murine epidermal growth factor (EGF). The conjugation between EGF and RNase was ascertained by SDS-PAGE using reduced and nonreduced conjugates. The EGF–RNase conjugate retained potent RNase activity and competed with 125I-EGF for binding to EGFR to the same extent as unconjugated EGF. Both the human and murine EGF–RNase conjugates showed dose-dependent cytotoxicity against EGFR- overexpressing A431 human squamous carcinoma cells with IC50 values of 3×10-7 M and 6×10-7 M, respectively, whereas free RNase had an IC50 of 10-4 M. Against the EGFR-deficient small-cell lung cancer cell line H69, the EGF–RNase conjugate had no cytotoxic effect. The Human EGF–RNase conjugate showed dose-dependent cytotoxicity against other squamous carcinoma cell lines (TE-5, TE-1) and breast cancer cell lines (BT-20, SK-BR-3, MCF-7) and the cytotoxicity of the conjugate correlated positively with the level of expression of EGFR by each cell line. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone on any cell line. Excess free EGF blocked EGF–RNase conjugate cytotoxicity against A431 cells. These results suggest that the EGF–RNase conjugate may be a more effective anticancer agent with less immunogenicity than coventional chimeric toxins.

Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The EGF-RNase conjugate showed dose-dependent cytotoxicity for EGF receptor-overexpressing A431 and TE-8 human squamous carcinoma cells with an IC50 of 2 x 10(-7)M and 10(-6)M, respectively, whereas the IC50 of RNase alone was almost 10(-4)M. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone. The conjugate showed no detectable cytotoxicity against EGF receptor-deficient small cell lung cancer cells (H69). Addition of excess EGF in the medium protected A431 cells from the EGF-RNase conjugate cytotoxicity. The cytotoxicity of the EGF-RNase conjugate was positively correlated with the EGF receptor numbers of each cell line. The chimeric toxin composed of only human proteins might be a more useful anti-cancer agent with less immunogenicity than the conventional chimeric toxins.

Betacellulin (BTC) was found to be expressed mainly in human pancreas and small intestine. This finding suggests that BTC possesses some specific function distinguished from the other members of epidermal growth factor (EGF) family. To clarify this function, the released form of human BTC has been expressed in E.coli, purified, and characterized. The recombinant human BTC was produced as an inclusion body. This material was dissolved in guanidine-HCl under reducing conditions, refolded, and purified through sequential liquid chromatography. Purified BTC was electrophoresed under reducing conditions and a molecular size of 18 kDa was determined, which is the supposed size of a dimer of the peptide. However, chemical analysis failed to show a covalently linked dimer. The molecular mass of BTC analyzed by mass spectrometry revealed it to be 9 kDa, which is consistent with theoretical value for a monomer. Recombinant BTC showed growth promoting activity for mouse fibroblasts and rat aortic smooth muscle cells which was equivalent to EGF On the other hand, BTC was found to exhibit a growth inhibitory effect on the cells overexpressing EGF receptor.

Background. The results of recent studies have suggested that p53 gene abnormalities are associated with carcinogenesis in several neoplasms. It is believed that bilateral breast carcinomas develop as a result of a different carcinogenetic mechanism and genetic environment from those of unilateral lesions. Methods. p53 Gene abnormalities in bilateral primary breast cancer were detected by polymerase chain reaction-single strand comformation polymorphism (PCR-SSCP) analysis. A total of 76 paraffin embedded tissue specimens from 38 patients with bilateral primary breast cancer were examined, and 62 patients with unilateral breast cancer were analyzed as control subjects. The bilateral tumors were defined as primary, based on clinical parameters and the presence of an intraductal component. There were 13 patients with synchronous bilateral breast cancer and 25 with metachronous bilateral breast cancer. Results. p53 Gene abnormalities were detected in 50% of the bilateral and 25.8% of the unilateral cases, and the difference was significant (P < 0.01, chi-square test). Abnormalities were detected in 56% of the metachronous cases, representing a much higher incidence than that of the unilateral cases (P < 0.001, chi-square test). The incidence of p53 gene abnormalities in the first and second tumors of the metachronous cases was 44% and 68%, respectively. The percentage of patients with a p53 gene abnormality and positive family history was higher for those with bilateral than with unilateral breast cancer (P < 0.01, chi-square test). Conclusion. These findings indicate that the genetic changes and mechanism of carcinogenesis in bilateral and unilateral breast cancer are different.

Hepatitis B virus (HBV) envelope L proteins, when synthesized in yeast cells, form a hollow bionanocapsule (BNC) in which genes (including large plasmids up to 40 kbp), small interfering RNA (siRNA), drugs, and proteins can be enclosed by electroporation. BNCs made from L proteins have several advantages as a delivery system: Because they display a human liver-specific receptor (the pre-S region of the L protein) on their surface, BNCs can efficiently and specifically deliver their contents to human liver-derived cells and tissues ex vivo (in cell culture) and in vivo (in a mouse xenograft model). Retargeting can be achieved simply by substituting other biorecognition molecules such as antibodies, ligands, receptors, and homing peptides for the pre-S region. In addition, BNCs have already been proven to be safe for use in humans during their development as an immunogen of hepatitis B vaccine. This protocol describes the loading of BNCs and their use in cell culture and in vivo.

消化器癌における分子生物学的パラメーターの予後因子としての有用性を明らかにすることを目的として, 癌遺伝子増幅の有無と予後や再発など癌患者の臨床像との関連を解析した.その結果食道癌では, int-2およびc-erb B癌遺伝子増幅が, 胃癌ではint-2およびc-erb B-2癌遺伝子増幅が高率に認められたが, 大腸癌や肝細胞癌では, いずれの癌遺伝子も増幅頻度は10%以下であった.食道癌, 胃癌ではint-2増幅群で術後累積生存率が低下し, さらに食道癌では遠隔臓器転移が, 胃癌では腹膜転移と遠隔臓器転移が非増幅群に比較して有意に高率であった.c-erb B癌遺伝子増幅は食道癌で高率にみられたが, これらの症例ではいずれも手術時にリンパ節転移がみられ, しかも術後5年生存率は低下していた.以上, 分子生物学的手法により癌遺伝子増幅を検索することにより, 癌患者の治療上重要な情報を得ることが可能であり, 有用な腫瘍マーカーであることが明らかにされた.

For the targeted delivery of genes and drugs to the human liver, hepatitis B virus (HBV) envelope L particles, which form hollow nanoparticles and display a peptide that is indispensable for liver-specific infection by HBV in humans, should be a useful tool. To test the efficacy of these particles in vivo, in the present study we generated a small animal model harboring a functional human liver tissue xenograft. An anti-asialo GM1 antibody was administered to SCID mice to induce the depletion of natural-killer-cell-dependent immune responsibility and then the mice underwent transplantation of a noncancerous liver tissue originating in humans into the kidney capsule. Interestingly, human liver tissues were engrafted in 58% of the animals at least for 14 days shown by a human hepatocyte-specific antibody. The engineered HBV nanoparticles which contained fluorescent chemicals could selectively bind to the xenograft in these immune-deficient mice when they were administered systemically. These results suggested that the model animal was usable to demonstrate the efficacy of the nanoparticles that could deliver chemicals specific to the normal human liver tissue by systemic administration, which will facilitate the study of human liver cell biology, drug metabolism and infections with hepatotropic viruses.