Functional Analysis of Human Hepatocytes Isolated From Chimeric Mouse Liver.

Chimeric mice with humanized liver were first established by transplanting primary human hepatocytes (PHHs) isolated from a Japanese 27-year-old donor into complementary DNA-urokinase-type plasminogen activator/severe combined immunodeficiency mice. The PHHs from the Japanese donor increased more than 100-fold in the mouse liver, and human hepatocytes purified from the chimeric mouse liver (hcPHs) were successfully transplanted into second-passaged mice. These PHHs and hcPHs can produce human albumin and preserve many liver-specific enzyme genes, which are important for liver function. Interestingly, hepatitis B virus can be infected with these chimeric mice; hepatitis B viral DNA and hepatitis B surface antigen levels were detectable. In conclusion, hcPHs can be an ideal cell source for analysis of human hepatocytes.

Ischemia reperfusion-facilitated sinusoidal endothelial cell injury in liver transplantation and the resulting impact of extravasated platelet aggregation.

BACKGROUND: The exact sequence of events leading to ultimate hepatocellular damage following ischemia/reperfusion (I/R) is incompletely understood. In this article, we review a mechanism of organ dysfunction after hepatic I/R or immunosuppressive treatment, in addition to the potential of liver sinusoidal endothelial cell (LSEC) protection and antiplatelet treatment for the suppression of hepatocellular damage. METHODS: A review of the literature, utilizing PubMed-NCBI, was used to provide information on the components necessary for the development of hepatocellular damage following I/R. RESULTS: It is well-established that LSECs damage following hepatic I/R or immunosuppressive treatment followed by extravasated platelet aggregation (EPA) is the root cause of organ dysfunction in liver transplantation. We have classified three phases, from LSECs damage to organ dysfunction, utilizing the predicted pathogenic mechanism of sinusoidal obstruction syndrome. The first phase is detachment of LSECs and sinusoidal wall destruction after LSECs injury by hepatic I/R or immunosuppressive treatment. The second phase is EPA, accomplished by sinusoidal wall destruction. The various growth factors, including thromboxane A2, serotonin, transforming growth factor-beta and plasminogen activator inhibitor-1, released by EPA in the Disse's space of zone three, induce portal hypertension and the progression of hepatic fibrosis. The third phase is organ dysfunction following portal hypertension, hepatic fibrosis, and suppressed liver regeneration through various growth factors secreted by EPA. CONCLUSION: We suggest that EPA in the space of Disse, initiated by LSECs damage due to hepatic I/R or immunosuppressive treatment, and activated platelets may primarily contribute to liver damage in liver transplantation. Endothelial protective therapy or antiplatelet treatment may be useful in the treatment of hepatic I/R following EPA.

Severe veno-occlusive disease/sinusoidal obstruction syndrome after deceased-donor and living-donor liver transplantation.

Veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) occurring after liver transplantation is a relatively rare complication but it often takes a life-threatening course. However, the detailed etiology and mechanism of VOD/SOS after liver transplantation (LT) remains unclear. We report two cases with rapidly progressive VOD/SOS after ABO-identical LT resistant to various therapies. In case 1, in which the patient underwent deceased-donor LT, the first episode of acute allograft rejection was triggered VOD/SOS, and the presence of donor non-specific anti-HLA antibodies was confirmed. The recipient died with graft failure on day 46 after transplantation. Case 2, in which the patient underwent living-donor LT from the mother, had neither rejection nor mechanical venous obstruction, but condition of the patient rapidly worsened and he died on day 13 after transplantation. This recipient's direct cross-match test for the donor's B lymphocyte was strongly positive, but that for T lymphocyte was negative. In both cases, neither stenosis of hepatic vein outflow tract nor C4d deposition in post-transplantation liver biopsy specimens and autopsy specimen was found. On the other hand, in both cases, the patient was transfusion unresponsive thrombocytopenia and hyperbilirubinemia persisted postoperatively, and glycoprotein Ⅰ bα was strongly stained in the neighboring centrilobular area (zone 3), especially in the space of Disse, and platelet phagocytosis was observed in Kupffer cells and hepatocytes around zone 3 such as clinical xenotransplantation of the liver in post-transplantation liver biopsy specimens. From the viewpoint of graft injury, VOD/SOS was considered that sustained sinusoidal endothelial cells injury resulted in bleeding in the space of Disse and led to around centrilobular hemorrhagic necrosis, and the fundamental cause was damage around centrilobular area including sinusoid by acute cellular rejection, antibody-mediated rejection or ischemic reperfusion injury. The extrasinusoidal platelet activation, aggregation, and phagocytosis of platelets were some of the main reasons for VOD/SOS and transfusion-resistant thrombocytopenia.

Chronic immune-mediated reaction syndrome as the cause of late graft mortality in living-donor liver transplantation for primary biliary cirrhosis.

INTRODUCTION: Few studies to date have investigated the causes of late graft mortality after living-donor liver transplantation (LDLT) for primary biliary cirrhosis (PBC). PATIENTS AND METHODS: Fifty-five LDLTs for PBC were retrospectively reviewed. Factors prognostic of graft survival after LDLT were investigated, and histologic findings in patients with late graft loss were assessed. RESULTS: The 1-, 5-, and 10-year cumulative graft survival rates were 85.1%, 82.5%, and 66.9%, respectively. Multivariate Cox regression analysis found that male donor and ≥ 4 HLA mismatches were independently associated with poor graft survival. Among the 13 grafts lost, 5 were lost >1 year after LDLT, including 1 each due to chronic rejection, veno-occlusive disease, and obliterative portal venopathy, and 2 to other causes. Pathologic reviews of the serial biopsy specimens and explanted grafts from these 5 patients, with graft rejections from "chronic immune-mediated reaction syndrome," showed reciprocal changes over time. No patient died of recurrent PBC. CONCLUSIONS: Male donor and ≥ 4 HLA mismatches were independent factors associated with poor graft survival. Late graft mortality after LDLT for PBC in some patients was due to chronic immune-mediated reaction syndrome, including chronic rejection, veno-occlusive disease, and obliterative portal venopathy, but not to recurrent PBC.

Revisiting the safety of living liver donors by reassessing 441 donor hepatectomies: is a larger hepatectomy complication-prone?

Donor safety is of paramount importance in performing living donor liver transplantation (LDLT). We retrospectively reviewed donor medical records to confirm whether larger donor hepatectomy is absolutely complication-prone. A total of 441 living donor hepatectomies were performed between October 1996 and July 2012 in our institute, which were divided into three eras (Era I, October 1996 to March 2004; Era II, April 2004 to March 2008; Era III, April 2008 to July 2012) and the incidences of postoperative complications were compared among the three types of hepatectomy-right hepatectomy (RH), left hepatectomy (LH) and left lateral segmentectomy (LLS). Although severe complications (Clavien's grade 3 or more) frequently occurred in RH in Eras I and II (15.4% and 10.7%, respectively), the incidence in Era III decreased to the comparable level observed in LH and LLS (5.4% in RH, 2.3% in LH and 5.3% in LLS). The incidence of postoperative complications did not relate to the type of hepatectomy selected in the latest era. Since most complications after hepatectomy were considered preventable, step-by-step meticulous surgical procedures are a prerequisite for further assuring donor safety irrespective of the type of hepatectomy selected.

Thoracoscopic esophagectomy with extended lymph node dissection in the left lateral position: technical feasibility and oncologic outcomes.

The aim of this study was to estimate the technical and oncologic feasibility of video-assisted thoracoscopic radical esophagectomy (VATS) in the left lateral position. From January 2003 to December 2011, 132 patients with esophageal cancer underwent VATS. The mean duration of the thoracic procedure and the entire procedure was 294 ± 88 and 623 ± 123 minutes, respectively. Mean blood loss during the thoracic procedure and the entire procedure was 313 ± 577 and 657 ± 719 g, respectively. The mean number of dissected thoracic lymph nodes was 32.6 ± 12.9. There were four in-hospital deaths (3.0%); two patients (1.5%) died of acute respiratory distress syndrome and two patients (1.5%) died of tumor progression. Postoperative unilateral or bilateral recurrent laryngeal nerve (RLN) palsy, or pneumonia was found in 33 (25.0%), 21 (15.9%), and 27(20.5%) patients, respectively. The patients were divided into the first 66 patients who underwent VATS (Group 1) and the subsequent 66 patients (Group 2). The numbers of cases who underwent neoadjuvant or induction chemotherapy for T4 tumor and intrathoracic anastomosis were higher in Group 2 than in Group 1. The duration of the procedure, amount of blood loss, and the number of dissected thoracic lymph nodes were not different between the two groups. The total number of dissected lymph nodes was higher in Group 2 than in Group 1 (72.6 ± 27.8 vs. 62.6 ± 21.6, P = 0.023). The rate of bilateral RLN palsy was less in Group 2 than in Group 1 (7.6% vs. 24.2%, P = 0.042). The mean follow-up period was 38.7 months. Primary recurrence consisted of hematogenous, lymphatic, peritoneal dissemination, pleural dissemination, and locoregional in 15 (11.3%), 20 (15.1%), 3 (2.3%), 4 (3.0%), and 5 patients (3.8%), respectively. The rate of regional lymph node recurrence within the dissection field was only 4.5%. The prognosis of patients with lymph node metastasis was significantly poorer than that of patients without lymph node metastasis. However, the prognosis of the 11 cases that had metastasis only around RLNs was similar to that of node-negative cases. Thirteen patients with pathological remnant tumor (R1 or R2) did not survive longer than 5 years at present. The overall 5-year survival rate of stage I, II, and III disease after curative VATS was 82.2%, 77.0%, and 52.3%, respectively. Expansion of VATS criteria for patients after induction chemotherapy for T4 tumor or thoracoscopic anastomosis did not adversely affect the surgical results by experience. Although the VATS procedure is accompanied by a certain degree of morbidity including RLN palsy and pulmonary complications, VATS has an excellent locoregional control effect. In addition, the favorable survival after VATS shows that the procedure is oncologically feasible.

Learning of thoracoscopic radical esophagectomy: how can the learning curve be made short and flat?

Attainment of proficiency in video-assisted thoracoscopic radical esophagectomy (VATS) for thoracic esophageal cancer requires much experience. We have mastered this procedure safely under the direction of an experienced surgeon. After adoption of the procedure, the educated surgeon directed induction of this surgical procedure at another institution. We evaluated the efficacy of instruction during the induction period by comparing the results at the two institutions in which VATS had been newly induced. We defined the induction period as the time from the beginning of VATS to the time when the last instruction was carried out. From January 2003 to December 2007, 53 patients were candidates for VATS at Kanazawa University (institution 1). Of these, 46 patients underwent curative VATS by a single operator. We divided this period into three parts: the induction period of VATS, post-induction period, and proficient period when the educated surgeon of institution 1 directed the procedure at Maebashi Red Cross Hospital (institution 2). At institution 1, 12 VATS were scheduled, and nine procedures (75%) (group A) including eight instructions were completed during the induction period (from January 2003 to August 2004). Thereafter, VATS was performed without instruction. In the post-induction period, nine VATS were scheduled, and eight procedures (88.8%) (group B) were completed from September 2004 to August 2005. Subsequently, 32 VATS were scheduled, and 29 procedures (90.6%) (group C) were completed during the proficient period (from September 2005 to December 2007). The surgeon at Maebashi Red Cross Hospital (institution 2) started to perform VATS under the direction of the surgeon who had been educated at institution 1 from September 2005. VATS was completed in 13 (76.4%) (group D) of 17 cases by a single surgeon including seven instructions during the induction period at institution 2 from September 2005 to December 2007. No lethal complication occurred during the induction period at both institutions. We compared the results of VATS among four groups from the two institutions. There were no differences in the background and clinicopathological features among the four groups. The number of dissected lymph nodes and amount of thoracic blood loss were similar in the four groups (35 [22-52] vs 41 [26-53] vs 32 [17-69] vs 29 [17-42] nodes, P = 0.139, and 170 [90-380] vs 275 [130-550] vs 220 [10-660] vs 210 [75-543] g, P = 0.373, respectively). There was no difference in the duration of the thoracic procedure during the induction period at the two institutions. However, the duration of the procedure was significantly shorter in the proficient period of institution 1 (group C: 266 [195-555] minutes) than in the induction period of both institutions (group A: 350 [280-448] minutes [P = 0.005] and group D: 345 [270-420] mL [P = 0.002]). There were no surgery-related deaths in any of the groups. The incidence of postoperative complications did not differ among the four groups. Thoracoscopic radical esophagectomy can be mastered quickly and safely with a flat learning curve under the direction of an experienced surgeon. The educated surgeon can instruct surgeons at another institution on how to perform thoracoscopic esophagectomy. The operation time of thoracoscopic surgery is shortened by experience.

Prospective randomized controlled study on the effects of perioperative administration of a neutrophil elastase inhibitor to patients undergoing video-assisted thoracoscopic surgery for thoracic esophageal cancer.

Sivelestat sodium hydrate (Ono Pharmaceutical Co., Osaka, Japan) is a selective inhibitor of neutrophil elastase (NE) and is effective in reducing acute lung injury associated with systemic inflammatory response syndrome (SIRS). We conducted a prospective randomized controlled study to investigate the efficacy of perioperative administration of sivelestat sodium hydrate to prevent postoperative acute lung injury in patients undergoing thoracoscopic esophagectomy and radical lymphadenectomy. Twenty-two patients with thoracic esophageal cancer underwent video-assisted thoracoscopic esophagectomy with extended lymph node dissection in our institution between April 2007 and November 2008. Using a double-blinded method, these patients were randomly assigned to one of two groups preoperatively. The active treatment group received sivelestat sodium hydrate intravenously for 72 hours starting at the beginning of surgery (sivelestat-treated group; n= 11), while the other group received saline (control group; n= 11). All patients were given methylprednisolone immediately before surgery. Postoperative clinical course was compared between the two groups. Two patients (one in each group) were discontinued from the study during the postoperative period because of surgery-related complications. Of the remaining 20 patients, 2 patients who developed pneumonia within a week after surgery were excluded from some laboratory analyses, so data from 18 patients (9 patients in each group) were analyzed based on the arterial oxygen pressure/fraction of inspired oxygen ratio, white blood cell count, serum C-reactive protein level, plasma cytokine levels, plasma NE level, and markers of alveolar type II epithelial cells. In the current study, the incidence of postoperative morbidity did not differ between the two groups. The median duration of SIRS in the sivelestat-treated group was significantly shorter than that in the control group: 17 (range 9-36) hours versus 49 (15-60) hours, respectively (P= 0.009). Concerning the parameters used for the diagnosis of SIRS, the median heart rates on postoperative day (POD) 2 were significantly lower in the sivelestat-treated group than in the control group (P= 0.007). The median arterial oxygen pressure/fraction of inspired oxygen ratio of the sivelestat-treated group were significantly higher than those of the control group on POD 1 and POD 7 (POD 1: 372.0 [range 284.0-475.0] vs 322.5 [243.5-380.0], respectively, P= 0.040; POD 7: 377.2 [339.5-430.0] vs 357.6 [240.0-392.8], P= 0.031). Postoperative white blood cell counts, serum C-reactive protein levels, plasma interleukin-1beta, tumor necrosis factor-alpha levels, and plasma NE levels did not differ significantly between the two groups at any point during the postoperative course, nor did serum Krebs von den Lungen 6, surfactant protein-A, or surfactant protein-D levels, which were used as markers of alveolar type II epithelial cells to evaluate the severity of lung injury. Plasma interleukin-8 levels were significantly lower in the sivelestat-treated group than in the control group on POD 3 (P= 0.040). In conclusion, perioperative administration of sivelestat sodium hydrate (starting at the beginning of surgery) mitigated postoperative hypoxia, partially suppressed postoperative hypercytokinemia, shortened the duration of SIRS, and stabilized postoperative circulatory status after thoracoscopic esophagectomy.

Duodenogastric reflux sustains Helicobacter pylori infection in the gastric stump.

BACKGROUND: Duodenogastric reflux (DGR) and Helicobacter pylori infection have been suspected of being contributing agents to the genesis of gastritis and subsequent cancer, but compelling, conclusive data about the exact relationship have been lacking. METHODS: We investigated the effect of DGR on H. pylori infection in 95 gastrectomized subjects divided into four groups according to type of reconstruction: the jejunal pouch interposition group (JPI, n = 36); the Roux-en-Y group (RY, n = 17); the Billroth I group (B-I, n = 20); and the Billroth II group (B-II, n = 22). The following items were examined for each group: the duration of DGR; the prevalence of H. pylori infection; other bacterial identification and quantity; and the severity of gastritis. RESULTS: The percent of total time of DGR was lower in the JPI (7%) and RY groups (28%) than in the B-I (59%) and B-II groups (88%) (P < 0.02). The prevalence of H. pylori infection was lower in the JPI (28%) and RY groups (29%) than in the B-I (60%) and B-II groups (73%) (P < 0.02). Inversely, the JPI and the RY groups had a higher quantity of other bacteria than the B-I group (P = 0.02). For all four groups, the stomachs infected with H. pylori were fewer than those tested negative for the organism (P < 0.0001). Inflammation scores were lower in both the JPI and RY groups than in the B-I and B-II groups (P < 0.05, respectively). CONCLUSIONS: Duodenogastric reflux facilitates the survival of H. pylori in the gastric stump after a distal gastrectomy.

Subtotal peritonectomy with chemohyperthermic peritoneal perfusion for peritonitis carcinomatosa in gastrointestinal cancer.

Treatment for peritonitis carcinomatosa in gastrointestinal cancer remains to be established though it is one of the commonest causes of cancer death. Subtotal peritonectomy (SP) with chemohyperthermic peritoneal perfusion (CHPP) was developed for the new therapeutic strategy for peritoneal dissemination in gastrointestinal cancer in our department. SP includes resection of stomach, colon, small bowel, spleen, gall bladder, and parietal peritoneum. CHPP was carried out by heated saline containing 25 mg/l cisplatin, 10 mg/l mitomycin C, and 20 mg/l etoposide. Intraperitoneal temperature was maintained at 42 degrees C for 60 min. Fifteen gastric cancer and three colon cancer patients with severe peritoneal dissemination underwent these procedures. The averages of operating time, intraoperative bleeding volume, and total perioperative transfused blood volume were 9 h, 4400 ml, and 5600 ml, respectively. The patients estimated as complete resection and residual disease by histopathological study numbered 11 and 7. There was no treatment-related deaths though bleeding occurred in 5 patients; perforation in 2 patients; and abscesses in 2 patients. The 1-year survival rate (1ysr) and the 2-year survival rate (2-ysr) of all the patients were 57% and 21%, respectively. The 1-ysr and the 2-ysr of the patients who underwent complete resection were 67% and 40% significantly greater than the 43% and 0% of the patients who had residual tumors (p=0.02). The combination therapy of SP and CHPP is feasible in spite of its morbidity and has great possibilities in complete resection of peritoneal dissemination and prolongation of patient's survival.

[Chemohyperthermic peritoneal perfusion and subtotal peritonectomy for peritonitis carcinomatosa in gastroenteric cancer].

A new operative procedure, called subtotal peritonectomy (SP), in combination with chemohyperthermic peritoneal perfusion, was developed for the treatment of peritonitis carcinomatosa in gastrointestinal cancer. SP includes resection of primary lesion, colon, small bowel, spleen, and gall bladder and parietal peritonectomy. Six patients with gastric cancer and two patients with colon cancer underwent these procedures. A great deal of discharge from the peritoneal cavity, an increase in systemic vascular resistance index, and a decrease in central venous pressure represented much decrease in circulatory volume on days 1 to 2 postoperatively. This state improved at 3 to 4 days after operation. Histopathological study revealed multiple peritoneal seedings with negative surgical margins in all patients. There were no related deaths though bleeding, perforation, and abscess occurred in two patients each. One patient died of peritoneal recurrence after one year, but the other have survived.