Takafumi Machimoto

Activation‐induced cytidine deaminase links bile duct inflammation to human cholangiocarcinoma

Chronic inflammation plays a critical role in oncogenesis in various human organs. Epidemiological studies have demonstrated that patients with primary sclerosing cholangitis have a predisposition to develop cholangiocarcinoma (CC). However, the molecular mechanisms that account for the development of bile duct carcinomas are not well defined. We recently provided evidence that activation‐induced cytidine deaminase (AID), a member of the DNA/RNA editing enzyme family, is implicated in human tumorigenesis via its mutagenic activity. We found here that ectopic AID production is induced in response to tumor necrosis factor‐α (TNF‐α) stimulation via the IkappaB kinase‐dependent nuclear factor‐κB (NF‐κB) activation pathway in human cholangiocarcinoma‐derived cells. Aberrant expression of AID in biliary cells resulted in the generation of somatic mutations in tumor‐related genes, including p53, c‐myc, and the promoter region of the INK4A/p16 sequences. In human tissue specimens, real‐time reverse transcription polymerase chain reaction (RT‐PCR) analyses revealed that AID was increased significantly in 28 of 30 CC tissues (93%), whereas only trace amounts of AID were detected in the normal liver. Immunohistochemistry showed that all of the CC tissue samples examined showed overproduction of endogenous AID protein in cancer cells. Moreover, immunostaining for AID was detectable in 16 of 20 bile epithelia in the tissues underlying primary sclerosing cholangitis. Conclusion: The proinflammatory cytokine‐induced aberrant production of AID might link bile duct inflammation to an enhanced genetic susceptibility to mutagenesis, leading to cholangiocarcinogenesis. (HEPATOLOGY 2008;47:888–896.)

Thy1-positive mesenchymal cells promote the maturation of CD49f-positive hepatic progenitor cells in the mouse fetal liver.

Previously, we reported a system to enrich mouse fetal hepatic progenitor cells (HPCs) by forming cell aggregates. In this study, we sorted two cell populations, CD49f+Thy1−CD45− cells (CD49f‐postive cells) and CD49f±Thy1+CD45− cells (Thy1‐positive cells), from the cell aggregates using a flow cytometer. CD49f‐positive cells stained positive for endodermal specific markers such as α‐fetoprotein (AFP), albumin (ALB), and cytokeratin 19 (CK19), and are thus thought to be HPCs. However, Thy1‐positive cells were a morphologically heterogeneous population; reverse‐transcription polymerase chain reaction (RT‐PCR) and immunocytochemical analyses revealed the expression of mesenchymal cell markers such as α‐smooth muscle actin, desmin, and vimentin, but not of AFP, ALB, or CK19. Therefore, Thy1‐positive cells were thought to be of a mesenchymal lineage. When these two cell populations were co‐cultured, the CD49f‐positive colonies matured morphologically and stored a significant amount of glycogen. Furthermore, real‐time RT‐PCR demonstrated an increased expression of tyrosine amino transferase and tryptophan oxygenase mRNA, and transmission electron microscopy confirmed that co‐cultured cells produced mature hepatocytes. However, when CD49f‐positive cells were cultured alone or when the two populations were cultured separately, the CD49f‐positive cells did not mature. These results indicate that CD49f‐positive cells are primitive hepatic endodermal cells with the capacity to differentiate into hepatocytes, and that Thy1‐positive cells promote the maturation of CD49f‐positive cells by direct cell‐to‐cell contact. In conclusion, we were able to isolate CD49f‐positive primitive hepatic endodermal cells and Thy1‐positive mesenchymal cells and to demonstrate the requirement of cell‐to‐cell contact between these cell types for the maturation of the hepatic precursors. (HEPATOLOGY 2004;39:1362–1370.)

Transplantation of Embryonic Stem Cell‐Derived Endodermal Cells into Mice with Induced Lethal Liver Damage

ESCs are a potential cell source for cell therapy. However, there is no evidence that cell transplantation using ESC‐derived hepatocytes is therapeutically effective. The main objective of this study was to assess the therapeutic efficacy of the transplantation of ESC‐derived endodermal cells into a liver injury model. The β‐galactosidase‐labeled mouse ESCs were differentiated into α‐fetoprotein (AFP)‐producing endodermal cells. AFP‐producing cells or ESCs were transplanted into transgenic mice that expressed diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Selective damage was induced in the recipient hepatocytes by the administration of DT. Although the transplanted AFP‐producing cells had repopulated only 3.4% of the total liver mass 7 days after cell transplantation, they replaced 32.8% of the liver by day 35. However, these engrafted cells decreased (18.3% at day 40 and 7.9% at day 50) after the cessation of DT administration, and few donor cells were observed by days 60–90. The survival rate of the AFP‐producing cell‐transplanted group (66.7%) was significantly higher in comparison with that of the sham‐operated group (17.6%). No tumors were detected by day 50 in the AFP‐producing cell‐transplanted group; however, splenic teratomas did form 60 days or more after transplantation. ESC transplantation had no effect on survival rates; furthermore, there was a high frequency of tumors in the ESC‐transplanted group 35 days after transplantation. In conclusion, this study demonstrates, for the first time, that ESC‐derived endodermal cells improve the survival rates after transplantation into mice with induced hepatocellular injury.

Contribution of 18F‐fluorodeoxyglucose positron emission tomography to the diagnosis of early pancreatic carcinoma

BACKGROUND/PURPOSE Pancreatic carcinoma has a poor prognosis, and early detection is essential to allow potentially curative resection. Despite the wide array of diagnostic tools available, the detection of small pancreatic tumors remains difficult. The aim of this study was to investigate the contribution of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to the diagnosis of early pancreatic cancer. METHODS FDG-PET was performed in 56 patients with pancreatic cancer who underwent curative surgery. The standardized uptake value (SUV) for FDG was calculated in each patient and the relationships between the SUV and various clinicopathological factors were analyzed. RESULTS The tumors ranged from 0.8 to 6.5 cm in diameter. When the cutoff value for the SUV was set at 2.5, 51 of the 56 patients (91%) had a positive FDG-PET study. The SUV did not show a significant difference in relation to tumor differentiation or pTS and pT factors. There was also no correlation between the SUV and the maximum tumor diameter (r = 0.22; P = 0.1). Five tumors had an SUV below the cutoff value, and all of these lesions had intermediate or scirrhous stroma rather than medullary stroma. CONCLUSIONS These results indicate that FDG-PET is useful for the detection of small early pancreatic cancers.

Alpha-fetoprotein-producing pancreatic cancer cells possess cancer stem cell characteristics

We aimed to demonstrate the existence of cancer stem cells in human pancreatic cancer, and to clarify that they are alpha-fetoprotein (AFP) producing cells. Six cell lines derived from human pancreatic cancers were examined, and AsPC-1 and PANC-1 were noted to express AFP. Single cell culture assays and xenotransplantation revealed that the AFP-producing cells had the capacity for self-renewal and differentiation, and that these cells were tumorigenic. Furthermore, they were resistant to anti-cancer agents. The ABCA12 transporter was expressed in the AFP-producing cells at a level more than twice as high as that in the non-AFP-producing cells. The AFP-producing cells were shown to be putative pancreatic cancer stem cells. Furthermore, the expression of ABCA12 appears to be associated with drug resistance.

Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model.

Background. The use of cell transplantation as an alternative therapy for orthotopic liver transplantation has been widely anticipated due to a chronic donor shortage. We previously reported the method used to enrich hepatic progenitor cells (HPCs) forming cell aggregations. In this study, we transplanted HPCs into the liver injury model mice to determine whether HPC transplantation may improve the liver dysfunction. Methods. We obtained donor cells from E13.5 fetal livers of green fluorescent protein (GFP) transgenic mice. We transplanted GFP-positive fetal liver cells into the transgenic mice which express diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Subsequently, we induced selective liver injury to recipient mice by DT administration. We then evaluated the engraftment of the transplanted cells and their effect on survivorship. Results. The low dose of DT induced sublethal liver injury and the high dose of DT was lethal to the liver injury model mice. The transplanted GFP-positive cells were engrafted into the recipient livers and expressed albumin, resembling mature hepatocytes. They continued to proliferate, forming clusters. The survival rate at 25 days after transplantation of the cell-transplanted group (8 of 20; 40.0%) was improved significantly (P=0.0047) in comparison to that of the sham-operated group (0 of 20; 0%). Conclusions. The transplanted cells were engrafted and repopulated the liver of recipient mice, resulting in the improvement of the survival rate of the liver injury model mice. We therefore propose that HPCs are a desirable cell source for cell transplantation.

Comparative study of transplantation of hepatocytes at various differentiation stages into mice with lethal liver damage.

Hepatocyte transplantation utilizing induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) has been expected to provide an alternative to liver transplantation. However, it remains uncertain precisely which cell type is the best suited for cell transplantation. In particular, it is unclear whether mature hepatocytes, which have sufficient liver function, or immature hepatic progenitor cells, which have a higher proliferative capacity, will provide a better outcome. The main objective of this study was to investigate the therapeutic efficacy of the transplantation of hepatocytes at various differentiation stages. We utilized transgenic mice that expressed diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. ESC-derived endodermal cells, fetal hepatocytes, and adult hepatocytes were transplanted into these mice with experimentally induced lethal acute liver injury caused by DT administration. The transplanted cells were marked by enhanced green fluorescent protein. We evaluated their effects on survival. At 35 days after transplantation, the survival rate of the adult hepatocyte-transplanted group (8/20, 40%) was significantly improved in comparison to that of the sham-operated group (2/25, 8%), the fetal hepatocyte-transplanted group (1/20, 5%), and the ESC-derived endodermal cell-transplanted group (0/21, 0%). The adult hepatocytes proliferated in the recipient livers and replaced a large part of their parenchyma. The transplantation of adult hepatocytes for acute liver failure significantly improved the survival rate in comparison to that of transplantation of immature cells, thus suggesting that ESCs and iPSCs should be differentiated into mature hepatocytes before cell transplantation for acute liver failure.

Impact of stepwise introduction of esophagojejunostomy during laparoscopic total gastrectomy: a single-center experience in Japan

Background The number of laparoscopic gastrectomies performed in Japan is increasing with the development of laparoscopic and surgical instruments. However, laparoscopic total gastrectomy is developing relatively slowly because of technical difficulties, particularly in esophagojejunostomy. Methods We retrospectively reviewed 83 patients with early gastric cancer in the upper portion of the stomach who underwent laparoscopic total gastrectomy between April 2007 and March 2016. We classified the patients into three periods, mainly on the basis of the esophagojejunostomy procedures performed: first period, various conventional procedures based on the physicians’ choice (n=14); second period, transoral method (n=51); and third period, fully intracorporeal technique (n=18). We evaluated the clinical impact of a stepwise introduction of unfamiliar new methods during laparoscopic total gastrectomy. Results Between the first and second periods, there were significant differences in the blood loss volume, number of harvested lymph nodes, frequency of conversion to open surgery, and postoperative hospital stay. The number of harvested lymph nodes was significantly higher in the third than in the second period, with no detriment to other intraoperative or postoperative factors. Conclusion The use of a unified surgical method for esophagojejunostomy seems to be the key to a successful and advantageous laparoscopic total gastrectomy. Stepwise introduction of a well-established technique of esophagojejunostomy during laparoscopic total gastrectomy will benefit patients, as shown, for example, by the higher number of dissected lymph nodes in the present study. However, a protracted learning curve is required.

Protocol for laparoscopic cholecystectomy: Is it rocket science?

Laparoscopic cholecystectomy (LC) does not require advanced techniques, and its performance has therefore rapidly spread worldwide. However, the rate of biliary injuries has not decreased. The concept of the critical view of safety (CVS) was first documented two decades ago. Unexpected injuries are principally due to misidentification of human factors. The surgeon’s assumption is a major cause of misidentification, and a high level of experience alone is not sufficient for successful LC. We herein describe tips and pitfalls of LC in detail and discuss various technical considerations. Finally, based on a review of important papers and our own experience, we summarize the following mandatory protocol for safe LC: (1) consideration that a high level of experience alone is not enough; (2) recognition of the plateau involving the common hepatic duct and hepatic hilum; (3) blunt dissection until CVS exposure; (4) Calot’s triangle clearance in the overhead view; (5) Calot’s triangle clearance in the view from underneath; (6) dissection of the posterior right side of Calot’s triangle; (7) removal of the gallbladder body; and (8) positive CVS exposure. We believe that adherence to this protocol will ensure successful and beneficial LC worldwide, even in patients with inflammatory changes and rare anatomies.

Abdominal Wall Recurrence of Hilar Bile Duct Cancer 12 Years After a Curative Resection: Report of a Case

A 74-year-old woman was diagnosed with hilar bile duct cancer, and underwent a curative resection of the bile duct and the left and caudate lobes of the liver in 1995. Ten years later (April 2005), she noted a small mass in the abdominal wall. The mass slowly enlarged to reach 4 cm in diameter by January 2007. With a diagnosis of a possible recurrence of bile duct cancer, a laparotomy was thus performed. The abdominal wall tumor was buried in the rectus abdominis muscle and was tightly attached to the ileum. The lesion was resected en bloc with the associated rectus muscle and ileocecal region. A histopathological examination of the resected specimen revealed tubular adenocarcinoma that closely resembled the original primary bile duct cancer. In addition, the immunohistochemical staining pattern of the abdominal tumor was identical to that of the original bile duct cancer. This indicated that the abdominal tumor represented a local recurrence (probably due to peritoneal implantation) at 12 years after the resection of the hilar bile duct cancer. This case emphasizes that long-time surveillance is required for patients with bile duct cancer, even if they have survived without recurrence for more than 5 years after a curative resection.