Shujiro Yazumi

Circulating naïve and CD4+CD25high regulatory T cells in patients with autoimmune pancreatitis.

Objective: Autoimmune pancreatitis (AIP) is a new clinical entity of pancreatic disorder. There are several immunologic and histological abnormalities specific for the disease, including increased levels of serum IgG4 and infiltration of lymphocytes and IgG4-positive plasmacytes. The role of IgG4 is unclear. Recently, regulatory T cells (Tregs) have been reported to be involved in the development of various autoimmune diseases as well as B cell shifting to IgG4-producing plasmacytes. To clarify the role of Tregs in the pathophysiology of AIP, we analyzed circulating Tregs in AIP. Methods: We recruited 27 patients with AIP for this study. For comparison, we also recruited 23 patients with other pancreatic disease and 32 healthy subjects as controls. We analyzed Tregs as CD4+CD25high and CD4+CD25+CD45RA+ (naïve) from peripheral blood by flow cytometry. Results: In peripheral blood, CD4+CD25high Tregs were significantly increased in AIP patients (3.01% ± 1.77%) compared with alcoholic chronic pancreatitis (CP) (1.65% ± 0.58%), idiopathic CP (1.53% ± 0.56%), and healthy control (1.72% ± 0.81%, P < 0.05). Naïve Tregs significantly decreased in AIP (0.32% ± 0.22%) compared with healthy control (0.83% ± 0.65%) and CP group (alcoholic and idiopathic CP; 0.52% ± 0.40%, P < 0.05). In untreated AIP patients, the number of CD4+CD25high Tregs and IgG4 are correlated (R = 0.53, P < 0.05). Conclusions: Increased numbers of CD4+CD25 high Tregs may influence IgG4 production in AIP, whereas decreased numbers of naïve Tregs may be involved in the pathogenesis of AIP.

Endoscopic management of biliary strictures after duct-to-duct biliary reconstruction in right-lobe living-donor liver transplantation

Background. The aims of this study were to characterize the features of the biliary strictures that occur after duct-to-duct biliary reconstruction during right-lobe living-donor liver transplantation (LDLT) and to evaluate the feasibility of correcting such stricture endoscopically by inserting an “inside stent,” that is, a short internal stent, above the sphincter of Oddi. Methods. Biliary stricture occurred in 26 (35.6%) of 73 consecutive patients who underwent right-lobe LDLT with duct-to-duct biliary reconstruction from July 1999 through October 2001 and survived for more than 3 months. Of the 26 patients who had biliary stricture, 22 were referred for endoscopic retrograde cholangiography (ERC) and 4 for percutaneous cholangiography. Results. ERC disclosed biliary stricture in 19 (86.4%) of the 22 patients who underwent the procedure. One patient had an unbranched stricture, 16 had a fork-shaped stricture, 1 had a trident-shaped stricture, and 1 had a stricture with more than three branches. Fourteen (73.7%) of the patients with strictures were treated endoscopically by inserting inside stents ranging from 7 F to 12 F in size, three underwent a Roux-en-Y hepaticojejunostomy to repair their stricture, and two were closely observed as outpatients. Of the 14 patients who were treated with the inside-stent, only 1 had acute cholangitis immediately after the procedure and underwent a Roux-en-Y hepaticojejunostomy. The other 13 patients who were treated with the inside stent have not required surgical repair for as long as an average of 586 days. Conclusion. Endoscopic placement of an inside stent is useful for treating biliary strictures in patients who have undergone right-lobe LDLT with duct-to-duct reconstruction.

Abnormal expression of BRCA1 and BRCA1‐interactive DNA‐repair proteins in breast carcinomas

Breast cancer is one of the most common malignancies among women. The molecular mechanisms involved in breast carcinogenesis, however, remain to be elucidated. Although somatic mutation of BRCA1 is rare, BRCA1 protein expression is reduced in about 30% of sporadic breast carcinomas (Yoshikawa et al., Clin. Cancer Res., 5:1249–1261, 1999), indicating its possible involvement even in sporadic breast carcinogenesis. Among the BRCA1‐interactive proteins are hRAD51 (a human homologue of Escherichia coli rec A protein), BARD1 (BRCA1‐associated RING domain 1) and p53, all of which are involved in DNA repair. We have analyzed the expression patterns of the hRAD51, BARD1 and p53 proteins in five breast cancer cell lines, including a BRCA1‐deficient cell line, and in 179 breast cancer tissue samples from Japanese women, including 113 sporadic, 47 hereditary (i.e., BRCA1 status unknown), and 19 BRCA1‐associated cases. Of the 179 breast carcinomas, fifty‐four (30%) exhibited reduced hRAD51 expression, and sixty‐two (35%) exhibited p53 overexpression. On the other hand, reduced expression level of BARD1, and of hMSH2 and hMLH1, which are components of DNA mismatch‐repair pathway and are involved in colorectal carcinogenesis, was observed respectively in only 10 (6%), 8 (5%) and 3 (2%) cases. The overall frequency of sporadic breast carcinomas with abnormal expression of either BRCA1 or the BRCA1‐interactive proteins was 67% (76/113). These results indicate that there may be an important role for the BRCA1‐associated DNA‐repair pathway, not only in BRCA1‐associated breast carcinomas, but also in sporadic breast carcinomas. Int. J. Cancer 88:28–36, 2000.

Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines

RUNX3, a Runt domain transcription factor involved in TGF-β signaling, is a candidate tumor-suppressor gene localized in 1p36, a region commonly deleted in a wide variety of human tumors, including those of the stomach, bile duct, and pancreas. Recently, frequent inactivation of RUNX3 has been demonstrated in human gastric carcinomas. In this study, to examine the involvement of RUNX3 abnormalities in tumorigenesis of bile duct as well as pancreatic cancers, we investigated not only the expression but also methylation status of RUNX3 in 10 human bile duct and 12 pancreatic cancer cell lines. Seven (70%) of the bile duct and nine (75%) of the pancreatic cancer cell lines exhibited no expression of RUNX3 by both Northern blot analysis and the reverse transcriptase polymerase chain reaction. All of the 16 cell lines that did not express RUNX3 also showed methylation of the promoter CpG island of the gene, whereas the six cell lines that showed RUNX3 expression were not methylated or only partially methylated in the RUNX3 promoter region. Moreover, treatment with the methylation inhibitor 5′-aza-2′-deoxycitidine activated RUNX3 mRNA expression in all of 16 cancer cell lines that originally lacked RUNX3 expression. Finally, hemizygous deletion of RUNX3, as detected by fluorescence in situ hybridization, was found in 15 of the 16 cancer cell lines that lacked RUNX3 expression. These data suggest that the inactivation of RUNX3 plays an important role in bile duct and pancreatic carcinogenesis, and that methylation is a common mechanism by which the gene is inactivated.

Covered self-expandable metal stents with an anti-migration system improve patency duration without increased complications compared with uncovered stents for distal biliary obstruction caused by pancreatic carcinoma: a randomized multicenter trial.

OBJECTIVES:The requirements of biliary stents used in the palliation of malignant biliary obstruction are a long duration of patency and minimal adverse effects. Covered self-expandable metal stents (SEMSs) have been shown to prevent tumor ingrowth, which is the most frequent complication of uncovered SEMSs. However, because they are prone to migration, the superiority of covered SEMS has yet to be convincingly demonstrated. The aim of this study was to evaluate the superiority of covered over uncovered SEMSs in the palliation of distal biliary obstruction due to unresectable pancreatic carcinoma, using both stent types with relatively low axial force and uncovered flared ends to prevent their migration.METHODS:From April 2009 to December 2010, 120 patients who were admitted to 22 tertiary-care centers because of distal biliary obstruction from unresectable pancreatic carcinomas were enrolled in this prospective randomized multicenter study. Patients were randomly assigned to receive a covered or uncovered SEMS deployed at the site of the biliary stricture during endoscopic retrograde cholangiopancreatography. Stent patency time, patient survival time, patient survival time without stent dysfunction (time to stent dysfunction or patient death), cause of stent dysfunction (ingrowth, overgrowth, migration, or sludge formation), and serious adverse events were compared between covered and uncovered SEMS groups.RESULTS:Patient survival time in the two groups did not significantly differ (median: 285 and 223 days, respectively; P=0.68). Patient survival time without stent dysfunction was significantly longer in the covered than in the uncovered SEMS group (median: 187 vs. 132 days; P=0.043). Stent patency was also significantly longer in the covered than in the uncovered SEMS group (mean±s.d.: 219.3±159.1 vs. 166.9±124.9 days; P=0.047). Reintervention for stent dysfunction was performed in 14 of 60 patients with covered SEMSs (23%) and in 22 of 60 patients with uncovered SEMSs (37%; P=0.08). Stent dysfunction was caused by tumor ingrowth, tumor overgrowth, and sludge formation in 0 (0%), 3 (5%), and 11 (18%) patients in the covered SEMSs group, and in 15 (25%), 2 (3%), and 6 (10%) patients in the uncovered SEMSs group, respectively. Stent migration was not observed in either group. Rates of tumor overgrowth and sludge formation did not significantly differ between the two groups, whereas the rate of tumor ingrowth was significantly lower in the covered than in the uncovered SEMS group (P<0.01). Acute pancreatitis occurred in only one patient in the covered SEMS group. Acute cholecystitis occurred in one patient in the covered SEMS group and in two patients in the uncovered SEMS group. There was no significant difference between the two groups in the incidence of serious adverse events.CONCLUSIONS:By preventing tumor ingrowth and migration, covered SEMSs with an anti-migration system had a longer duration of patency than uncovered SEMSs, which recommends their use in the palliative treatment of patients with biliary obstruction due to pancreatic carcinomas.

Frequent downregulation of the runt domain transcription factors RUNX1, RUNX3 and their cofactor CBFB in gastric cancer

Our previous studies suggest that lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer, but potential roles of other members of the RUNX family genes have not yet been reported. We examined the expression of 3 Runt‐related (RUNX) genes, RUNX1, RUNX2 and CBFB, in gastric cancer cell lines and primary gastric cancer specimens and compared them to those of RUNX3 reported earlier in conjunction with clinicopathologic factors. Expression of RUNX family genes in 9 gastric cancer cell lines, 56 primary gastric cancer specimens and surrounding normal gastric mucosa were estimated by Northern blot analysis, quantitative RT‐PCR and in situ hybridization. Northern blot analysis in gastric cancer cell lines showed downregulation of RUNX1 and RUNX3 in 67% and 78% of the cell lines tested, respectively. The ratio of the average RUNX mRNA/β‐actin mRNA ratio (×103) for RUNX1 was 48.0 ± 21.1 vs. 21.4 ± 8.1; RUNX2, 1.1 ± 0.3 vs. 1.0 ± 0.2; RUNX3, 9.2 ± 6.3 vs. 3.1 ± 1.3 and CBFB, 42.0 ± 19.4 vs. 21.0 ± 8.4 (normal vs. tumor, respectively, average ±SD). The basal RUNX2 expression was very weak, and there was no significant change in gastric cancers. Both RUNX1 and RUNX3 showed remarkable downregulation in 62% and 69%, respectively, of surgically resected specimens compared to surrounding mucosa analyzed by quantitative RT‐PCR (p < 0.01). Furthermore, CBFB, the gene encoding the cofactor of RUNX1, ‐2, ‐3, was also downregulated in significant fraction (32%, p < 0.05). The percentage of downregulation of RUNX1, RUNX3 and CBFB increased as the cancer stage progressed. Tricostatin A and 5′‐azacitidin reactivate RUNX3 expression, but they could not reactivate expression of RUNX1 and CBFB in gastric cancer cells, suggesting that the downregulation was due to mechanisms other than methylation of the promoter region. These findings suggest that RUNX1 and CBFB in addition to RUNX3 play some roles in gastric cancers and that roles of RUNX gene family in gastric cancer are more widespread and complex than previously realized.

Possible Involvement of RUNX3 Silencing in the Peritoneal Metastases of Gastric Cancers

Purpose: Our previous results suggested that a lack of RUNX3 function contributed to human gastric carcinogenesis, but the role of RUNX3 in progression and metastasis remains unclear. We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray. Experimental Design and Results: Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100%) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-β (TGF-β)–induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of ∼21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified ∼28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A1 1, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in the TGF-β signaling pathway. Conclusion: These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.

Biliary complications after a right-lobe living donor liver transplantation

Right-lobe living donor liver transplantation (RL-LDLT) has become an acceptable procedure for adult patients with end-stage liver disease in this decade. However, biliary complications in RL-LDLT remain a serious problem: the incidence of anastomotic biliary leakage and stricture after RL-LDLT is reported to be 4.7%–18.2% and 8.3%–31.7%, respectively. The incidence varies according to the type of biliary reconstructions between Roux-en-Y hepaticojejunostomy and duct-to-duct biliary reconstruction. The anatomical biliary diversity of a right-lobe graft makes it difficult to reconstruct the biliary system. Indeed, most biliary strictures in patients with duct-to-duct reconstruction develop in multibranched fashion. In this regard, endoscopic biliary stenting appears to be efficacious for treating multibranched biliary strictures because multiple stenting permits the drainage of each segmental branch of the stricture. In this review, we describe various aspects of biliary complications occurring in RL-LDLT and their treatment.

Dynamics of Hepatitis B Virus Quasispecies in Association with Nucleos(t)ide Analogue Treatment Determined by Ultra-Deep Sequencing

Background and Aims Although the advent of ultra-deep sequencing technology allows for the analysis of heretofore-undetectable minor viral mutants, a limited amount of information is currently available regarding the clinical implications of hepatitis B virus (HBV) genomic heterogeneity. Methods To characterize the HBV genetic heterogeneity in association with anti-viral therapy, we performed ultra-deep sequencing of full-genome HBV in the liver and serum of 19 patients with chronic viral infection, including 14 therapy-naïve and 5 nucleos(t)ide analogue(NA)-treated cases. Results Most genomic changes observed in viral variants were single base substitutions and were widely distributed throughout the HBV genome. Four of eight (50%) chronic therapy-naïve HBeAg-negative patients showed a relatively low prevalence of the G1896A pre-core (pre-C) mutant in the liver tissues, suggesting that other mutations were involved in their HBeAg seroconversion. Interestingly, liver tissues in 4 of 5 (80%) of the chronic NA-treated anti-HBe-positive cases had extremely low levels of the G1896A pre-C mutant (0.0%, 0.0%, 0.1%, and 1.1%), suggesting the high sensitivity of the G1896A pre-C mutant to NA. Moreover, various abundances of clones resistant to NA were common in both the liver and serum of treatment-naïve patients, and the proportion of M204VI mutants resistant to lamivudine and entecavir expanded in response to entecavir treatment in the serum of 35.7% (5/14) of patients, suggesting the putative risk of developing drug resistance to NA. Conclusion Our findings illustrate the strong advantage of deep sequencing on viral genome as a tool for dissecting the pathophysiology of HBV infection.

Regression of fundic gland polyps following acquisition of Helicobacter pylori

The prevalence of Helicobacter pylori infection is very low in patients with fundic gland polyps (FGPs) of the stomach. We report here two cases with multiple FGPs that regressed following new H pylori acquisition. Patient Nos I and II had multiple FGPs in normal fundic mucosa without inflammatory changes or atrophy. Both were not infected with H pylori. Following acquisition of H pylori infection however, all FGPs in both patients completely disappeared except for one FGP in patient No I. Although the size of the remaining polyp in patient No I was greatly reduced after H pylori acquisition, it became enlarged again after eradication. Interestingly, in the remaining polyp, we found an activating β-catenin gene mutation whereas no such mutations were detected in FGPs of patient No II. Thus H pylori infection may have an inhibitory effect on the development of FGPs.