Search

Blood vessels deliver oxygen and nutrients to tissues, and vascular networks are spatially organized to meet the metabolic needs for maintaining homeostasis. In contrast, the vasculature of tumors is immature and leaky, resulting in insufficient delivery of nutrients and oxygen. Vasculogenic processes occur normally in adult tissues to repair “injured” blood vessels, leading us to hypothesize that bone marrow mononuclear cells (BMMNC) may be able to restore appropriate vessel function in the tumor vasculature. Culturing BMMNCs in endothelial growth medium resulted in the early outgrowth of spindle-shaped attached cells expressing CD11b/Flt1/Tie2/c-Kit/CXCR4 with proangiogenic activity. Intravenous administration of these cultured vascular proangiogenic cells (VPC) into nude mice bearing pancreatic cancer xenografts and Pdx1-Cre;LSL-KrasG12D;p53lox/+ genetically engineered mice that develop pancreatic ductal adenocarcinoma significantly reduced areas of hypoxia without enhancing tumor growth. The resulting vasculature structurally mimicked normal vessels with intensive pericyte coverage. Increases in vascularized areas within VPC-injected xenografts were visualized with an ultrasound diagnostic system during injection of a microbubble-based contrast agent (Sonazoid), indicating a functional “normalization” of the tumor vasculature. In addition, gene expression profiles in the VPC-transplanted xenografts revealed a marked reduction in major factors involved in drug resistance and “stemness” of cancer cells. Together, our findings identify a novel alternate approach to regulate abnormal tumor vessels, offering the potential to improve the delivery and efficacy of anticancer drugs to hypoxic tumors. Cancer Res; 70(15); 6283–92. ©2010 AACR.

Supplementary Figure 2 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Movie 1 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Table 1 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Figure 1 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Figure Legends 1-3 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Figure 3 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Supplementary Table 2 from Transplanting Normal Vascular Proangiogenic Cells to Tumor-Bearing Mice Triggers Vascular Remodeling and Reduces Hypoxia in Tumors

Trial design: Elimination of small colorectal polyps with cold snare polypectomy (CSP) is reported to be as safe as hot snare polypectomy (HSP). The effectiveness of CSP has not been clearly defined, and the incidence of long-term recurrence has not been determined. We conducted a randomized control study and one-year follow-up study to assess their safety and efficacy. Methods: Patients with small colorectal polyps were randomized to receive CSP or HSP. Polypectomy was performed to determine the pathological curability, and patients completed a questionnaire about the tolerability of the procedure. Follow-up colonoscopy was performed to determine the local recurrence of adenoma. The major outcome was the non-inferiority of CSP to HSP in the rate of delayed bleeding and minor outcomes, including the incidence of immediate bleeding and perforation, procedural time, and the resection rate. Results: A total of 119 participants were recruited in this randomized study and underwent polypectomy. Among the 458 polyps, 332 eligible polyps were analyzed. The rate of adverse events was 0.6% (1/175) for CSP and 0% (0/157) for HSP, which showed the non-inferiority of CSP. While the complete resection rate of CSP was very high (100%), the R0 rate was not satisfactory (horizontal margin, 65.5%; vertical margin, 89.1%). Two local recurrences (2.5%) were observed in the follow-up of 80 adenomas treated with CSP. No recurrence was found in 79 lesions in the HSP group, which was not significant (P = .06). Conclusions: Colorectal polyps were safely resected using CSP, similar to HSP. Most would agree to say that CSP is considered safer than HSP. The main question is then related to efficacy. Our results of the present study demonstrate that recurrence after CSP should be carefully managed for curative treatment.

Eradication therapies for Helicobacter pylori infection are advancing as new acid inhibitory reagents approved. The aim of this study was to assess the efficacy and safety of vonoprazan-based triple treatment. Triple therapy with vonoprazan and two antibiotics (amoxicillin and clarithromycin or metronidazole) received focus in this analysis. We performed a multicenter retrospective study of patients who received vonoprazan-based eradication therapy between February 2015 and February 2016 and conducted a review of the literature. The eradication rate among the 799 patients in our multicenter study was 94.4% (95% confidence interval [CI] 92.6–96.2%) in the per-protocol analysis for first-line treatment (with vonoprazan 20 mg, amoxicillin 750 mg, and clarithromycin 200 or 400 mg, twice a day for 7 days) and 97.1% (95% CI 93.0–101.1%) for second-line treatment (with vonoprazan 20 mg, amoxicillin 750 mg, and metronidazole 250 mg, twice a day for 7 days). The overall incidence of adverse events was 4.4% in an intention-to-treat analysis with no patients hospitalized. In a literature review, six reports, in which 1380 patients received vonoprazan-based first-line eradication therapy, were included and were all reported by Japanese researchers. The eradication success rates in per-protocol analysis were between 85 and 93%, which was roughly the same among the studies. Vonoprazan-based triple therapy was effective and safe for Helicobacter pylori eradication in real-world experience, confirmed by a multicenter study and a review of the pertinent literature.

We herein report a rare case of ileal adenocarcinoma that was completely removed by endoscopic submucosal dissection (ESD) without any complications. An 80-year-old man was referred to our hospital to undergo treatment for an ileal tumor. Conventional colonoscopy showed a reddish depressed lesion that was classified as type 0-IIc according to the Paris classification. The ileal tumor was successfully removed en bloc by ESD with a negative surgical margin. The histological findings showed a well-differentiated adenocarcinoma with no submucosal or lymphovascular invasion. Colonoscopy and CT performed one year after ESD showed no local recurrence, stenosis, or lymph node metastasis.

We herein report the rare case of a 76-year-old woman who underwent cholecystectomy with bile duct resection for advanced gallbladder cancer associated with pancreaticobiliary maljunction (PBM) and subsequently developed multiple cancers of the pancreaticobiliary system (the distal bile duct, intrahepatic duct and pancreatic duct) after the operation. We performed conventional endoscopic retrograde cholangiopancreatography (ERCP) using a side-viewing scope to evaluate the masses in the distal bile duct and the pancreatic duct. We also performed ERCP using double-balloon enteroscopy (DBE) to observe the mass in the intrahepatic duct. It was possible to directly observe the lesion using DBE and to perform a biopsy under visual control. All lesions were correctly diagnosed by the combination of ERCP using different endoscopes. The present case suggests that it is necessary to pay close attention (with regard to carcinogenesis) to the whole pancreaticobiliary system in patients with PBM. In addition, the combination of ERCP using DBE and a side-viewing scope may be useful for making a precise diagnosis in patients with altered biliary anatomy who have multiple cancers of the pancreaticobiliary system.

Epidermoid cysts arising from both the pancreas and spleen are rare. We herein report a case of a surgically resected epidermoid cyst in the pancreatic tail invaginated from the spleen. A multi-locular cyst, 2 cm in diameter, without a solid component was discovered incidentally in the pancreatic tail. During the 11-year follow-up, the emergence of satellite cystic lesions with distinct appearances was seen, and surgical resection was selected despite the lack of any associated symptoms or evidence of cytological abnormalities. Histologically, these cysts were lined with benign multi-layered flattened epithelium surrounded by a thin layer consisting of cells positive for CD8 and CD68 and connecting to the spleen.

Pertuzumab is a humanized monoclonal antibody that binds to HER2 at an epitope that prevents HER2 from dimerizing with ligand-activated HER-family receptors. To assess the potential of pertuzumab as a new therapy, the expression status of HER family members was determined in biliary tract carcinoma (BTC), and the antitumor activity of pertuzumab was investigated by assessing the inhibition of BTC cell growth.The expression status of HER family members in 113 archival specimens of BTC was analyzed by using immunohistochemistry and fluorescence in situ hybridization. Using ten BTC cell lines, heregulin-alpha (HRG-α) stimulated cell proliferation and its inhibition by pertuzumab was tested in vitro. The phosphorylated HER family proteins and their respective downstream molecules were analyzed. In vivo antitumor activity of pertuzumab was evaluated in a xenograft model.Protein overexpression of HER2 and/or HER3 was observed in 23-34 % of the specimens and gene amplification in 17-27 %. Seven of the ten cell lines showed HER2 and/or HER3 protein overexpression and gene amplification, and HRG-α stimulated cell proliferation was observed in four of the ten cell lines. In a BTC cell line co-overexpressing HER2 and HER3, pertuzumab potently inhibited the HRG-α stimulated cell proliferation in a dose-dependent manner, and completely blocked the phosphorylation of HER3. Suppression of downstream pathway molecules including p-AKT was also observed. Pertuzumab inhibited the in vivo growth of subcutaneous tumors, and increased the number of apoptotic cancer cells.Pertuzumab exerts potent antitumor activity in BTC cells co-overexpressing HER2 and HER3. Pertuzumab provides a new therapeutic option against BTC.

The purpose of this study was to describe the cyst infection of intraductal papillary mucinous neoplasm in 2 patients. The patients were 62- and 74-year-old men. The initial symptom was acute febrile abdominal pain. Laboratory tests revealed severe infection (C-reactive protein concentrations were 23.3 µg/mL in patient 1 and 22.3 µg/mL in patient 2) and multilocular cystic masses (the diameters were 70 mm in patient 1 and 50 mm in patient 2) at the pancreatic head that involved peripancreatic vessels were demonstrated by computed tomography. Laboratory and radiographic findings were markedly improved by endoscopic transpapillary drainage. The enteric bacteria were detected in the drainage specimens. Curative resection was achieved, and histological findings indicated a carcinoma in situ in patient 1 and an invasive carcinoma in patient 2. Neither hyperamylasemia nor histological fat necrosis, frequently observed in acute pancreatitis, was evident. Both patients were free from recurrence after surgery (17 months in patient 1, and 18 months in patient 2). Cyst infection is an unknown complication of intraductal papillary mucinous neoplasm. Transpapillary drainage is highly recommended as an initial intervention. It is difficult to distinguish between cyst infection and unresectable invasive carcinoma with imaging modalities; however, surgical intervention after drainage may contribute to long-term survival.

Human peripheral blood mononuclear cells (PB-MNCs) have angiogenic properties, which make them promising cells for use in angiogenic therapy approaches in regenerative medicine. To explore an efficient method for expanding pro-angiogenic cells from PB-MNCs, we developed a novel serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and thrombopoietin (TPO). Using this ex vivo culture, we obtained floating spheres composed mainly of CD11b(+) monocytes expressing c-Mpl (TPO receptor) and which exhibited acetylated low-density lipoprotein uptake and phagocytosis. Expression of IL-8, CXCR4, and vasohibin-2 mRNA was upregulated in these cells. In the presence of TPO, the number and size of the spheres were increased. In a nude mouse hind-limb ischemia model, the intramuscular injection of spheroid cells treated with TPO rescued blood perfusion more effectively than that without TPO. These results indicate that the ex vivo addition of TPO augments the pro-angiogenic activity of peripheral CD11b(+) monocytes, suggesting that this method shows promise for uses in human cell therapy aimed at the induction of vascular regeneration by activating the angiogenic properties of human peripheral blood-derived monocytes.

Cholangiocarcinoma (CC) is a lethal malignancy because it exhibits asymptomatic growth infiltrating the surrounding structures and therefore is usually detected at an advanced stage. The mainstay of treatment for CC is complete resection with negative surgical margins. Therefore, its diagnosis at a relatively early stage is demanded for performing relevant surgical resection. Since the definitive CC diagnosis depends on invasive methods such as biliary cytology and biopsy, a noninvasive assay with high diagnostic accuracy is keenly required. We therefore developed a CC marker with high specificity by the Wisteria floribunda agglutinin (WFA)-assisted glycoproteomics approach. WFA-positive glycoproteins were enriched by the direct dissection of the WFA-stained CC tissue region and following WFA-agarose column chromatography. Subsequent analysis by mass spectrometry identified 71 proteins as candidate markers. Screening of these candidates by gene expression profiling and immunohistochemistry resulted in the selection of L1 cell adhesion molecule (L1CAM) as the most specific CC marker. We confirmed the importance of WFA-positivity for L1CAM using both bile and serum of CC and benign bile duct disease patients. Specifically, WFA-positive L1CAM was enriched from serum by the WFA-assisted affinity capturing, with which CC was efficiently distinguished from benign. In the primary verification study using bile from CC patients (n = 29) and that of benign bile duct disease (n = 29), WFA-positive L1CAM distinguished CC with high specificity (sensitivity = 0.66, specificity = 0.93, overall accuracy = 0.79, area under the receiver operating curve [AUC] = 0.82). The combined use of the WFA-positive L1CAM assay with the high sensitive assay detecting WFA-positive sialylated mucin 1 sufficiently improved the diagnostic accuracy of CC (overall accuracy = 0.84, AUC = 0.93). This combination will possibly be a precise procedure for CC diagnosis compared with conventional diagnostic techniques. Biological significance In this study, we constructed the system for verification of the candidate molecules that exhibit disease specific glyco-alterations and discovered a useful CC marker by the glycoproteomics-assisted strategy for biomarker discovery. Based on the strategy, we previously found that WFA is the best probe to detect CC-specific glycosylation and WFA-positive sialyl MUC1 as a possible biomarker candidate. While the diagnostic specificity of WFA-positive sialyl MUC1 was not superb, we proposed a new biomarker candidate WFA-positive L1CAM with high specificity in bile and serum to complement the previous one. We proved that the novel combination assay of WFA-L1CAM and WFA-sialyl MUC1 selected based on our strategy has the possibility to become a reliable serological test. This study represents application of our strategy, which can be extrapolated to discovery of marker candidates for other diseases.

N-Acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the β1-6 branching of N-acetylglucosamine on asparagine-linked oligosaccharides of cellular proteins, enhances the malignant behaviors of carcinoma cells in experimental models. The aim of this study was to determine clinical significance of GnT-V expression in human pT2 gallbladder carcinoma with simple in vitro experiments. Ninety patients with pT2 gallbladder carcinoma were included for this study. The in vitro and in vivo biological effects of GnT-V were investigated using gallbladder carcinoma cells with variable GnT-V expression levels induced by a small interfering RNA. Of the 90 cases, 57 showed positive staining and the remaining 33 demonstrated negative staining, the subcellular localization in the 57 cases was classified into the granular-type in 31 cases and the diffuse-type in 26 cases. In 76 cases with curative resection, postsurgical survival was significantly poorer in those showing positive staining than in those showing negative staining (P = 0.028). In all of the 76 cases, postsurgical recurrence was significantly more frequent in those showing diffuse-type localization than in those showing negative staining. Experimental analyses demonstrated that the down-regulation of GnT-V expression in gallbladder carcinoma cells induced suppression of cell growth in vitro. The expression levels of GnT-V in the cells were highly correlated with the rapid in vivo growth coupled with the enhanced angiogenesis, and the tendency to form liver metastasis. GnT-V expression in the subserosal layer of pT2 gallbladder carcinoma is correlated with the aggressiveness of the disease.

This is an Open Access journal, all articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

Squamous cell carcinoma of the extrahepatic bile duct is quite rare. A 77-year-old woman with jaundice and general fatigue was referred to our hospital. Multiphase contrast-enhanced computed tomography visualized a 17-mm solid mass in the junction of the cystic and common bile ducts. The patient underwent pylorus-preserving pancreaticoduodenectomy. The pathological findings demonstrated keratin-positive poorly differentiated squamous cell carcinoma of the extrahepatic bile duct (T3N0M0, stage IIIA). Although adjuvant chemotherapy with gemcitabine was administered, the patient exhibited local recurrence at the site of anastomosis of biliojejunostomy 20 months after resection and died 32 months after resection.

Cholangiocarcinoma (CC) is an aggressive malignant tumor for which useful markers are not presently available for early and precise diagnosis. The aim of this study was therefore to identify a high-performance diagnostic marker with a special focus on glyco-alteration of glycoproteins. In the course of study, we found that Wisteria floribunda agglutinin (WFA) is the best probe to differentiate intrahepatic cholangiocarcinoma (ICC) lesions from normal bile duct epithelia (BDE) (P < 0.0001). The subsequent histochemical study confirmed ICC-specific WFA staining on 165 tissue specimens. On the other hand, the WFA staining was shown to be closely associated with that of MY.1E12 established previously against sialylated mucin 1 (MUC1) by double-staining experiments. Moreover, glyco-alteration of MUC1 could be verified by western blotting of WFA-captured bile samples from patients with CC patients. Thus, we attempted to construct an enzyme-linked immunosorbent assay system for more convenient CC diagnosis, where WFA-coated plates, the specific monoclonal antibody MY.1E12, and the bile specimens from CC including ICC (n = 30) and benign diseases (n = 38) were combined. As a result, CC was clearly distinguished from benign diseases with statistical scores (sensitivity = 90.0%, specificity = 76.3%, and area under the curve = 0.85). As a particular note, the obtained sensitivity is the highest score among those having been so far reported. Conclusion: Our approach focusing significant glyco-alteration of a particular glycoprotein yielded a novel diagnostic system for CC with satisfactory clinical scores. HEPATOLOGY 2010

An ultra-sensitive method for glycan analysis targeting small tissue sections (1.5mm in diameter) is described as an application of a recently-established lectin microarray technology. The developed system achieved a high level of detection of a tissue section consisting of approximately 500 cells for differential profiling, where both N- and O-glycans attached to a pool of glycoproteins are subjected to multiplex analysis with 43 lectins. By using an optimized protocol for differential glycan analysis, sections of adenocarcinoma (n=28) and normal epithelia (n=12) of the colon were analyzed in an all-in-one manner. As a result, Wisteria floribunda agglutinin (WFA) was found to clearly differentiate cancerous from normal epithelia with P

Biliary tract cancer is still challenging to treat and manage due to its poor sensitivity to conventional therapies and the inability to prevent or detect the early tumor formation. The most well known risk factor for gallbladder cancer is the presence of chronic inflammation, usually related to gallstones. It has been suggested that cyclooxygenase-2 (COX-2) plays a variety of roles in the gastrointestinal tract, including pathogenic processes such as neoplasia. Recently, we have generated transgenic mice that overexpress rat ErbB-2 under the control of bovine keratin 5 promoter (BK5.ErbB-2 mice). Homozygous BK5.ErbB-2 mice develop adenocarcinoma of gallbladder with an ∼90% incidence. In addition to the activation of ErbB-2 and epidermal growth factor receptor, mRNA and protein levels of COX-2 were up-regulated in the gallbladder carcinomas that developed in these transgenic mice. The aim of this study was to examine the effects of a COX-2 inhibitor, CS-706, on the development of gallbladder carcinomas using the BK5.ErbB-2 mouse model. Ultrasound image analysis as well as histologic evaluation revealed a significant therapeutic effect of CS-706 on the gallbladder tumors, either as reversion to a milder phenotype or inhibition of tumor progression. The antitumor effect was associated with inhibition of prostaglandin E2 synthesis. CS-706 treatment also down-regulated the activation of ErbB-2 and epidermal growth factor receptor, resulting in decreased levels of phosphorylated Akt and COX-2 in gallbladder cancers of BK5.ErbB-2 mice. Based on our results, targeting COX-2 could provide a potentially new and effective therapy alone or in combination with other therapeutic agents for patients with biliary tract cancer. [Mol Cancer Ther 2007;6(6):1709–17]

The macrolide fungicide rapamycin has shown significant antiproliferative action toward a variety of tumor types. In this study, we used BK5.erbB2 transgenic mice as an animal model to examine the therapeutic effect of rapamycin as a potential treatment for gallbladder cancer. Homozygous BK5.erbB2 mice overexpressing the wild-type rat erbB2 gene in basal epithelial cells of the gallbladder have an ∼70% incidence of gallbladder adenocarcinoma by 2 to 3 months of age. Groups of mice (∼2–3 months of age) were treated with rapamycin by i.p. injection (once daily for 14 days) and then sacrificed 24 h after the last treatment. Rapamycin significantly reduced the incidence and severity of gallbladder carcinoma in BK5.erbB2 mice in a dose-dependent manner. Tumors responsive to treatment exhibited a higher number of apoptotic cells. Furthermore, rapamycin treatment led to decreased levels of phosphorylated p70 S6 kinase (Thr389) in gallbladder tissue as assessed by both Western blot and immunofluorescence analyses. Finally, immunofluorescence staining revealed elevated phosphorylated Akt (Ser473) and phosphorylated mammalian target of rapamycin (mTOR; Ser2448) in human gallbladder cancer compared with normal gallbladder tissue. Based on our results using a novel genetically engineered mouse model and the fact that the Akt/mTOR pathway is activated in human gallbladder cancer, rapamycin and related drugs may be effective therapeutic agents for the treatment of human gallbladder cancer. [Cancer Res 2007;67(8):3794–800]

Carcinomas of the gallbladder (GBCa) and bile ducts are aggressive tumors with poor survival and it is, therefore, essential to elucidate the molecular mechanisms of the various signaling pathways in order to develop effective therapies. In this study, tumor specimens from 40 GBCa patients, 12 extrahepatic bile duct carcinoma patients and 26 intrahepatic bile duct carcinoma patients from the USA and Japan were investigated for insulin-like growth factor I receptor (IGF-IR), mammalian target of rapamycin (mTOR) and rapidly accelerated fibrosarcoma-1 (Raf-1) expression by immunohistochemistry; in addition, the correlations with histological type, pathological stage and patient outcome were analyzed. Positive expression of IGF-IR, mTOR and Raf-1 were identified in 68, 73 and 85% of the specimens, respectively. There was no association with histological type and pathological stage, although the positive expression rate of Raf-1 was higher in advanced-stage GBCa. Moreover, patients with positive expression of IGF-IR exhibited significantly reduced survival compared to those with negative IGF-IR expression. In conclusion, IGF-IR, mTOR and Raf-1 were highly expressed in biliary tract cancer and targeted therapy against IGF-IR may be an effective strategy. Among these molecules, IGF-IR expression was found to be a useful biomarker for identifying patients who may benefit from additional treatment.

The cell cycle G 2 /M specific inhibitor cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is composed of CdtA, CdtB, and CdtC coded on the cdtA , cdtB , and cdtC genes that are tandem on the chromosomal cdt locus. A. actinomycetemcomitans CdtA has the lipid binding consensus domain, the so-called “lipobox”, at the N-terminal signal sequence. Using Escherichia coli carrying plasmid pTK3022, we show that the 16th residue, cysteine, of CdtA bound [ 3 H]palmitate or [ 3 H]glycerol. Further, posttranslational processing of the signal peptide, CdtA, was inhibited using globomycin, an inhibitor of lipoprotein-specific signal peptidase II. Fractionation and immunoblotting show the lipid-modified CdtA is present in the outer membrane. Immunoprecipitation and the pull-down assay of the CDT complex from E. coli carrying a plasmid containing cdtABC demonstrated that the CDT complex in the periplasm is composed of CdtA, CdtB, and CdtC and that the CDT complex in culture supernatant is an N-terminally truncated (36 to 43 amino acids) form of CdtA (CdtA′), CdtB, and CdtC. This suggests that CDT is present as a complex both in the periplasm and the supernatant where CdtA undergoes posttranslation processing to CdtA′ in the process of biogenesis and secretion of CDT holotoxin into the culture supernatant. Site-directed mutagenesis of the 16th cysteine residue to glycine in CdtA altered localization of CdtA in the cell and reduced the amount of CDT activity in the culture supernatant. This suggests that CDT forms a complex inside the periplasm for lipid modification where posttranslational processing of CdtA plays an important role for the efficient production of CDT holotoxin into the culture supernatant.

The significance of MUC 1 expression in the gallbladder tissues in relation to cancer and non-cancer disease is not well understood. The aim of this study was to clarify the significance of MUC 1 expression.A monoclonal antibody (CA 15--3; DF 3) was applied to stain MUC 1 core protein in surgical specimens.MUC 1 expression is significantly higher (p0.0001) in gallbladder cancer (69/88) compare to non-cancerous tissue, while, very trace in normal and inflammatory tissues. The expression rate was significantly lower (p0.0001) when the cancer did not penetrate the mucosal layer than when cancers did penetrate this layer. The MUC 1 expression rate was (4/14) in T1 tumours, (11/14) in T4, (40/45) in T3, and (14/15) in T2, respectively. Every cell of normal and inflammatory mucosa, and T1 cancers had the polarized pattern. The depolarized pattern was dominant in cancer cells from the advanced tumours of T2, T3 and T4. That is, (45/74) of cancer cells from the mucosal layer and (58/74) of penetrating cancer cells in submucosal layer had the depolarized pattern. There was no significant correlation of MUC 1 expression rate and staining pattern with cancer differentiation and microscopic venous invasion. On the other hand, lymphatic vessel invasion was significantly correlated with the staining pattern but not with expression rate.MUC 1 core protein expression rate and pattern are suggesting that MUC 1 core protein would be a marker of malignant transformation of gallbladder epithelium and its depolarized expression would also be a marker of invasion of gallbladder cancer.

Purpose and Experimental Design: Little is known about the molecular events leading to the development and progression of pathological tumor stage 2 (pT2) gallbladder carcinoma. An alteration in the site of O-glycosylation may be associated with malignant behavior of carcinoma cells by modulation of the biological properties of the target mucin. The UDP-N-acetyl-α-d-galactosamine-polypeptide N-acetylgalactosaminyltransferase isozyme 3 (GalNAc-T3) has the epithelial gland-specific expression and catalyzes mucin-type O-glycosylation. In this study, immunohistochemistry was performed to determine the expression level of GalNAc-T3 in 34 cases of pT2 gallbladder carcinoma to determine the correlation of the GalNAc-T3 expression level with mode of recurrence and postsurgical survival. Results: The expression levels of GalNAc-T3 protein and mRNA were increased in gallbladder carcinomas compared with the levels in adjacent noncancerous tissues and in intact gallbladders. Immunostaining of GalNAc-T3 was recognized in the cancerous epithelia, and the subcellular localization was classified into granular and diffuse types. In the 34 cases of pT2 carcinoma, the localization of GalNAc-T3 was granular type in 50% and diffuse type in 50% of the cases at the deepest invading sites in the subserosal layer. Postsurgical recurrence was significantly more frequent in cases showing diffuse-type localization of GalNAc-T3 at the deepest invading sites (65%) than in those showing granular-type localization (23%; P < 0.05). Postsurgical survival was significantly poorer in cases showing diffuse-type localization than in those showing granular-type localization (P = 0.033) Conclusions: In pT2 gallbladder carcinoma, the presence of diffuse-type localization of GalNAc-T3 in the subserosal layer is correlated with aggressiveness of the disease. This phenotype may serve as a unique biological feature associated with the malignant behavior.

The overall outcome of gallbladder carcinoma has not been favorable because of frequent recurrence at distant sites after surgery. A high-level expression of MUC1 recognized by a monoclonal antibody (mAb), MY.1E12, is correlated with poor survival in several types of carcinomas. There is a need to find new prognostic parameters that can give further insights into tumor aggressiveness of the disease. Immunohistochemistry was performed to determine the expression level of mAb MY.1E12-reactive-MUC1 (MY.1E12-MUC1) in 79 cases of gallbladder carcinoma of different depths of invasion and to determine the correlation of the expression level with clinicopathological findings. The cellular distribution of MY.1E12-MUC1 was heterogeneous among carcinomas of different depths of invasion. Immunohistochemical localization was classified into apical, cytoplasmic and stromal types based on the predominant cellular distribution. In 35 cases of pT2 carcinoma in which curative resections had been performed, the localization was apical type in 54%, cytoplasmic type in 66%, and stromal type in 56% of the cases at the deepest invading sites in the subserosal layer. Postsurgical recurrences at distant sites were seen in 11 of 18 cases of pT2 carcinoma (61%) that had more than or equal to 10% of the cancerous epithelia showing stromal localization of MY.1E12-MUC1 at the deepest invading sites (stromal group) and in 3 of 17 cases (18%) that had less than 10% of the cancerous epithelia showing stromal localization (nonstromal group). The postsurgical 5-year survival rate was significantly poorer in the former (54%) than in the latter (79%; P = 0.049). In 38 cases of pT3 and pT4 carcinomas, the frequency of synchronous distant organ metastasis at the time of diagnosis was significantly higher in cases in the stromal group (61%) than in cases in the nonstromal group (20%). Moreover, in 73 cases of pT2, pT3 and pT4 carcinomas, the expression rate of abnormal localization of E-cadherin was significantly higher in the stromal group (63%) than in the nonstromal group (30%). The MUC1 mRNA levels revealed by in situ hybridization would not be a determinant important for the stromal localization. The stromal localization of MY.1E12-MUC1 may be a phenotype serving as a unique biological feature associated with the tumor aggressiveness of gallbladder carcinoma, such as the tendency to form distant organ metastasis.

Background Long‐term survival has not been described following surgical resection for liver metastasis after radical resection of an advanced hilar bile duct carcinoma (Klatskin tumour). One such patient who developed liver metastasis after radical treatment for stage IVA (pTNM) hilar cholangiocarcinoma has survived 5.5 years after resection of the liver metastasis followed by chemotherapy. Case report A 50‐year‐old man developed a solid liver metastasis in segment VIII 17 months after radical resection of a stage IVA (pT3 pN1 M0) Klatskin tumour followed by postoperative radiotherapy (54 Gy) and systemic chemotherapy (oral UFT 450 mg/day plus intravenous cisplatin 20 mg on 5 consecutive days each month). The patient is alive at 7 years after the primary resection followed by resection of the liver metastasis plus further systemic chemotherapy comprising oral UFT combined with intravenous adriamycin (ADM) and mitomycin C (MMC). Conclusion Aggressive salvage resection surgery can be an effective component of a multidisciplinary treatment regimen, even for a postoperative liver metastasis that developed after radical resection of an advanced Klatskin tumour, provided that the metastasis is solid and has not failed local‐regional control.

Background & Aims: In hepatolithiasis, chronic proliferative cholangitis may influence the progression of the disease. Prostaglandin (PG) E 2 experimentally causes morphologic changes to intrahepatic bile ducts, analogous to the changes found in cholangitis. This study was designed to gain an understanding of the involvement of PGE 2 and PGE receptor (EP) subtypes in the development of cholangitis. Methods: The expression levels of secretory-type group IIA phospholipase A 2 (sPLA 2 -IIA) and cyclooxygenase (COX)-2 as well as EP subtypes were determined in the bile ducts with change of cholangitis. In in vitro experiments, growth promotion and mucin secretagogue properties of biliary epithelial cells in response to EP-selective agonists or antagonists were studied. Results: The messenger RNA (mRNA) level of sPLA 2 -IIA and the protein and mRNA levels of COX-2 were significantly increased in the bile ducts of patients with hepatolithiasis compared with the levels of the bile ducts of control subjects. These changes were associated with a concomitant increase in PGE 2 and total mucin concentrations in the bile. The mRNAs of EP subtypes EP 2 , EP 3 , and EP 4 but not EP 1 were amplified in the bile ducts. Treatment with an EP 4 -selective agonist (ONO-AE1-329) caused a dose-dependent increase in DNA synthesis, colony number, and mucin secretion in the cells. Conversely, treatment with an EP 4 -selective antagonist (ONO-AE3-208) abolished the biological effects of PGE 2 on the cells. Conclusions: In hepatolithiasis, an enhanced synthesis of sPLA 2 -/COX-2-derived PGE 2 and its actions mediated via the EP 4 receptor in the bile ducts may be of pathobiological significance for chronic proliferative cholangitis.

BACKGROUND: This study was performed to develop a clinically relevant porcine model of auxiliary partial orthotopic liver transplantation (APOLT) for fulminant hepatic failure. METHODS: FHF was induced by intraportal administration of alpha-amanitin and lipopolysaccharide. Thereafter, pigs were divided into four groups. Group 1 was an untreated, control group. In group 2, pigs underwent only a left hemihepatectomy. Pigs in groups 3 and 4 received APOLT after hemihepatectomy. Tacrolimus was administered to pigs in group 4, but not to those in group 3. RESULTS: Two-week survival rates were 0%, 20%, 40%, and 100% for groups 1, 2, 3, and 4, respectively. In group 4, after abolishing the graft function at 1 week, pigs survived for more than 2 weeks, and regeneration of the native liver was confirmed histologically. CONCLUSIONS: Pigs suffering from fulminant hepatic failure could achieve long survival and liver regeneration with a temporary support of the auxiliary graft.

Background: Many reconstruction procedures have been developed in efforts to resolve patients' complaints after total gastrectomy. However, there have been few reports of longterm comparisons between reconstruction procedures, especially with regard to the prevention of duodenal food passage. This study was undertaken to compare the longterm subjective and functional results among Roux-en-Y esophagojejunostomy (R-Y), R-Y with pouch (P-Y), and jejunal interposition with pouch (P-I) after total gastrectomy. Methods: Consecutive patients requiring curative total gastrectomy were enrolled in this prospective study by the envelope method. Results: Hospital stay was longer following a P-I than an R-Y or a P-Y. Over 50% of R-Y patients complained of heartburn, and 20% of R-Y patients showed dumping syndrome throughout the postoperative period, with this rate being significantly different from rates in the other two groups. P-Y patients complained of early satiety in the late postoperative period, while P-I patients complained of early satiety in the early postoperative period. The nutritional index in P-I patients was higher than those in patients with the other two procedures. Gastrointestinal and hepatobiliary dual scintigraphy (GHDS) showed that the rate of bile reflux with an R-Y was relatively high after surgery. Food reflux with a P-Y was increased (9.4% to 11.1%), but with a P-I food reflux was decreased at 3 years after surgery (13.3% to 9.9%). Patients with a P-Y had a faster recovery of body, weight in the early postoperative period; however, at 5 years after operation, body weight recovery with a P-I was greatest. Conclusion: Reconstruction should be performed with pouch formation after total gastrectomy with curative intent.

Postsurgical recurrence at distant sites frequently occurs in pathological tumor stage 2 (pT(2)) carcinoma of the gallbladder even though the carcinoma is limited to the gallbladder wall. Little is known, however, about the molecular events leading to its development and progression. A large body of evidence suggests that cyclooxygenase-2 (COX-2) is up-regulated in carcinoma tissues and plays roles in promoting cell-proliferation, growth and metastasis of carcinoma cells. In the present study, immunohistochemistry was performed to determine the expression levels of COX-2 in the subserosal layer of 33 cases of pT(2) gallbladder carcinoma in which curative resections had been performed and to determine the correlations of the expression levels of COX-2 with mode of recurrence and postsurgical survival. Immunostaining of COX-2 in the epithelia was recognized in more than 80% of normal epithelia, noncancerous pathological lesions of the gallbladder except for intestinal metaplasia and pT(1-4) carcinoma specimens. Intense staining was observed in large percentages of hyperplastic lesions (65%), pT(2) carcinoma specimens (76%) and pT(3) and pT(4) carcinoma specimens (64%) compared to the percentages of normal epithelia and other pathological lesions (0-25%). Intense staining was also observed in the adjacent stroma in pT(2) carcinoma specimens (33%) and in those in pT(3) and pT(4) carcinoma specimens (43%) but only in small percentages of the stroma adjacent to normal epithelia and pathological lesions (0-8%). In situ hybridization confirmed the existence of COX-2 mRNA in both the cancerous epithelia and adjacent stroma of pT(2)-pT(4) carcinomas. In 33 cases of pT(2) carcinoma, distant recurrence, i.e., liver metastasis, was seen in 3 of 9 cases of pT(2) carcinoma (33%, P0.05) with intense stromal staining in the subserosal layer and in 1 of 24 cases (4%) without intense staining, whereas no significant correlation was found between parameters of pathological malignancies (histological grade, lymphatic permeation, venous permeation and lymph node metastasis) and the intensity of stromal staining in the subserosal layer. The postsurgical survival outcome was significantly poorer in the former than in the latter (p = 0.010). In pT(2) gallbladder carcinoma, upregulation of COX-2 in the stroma adjacent to the cancerous epithelia in the subserosal layer correlates with the aggressiveness of the disease, such as the tendency to form distant recurrences. This phenotype may serve as a unique biological feature associated with the malignant behavior of pT(2) gallbladder carcinoma.

Purpose. Dysfunction of E-cadherin, a cell–cell adhesion molecule, correlates with the grade of dedifferentiation and/or invasiveness of rectal cancer. However, the relationship between E-cadherin expression in the primary tumor and the potential for metastasis has never been reported. Methods. E-cadherin expression in 43 primary rectal cancer, including 10 poorly differentiated type, and their associated metastatic lymph nodes (LN mets.) were immunohisto-chemically evaluated. Results. Heterogeneous immunostaining, suggestive of damage to the E-cadherin-mediated cell–cell adhesion system, was seen in 13 of the 28 LN mets positive primary lesions, but in 0 of the 15 LN mets negative primaries. Furthermore, the incidence of heterogeneous immunostaining differed significantly between poorly differentiated and differentiated cancers, being seen in 8 of 10 cases and 5 of 33 cases, respectively (P = 0.0003 by Fisher's exact test). Interestingly, most of the LN mets. foci (25 of 28 cases) showed homogeneous staining regardless of the E-cadherin staining pattern of the primary lesion. Conclusion. Heterogeneous immunostaining of E-cadherin in poorly differentiated rectal cancer was associated with lymph node metastasis. Its staining pattern in metastatic lymph nodes were, however, generally homogenous.

The molecular mechanism of gallbladder carcinogenesis and cancer growth remains unknown. BK5.erbB2 transgenic mice in which erbB2 is overexpressed and activated in the biliary epithelia develop adenocarcinoma of the gallbladder at a high incidence. Although it has been reported that erbB2 plays an important role in tumorigenesis, little is known about the involvement of its ligand(s). The expression level of Muc4, a potential functional ligand for erbB2, and its interaction with erbB2 in the gallbladder of BK5.erbB2 mice were determined. By immunohistochemistry and in situ hybridization, both Muc4 mRNA and protein levels were strongly expressed in the cancerous epithelia of gallbladder from BK5.erbB2 mice. Also, in the hyperplastic (precancerous) epithelia, the protein levels were modestly expressed. Immunostaining with Muc4 (ASGP2) Ab overlapped with that with erbB2 Ab in the apical membranous components of the cancerous epithelia, indicating the co-localization of Muc4 and erbB2. Immunoprecipitation experiments revealed an interaction between Muc4 and erbB2 in the gallbladders. The interaction was associated with the hyperphosphorylation of erbB2, MAPK and Akt, and also with the overexpression of cyclooxygenase-2. However, in other organs that overexpressed erbB2 (trachea, esophagus and forestomach), Muc4 was expressed in only trace or modest amounts, and erbB2 was not hyperphosphorylated. Collectively, Muc4 is upregulated and interacts with erbB2 in gallbladders from BK5.erbB2 mice. It is likely that Muc4 plays an important role during gallbladder carcinogenesis and/or cancer growth by potentiating erbB2 signaling.

Some patients with gastrointestinal schwannoma (GIS) have been previously reported in the literature. However, GIS of the colon is quite rare. In addition, it is sometimes difficult to differentiate neurogenic tumors from other soft tissue tumors. We herein describe two cases of schwannoma of the colon, while also reviewing the relevant Japanese literature. The first case, a 73-year-old woman underwent a sigmoidectomy with lymph node dissection following the diagnosis of submucosal tumor. In the second case, a submucosal tumor was located in the cecum of a 44-year-old man. An endoscopic tumor resection was performed in the second case. The resected tumors measured 3.6 and 1.0 cm in maximal diameter, respectively. Microscopically, the tumors consisted predominantly of spindle-shaped cells that proliferated in an interlaced fashion. Mitosis was rarely seen in these tumors. Immunohistochemically, the tumor cells were strongly positive for S-100 protein, weakly positive for glial fibrillary acidic protein, and negative for CD34, alpha-smooth-muscle actin, and cytokeratin (CAM 5.2) in both cases. The tumors in the two cases were both diagnosed to be benign schwannoma of the colon. In general, schwannoma of the gastrointestinal tract is considered to be benign and should therefore be distinguished from other spindle-cell tumors or malignancies. Once diagnosed as schwannoma, extensive surgery should be avoided. Actually, such patients tend to show a good postoperative course with no evidence of recurrence.

A 53-year-old man was admitted to the hospital because of anal bleeding and lumbago. Barium enema and colonoscopic examinations revealed sigmoid colon cancer, and computed tomography revealed metastasis to the caudate lobe of the liver. A low anterior resection and a resection of the caudate lobe of the liver were performed. Pathological diagnosis was poorly differentiated adenocarcinoma. (se, ly1, v2, ow(-), aw(-), ew(+)) After the operation that resulted in curability B, bleeding through the abdominal drains was observed, and severe thrombocytopenia and elevated fibrin/fibrinogen degradative products (FDP) were indicative of disseminated intravascular coagulation (DIC). Despite the treatment with gabexate mesilate, the bleeding tendency was not improved. Since a bone scintigraphy demonstrated diffuse abnormal uptake of isotope, a bone marrow biopsy was performed. As a results, DIC associated with systemic bone marrow metastasis was diagnosed. Sequential methotrexate and 5FU (MTX-5FU) therapy (MTX 30mg/m2, 5FU 600mg/m2) was introduced. After 7 courses of the chemotherapy, he recovered from DIC accompanied by an increase in platelet count and decreases in FDP and CEA (carcinoembryonic antigen) levels. It is suggested that the suquential MTX-5FU therapy was effective for DIC due to systemic bone marrow metastasis in the present case of sigmoid colon cancer.

Actinobacillus actinomycetemcomitans has been shown to produce a soluble cytotoxic factor(s) distinct from leukotoxin. We have identified in A. actinomycetemcomitans Y4 a cluster of genes encoding a cytolethal distending toxin (CDT). This new member of the CDT family is similar to the CDT produced by Haemophilus ducreyi . The CDT from A. actinomycetemcomitans was produced in Escherichia coli and was able to induce cell distension, growth arrest in G 2 /M phase, nucleus swelling, and chromatin fragmentation in HeLa cells. The three proteins, CDTA, -B and -C, encoded by the cdt locus were all required for toxin activity. Antiserum raised against recombinant CDTC completely inhibited the cytotoxic activity of culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. These results strongly suggest that the CDT is responsible for the cytotoxic activity present in the culture supernatant and cell homogenate fractions of A. actinomycetemcomitans Y4. This CDT is a new putative virulence factor of A. actinomycetemcomitans and may play a role in the pathogenesis of periodontal diseases.

水疱性類天疱瘡の治療中,大量下血を繰り返したMecke1憩室潰瘍の症例に対し,卵黄動脈を塞栓,止血し,血漿交換を行った後に回腸部分切除を施行した症例を経験したので報告する.症例は67歳,男性.水疱性類天疱瘡の診断でステロイドセミパルス療法後,下血を認め,ショック状態となった.緊急血管造影検査にて,胎生期の遺残である卵黄動脈からの造影剤の血管外漏出を認めたことから, Meckel憩室潰瘍と診断し,塞栓術にて止血した.塞栓術後,原疾患の増悪を認めたため,血漿交換療法を施行した.その結果,皮疹は消退し,ステロイドを減量することが可能となり,安全に回腸部分切除術を施行できた.

62歳,女性.左頚部腫瘤を主訴に来院.Virchowリンパ節と大動脈周囲リンパ節転移を伴う3型の直腸S状部低分化腺癌と診断された.CEAは45.8(正常値0.0～5.0)ng/ml.本例に,メトトレキセート(以下MTX)-5FU,5FU-ロイコボリン(以下LV)交互療法を3クール施行した.プロトコールは1コース56日間とし,前半4週に5FU急速静注のMTX-5FU交代療法を2回施行し,後半4週に5FU接続投与の5FU-LV療法を施行するものとした.2コース終了時において,原発巣は著効を示し,リンパ節転移は50%以上の縮小を認め,有効であった.CEAは9.5ng/mlへと低下した,この状態は4週間以上継続した.重篤な副作用は出現せず,performance statusは0のまま経過した.しかし,第3コースの半ばより,腫瘍は再度増大するようになり,全身状態の悪化とともに,治療開始から約9カ月後に死亡した.