Yasuyuki Ono

Simultaneous Quantification of Epstein-Barr Virus, Cytomegalovirus, and Human Herpesvirus 6 DNA in Samples from Transplant Recipients by Multiplex Real-Time PCR Assay

ABSTRACT We developed a multiplex real-time PCR assay using 6-carboxyfluorescein, 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, and carbocyanine 5-labeled probes to simultaneously quantify Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 (HHV-6) DNA. When previously tested and stored DNA samples were examined, results of the multiplex real-time PCR assay were as sensitive and specific as those of a single real-time PCR assay. The multiplex assay was used to quantify the EBV, CMV, and HHV-6 DNA in 46 transplant recipients. A total of 303 whole-blood and plasma specimens were collected and analyzed. According to the results of the multiplex assay, the detection rates for viral DNA in whole blood and plasma were 23.8% and 5.9% for EBV, 11.2% and 5.3% for CMV, and 12.5% and 2.0% for HHV-6, respectively. All forms of viral DNA were detected more frequently in whole blood than in plasma. During the symptomatic period, EBV DNA was detected in all whole-blood specimens but not in all plasma specimens. Furthermore, the EBV DNA load in whole blood was higher during the symptomatic period than during the asymptomatic period, whereas the EBV DNA load in plasma was similar for both periods. These results demonstrate that whole blood is more suitable for the quantification of EBV DNA in transplant patients. However, a cutoff value with clinical relevance still needs to be determined.

Attenuated nitrergic inhibitory neurotransmission to interstitial cells of Cajal in the lower esophageal sphincter with esophageal achalasia in children

Abstract Background : Esophageal achalasia (EA) is a rare disease in children, the etiology and pathogenesis of which remain controversial. Previous studies have suggested that a specific class of interstitial cells of Cajal (ICC) act as mediators in nitrergic inhibitory neurotransmission in the lower esophageal sphincter (LES). The aim of this investigation is to clarify the status of ICC and nitrergic inhibitory neurons in the LES of EA using immunohistochemistry.

Effectiveness and safety of immunization with live-attenuated and inactivated vaccines for pediatric liver transplantation recipients.

BACKGROUND Liver transplantation recipients are at high risk for severe complications due to infections because of being treated with immunosuppressive drugs that affect the immune system. Vaccination for liver transplantation candidates is generally recommended before surgery, but the opportunities for vaccination prior to transplantation in pediatric candidates are often limited by severe disease conditions. METHODS The participants in this study comprised 39 pediatric recipients of living donor liver transplantation performed between 2005 and 2013. Criteria for administering live-attenuated (measles, rubella, mumps, and varicella) and inactivated (hepatitis B, pertussis, and Japanese encephalitis) vaccines were as follows: (1) >1 year after transplantation; (2) no use of systemic steroids to treat acute rejection within the last 6 months; (3) serum trough concentration of tacrolimus <5 ng/mL; (4) no severe immunosuppression according to blood examinations; and (5) provision of written informed consent. Median age at transplantation was 17 months, and median period from transplantation to the beginning of immunization was 18 months. RESULTS Seroprotection rates for measles, rubella, mumps, varicella, hepatitis B, pertussis, and Japanese encephalitis after post-transplant immunization were 44% (11/25), 70% (19/27), 48% (12/25), 32% (6/19), 83% (19/23), 87% (13/15), and 88% (7/8), respectively. Seroprotection rates for measles, rubella, mumps, and varicella after second vaccination for recipients with primary vaccine failure after first vaccination were 100% (8/8), 50% (1/2), 71% (5/7), and 50% (5/10), respectively. While four recipients contracted mumps and eight contracted varicella before immunization, one recipient developed varicella after immunization. No serious systemic adverse events were observed in vaccinated recipients. CONCLUSIONS Seroprotection rates for measles, mumps, and varicella appeared low in children after the first post-transplantation vaccination. Immunizations with four live-attenuated and three inactivated vaccines were safe and effective for pediatric liver transplantation recipients who were not severely immunosuppressed.

Gene expression profiling reveals upregulated UCA1 and BMF in gallbladder epithelia of children with pancreaticobiliary maljunction.

Background:Pancreaticobiliary maljunction is usually associated with choledochal cysts and often causes biliary carcinoma; however, the mechanism of carcinogenesis remains unknown. No study has analyzed overall changes in genetic expression beginning during childhood in gallbladder epithelia with pancreaticobiliary maljunction. Patients and Methods:The genomewide expression of gallbladder epithelia was analyzed in 6 children with pancreaticobiliary maljunction and in 4 pediatric controls. Selected genes that were expressed differentially were further analyzed by the real-time reverse transcription-polymerase chain reaction (RT-PCR). The products of upregulated genes confirmed by real-time RT-PCR were immunohistochemically analyzed using gallbladders from 19 children with pancreaticobiliary maljunction, 5 pediatric controls, and 5 children with gallstones. Results:Microarray analysis identified 188 upregulated and 160 downregulated genes. RT-PCR confirmed upregulation in 5 of 6 genes and downregulation in 1 of 5 genes, including UCA1, DUOX2, DUOXA2, ID1, BMF, and GP2. Immunohistochemistry showed a significantly higher expression of BMF in the pancreaticobiliary maljunction patients than in the controls and gallstone patients. Conclusions:This study identified several deregulated genes in the gallbladder of children with pancreaticobiliary maljunction, which may contribute to the pathophysiology. UCA1, a noncoding RNA, is an oncofetal gene, and its upregulation may be important for biliary carcinogenesis. The elevated expression of BMF may function as an apoptotic activator in proliferative gallbladder epithelia.

Pancreaticobiliary Maljunction Without Bile Duct Dilatation in Children: Distinction From Choledochal Cyst

Objectives: Pancreaticobiliary maljunction without bile duct dilatation (nondilated type) is rare in children, and its definition remains unclear. There is controversy over treatment between pediatric and adult patients. We reevaluated our previous definition of the nondilated type in children (common bile duct diameter ≤6 mm on cholangiography). Patients and Methods: Of 150 children with pancreaticobiliary maljunction, 14 fulfilled the definition of the nondilated type. Clinical and cholangiographic findings were reviewed and compared with cholangiograms of 17 children without biliary diseases (controls). The age-related normal range of the choledochal diameter was defined in combination with previous data. Results: All of the patients had symptoms and signs similar to those of choledochal cysts. Cholangiographic features of choledochal cysts were detected in all of the patients (dilated common channel, 8; distal bile duct stenosis, 5; dilated cystic duct, 4; filling defect in the common channel, 8). The choledochal diameter in control children increased with age. Only 4 patients (29%) had a choledochal caliber within the normal range. Conclusions: Most pediatric cases reported as the nondilated type are slightly dilated (forme fruste) choledochal cysts and differ from the nondilated type seen in adults. The nondilated type in children should be defined on the basis of the age-related choledochal diameter.

A New Era of Laparoscopic Revision of Kasai Portoenterostomy for the Treatment of Biliary Atresia

Purpose. Kasai portoenterostomy is the standard therapy for biliary atresia (BA). If Kasai is unsuccessful, there is controversy over whether revision of Kasai restores adequate biliary drainage. Although there are several reports of laparoscopic Kasai (Lap-Kasai), none has described laparoscopic revision (Lap-revision). The purposes of this study were to evaluate the feasibility and efficacy of Lap-revision. Methods. 65 patients underwent open Kasai between November 2001 and November 2013, and 12 patients underwent Lap-Kasai between December 2013 to January 2015. The indications for revision included bile flow cessation and recurrent cholangitis. Clinical data were compared between open and laparoscopic revisions of Kasai. Results. Open revision of Kasai was performed in 20 patients after open Kasai, and Lap-revision was performed in 4 patients after Lap-Kasai. Lap-revision was completed without conversion or major complication in any patient. The bilirubin level was normalized by Lap-revision in all four patients, and three of them were alive with their native liver. Open and laparoscopic revisions of Kasai were comparable in terms of the operation time, blood loss, and surgical outcomes. Conclusion. Lap-revision is a feasible and effective method for the treatment of BA and might herald a new era for the treatment of this disease.

Fatty acid calcium stones in patients with pancreaticobiliary maljunction/choledochal cyst as another cause of obstructive symptoms besides protein plugs

Symptoms of choledochal cysts are caused by protein plugs, which clog up in the long common channel and increase pancreaticobiliary ductal pressure. We report that fatty calcium acid stones/debris are another previously unreported cause of obstructive symptoms in 2 cases with choledochal cyst.

Histological assessment of bile lake formation after hepatic portoenterostomy for biliary atresia

Bile lakes develop after hepatic portoenterostomy in some patients with biliary atresia, and have been regarded as an indication of poor prognosis. We reported that bile lakes have no epithelium of the bile duct on their wall, and are surrounded by bile ducts; however, the mechanism of bile lake formation is little known. We investigated histologically how bile ducts are formed using whole removed liver, and the characteristics of bile ducts around bile lakes. From April 1980 to July 2006, we encountered 84 patients with biliary atresia. Bile lakes were analyzed histologically in 11 patients who underwent liver transplantation in our hospital. Bile lakes had a fibrotic cyst wall and lacked epithelia. In most cases, bile stasis, calculi formation, damaged bile ducts, and invasion of inflammatory cells were observed around the bile lakes. Bile ducts around bile lakes were not stained by CD56, but bile ducts around liver lobuli were stained by CD56. The present study speculates that bile lakes would arise from original bile ducts, which are damaged, and fuse together after calculi are formed in bile ducts.

Intrahepatic cystic lesions after hepatic portoenterostomy for biliary atresia with bile lake and dilated bile ducts.

Background: Intrahepatic cystic lesions develop after hepatic portoenterostomy in some patients with biliary atresia and have been regarded as an indication of poor prognosis; however, there is confusion about the terminology and concepts of the lesions. We investigated whether the cystic lesions constituted a single entity. Patients and Methods: From 1980 to 2005, we encountered 80 patients with biliary atresia. Abdominal ultrasonography disclosed intrahepatic cystic lesions in 20 (25%) patients after hepatic portoenterostomy. The cystic lesions were analyzed morphologically with radiologic imaging studies and histologically in 13 patients who underwent liver transplantation. Results: Fifteen patients had solitary simple cystic lesions, and 5 patients had multiple continuous oval or beaded lesions. Solitary lesions had a fibrotic cyst wall and lacked epithelia. Continuous lesions had a cyst wall covered with biliary epithelia. Thirteen patients with solitary cysts died or required liver transplantation. In 2 patients with continuous lesions, surgical reboring of the porta hepatis could eliminate cystic lesions and jaundice. Conclusions: Intrahepatic cystic lesions include 2 different conditions. Solitary cysts are retention pseudocysts, which should be referred to as a bile lake, and are associated with poor prognosis. Continuous beaded cysts are dilated bile ducts, which may be reversed. This distinction is important when considering the treatment strategy.

Prophylactic effect of H2 blocker for anastomotic stricture after esophageal atresia repair.

Anastomotic stricture is the main complication after esophageal atresia (EA) repair. In this study, we assessed the efficacy of long‐term prophylactic H2 blocker treatment in preventing stricture.