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By using the time lapse of both phase-contrast and fluorescent images, we examined the morphology of cells and the dynamics of gene expression (EGFP). We applied k-means clustering to the time course data of fluorescent intensity of EGFP and successfully found four subpopulations. Discriminating the appropriate clusters and investigating the details of them, we found that almost all cells express the transfected gene after cell division and also found there is a strong correlation between onset timing and cell division. This result suggested that it is possible to normalize the dynamics of gene by arranging the onset timing of gene expression or by arranging the cell division., (2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The operation for liver metastases from colorectal cancer is increasing due to a progress of chemotherapy. On the other hand, the live damage induced by chemotherapy affects on post operative course. We studied histopathological liver injury on the patients who received preoperative chemotherapy. Between January 2004 and May 2009, forty seven patients underwent hepatectomy for colorectal liver metastases after systemic chemotherapy, and the remnant liver was histopathologically investigated about sinusoidal dilatation and non-alcoholic steatohepatitis. As a result, chemotherapy including irinotecan or oxaliplatin was significantly associated with sinusoidal dilatation, and female gender or higher body mass index was correlated with non-alcoholic steatohepatitis. These results should be taken into consideration before liver resection in patients who have received preoperative chemotherapy.

Extrahepatic bile duct carcinoma is one of the most extremely aggressive cancers with poor prognosis after curative resection. Syndecan-1 and E-cadherin are transmembrane glycoproteins, and have important roles in cell-cell adhesion and tumor progression. In this study, we examined 84 surgically resected cases of extrahepatic bile duct adenocarcinoma to clarify clinicopathological significance of syndecan-1/E-cadherin expression. Reduced expressions of syndecan-1 and Ecadherin were found in 69.0% (58/84) and 46.4% (39/84) of the bile duct carcinomas. Reduced syndecan-1 expression was correlated with lymphatic/venous/nervous invasion (P < 0.0001), and was associated with short overall survival (P = 0.0002). Reduced E-cadherin expression was correlated with lymphatic and nervous invasion (P = 0.008, P < 0.0001, respectively), and was associated with short overall survival (P = 0.0038). The results indicated that reduced syndecan-1/E-cadherin expression may be good indicators of recurrence and prognosis in extrahepatic bile duct carcinoma.

We report a case of torsion of an accessory lobe of the liver (ALL) in a 14-year-old girl. The patient was admitted for acute abdominal pain and laparotomy revealed an ALL, the pedicle of which was elongated with 180 degrees torsion. The diagnosis was not made preoperatively because of the rarity of this condition. However, a close relationship between omphalocele repair and the development of ALL has been reported; thus, a history of omphalocele repair should alert the doctor to the possibility of this condition, which could be suggested by imaging findings preoperatively. Torsion of an ALL should be included in the differential diagnosis of a patient with a history of omphalocele, who presents with acute abdominal symptoms.

Nerve growth factor (NGF)-induced neurite maturation in PC12D cells involves neuritogenesis and neurite outgrowth. Actions of compounds affecting the neurite maturation are sometimes invisible behind the individually variable events in nature even in the clonal population. In this study, we designed a time-lapse imaging system to determine the timing of each event in individual PC12D cells. To examine the system, we analyzed the effect of staurosporine on the neurite maturation in PC12D cells. By using the system, we obtained four event timing data sets (stimulation by NGF with and without staurosporine at the concentrations of 0.01, 0.1, and 1 microM). A permutation test of these data sets revealed that staurosporine caused an early induction of neurite outgrowth during neurite maturation in PC12D cells. These results suggest that the time-lapse imaging system to determine the timing of each event in individual cells can provide a novel insight into features of a cell mass by single-cell analysis and is expected to be utilized for profiling of compounds that can serve as candidate drugs.

Introduction: Biliary atresia is the most common indication for orthotopic liver transplantation (OLT) in childhood. The purpose of this study was to determine predictive prognostic factors for children with biliary atresia related to the timing for OLT within 15 months after hepatoportoenterostomy (HPE)., Patients and Methods: We retrospectively analyzed the medical records of 25 children (7 boys and 18 girls) who underwent HPE because of biliary atresia between January 1990 and December 2005 at our center. Data examined included age and pathologic findings at HPE, Pediatric End-Stage Liver Disease score at first admission, whether phototherapy was given, liver function test results and total bilirubin level before and 30 days after HPE, and number of cholangitis events., Results: Twelve children were alive with their native liver, 8 had undergone living donor OLT (all children alive), and 5 had died without OLT. Five- and 10-year survival rates without OLT after HPE were 47.4% and 26.3%, respectively. At univariate analysis, the predictive prognostic factors for children with biliary atresia were total bilirubin level at 30 days after HPE and Pediatric End-Stage Liver Disease score before HPE. At multivariate analysis, the only prognostic factor was total bilirubin level at 30 days after HPE., Conclusions: In this study, the predictive prognostic factor was total bilirubin level at 30 days after HPE. Orthotopic liver transplantation within 15 months after HPE is needed in children with biliary atresia with a high total bilirubin level at 30 days after HPE.

Introduction: Pediatric hepatocellular carcinoma (HCC) is an uncommon disease with a poor prognosis. There are few reports about liver transplantation for pediatric adult-type HCC. We experienced a case of living donor liver transplantation (LDLT) for a child with recurrent pediatric adult-type HCC., Case Report: A 12-year-old boy was admitted to the Department of Pediatrics in our institution due to HCC in May 2005. He underwent hepatectomy after 3 courses of chemotherapy in July 2005. After the operation, he had 2 more courses of the same chemotherapy. His posttheraputic course was uneventful for 1 year. However, his alpha-fetoprotein level increased and a computed tomography (CT) scan showed recurrent tumor in his remnant liver in October 2006. He underwent another chemotherapy session immediately. However, CT revealed multiple liver tumors after chemotherapy in December 2006. His mother requested to be an LDLT donor, which was performed on January 23, 2007. The donor operation was a right hepatic lobectomy. The postoperative course of the donor was unremarkable and she has now returned to work. The recipient's posttransplantation course was uneventful and he was discharged at postoperative day 53 and is currently doing well., Conclusion: Liver transplantation in conjunction with chemotherapy may have an increasing role in the management of pediatric HCC.

Cell migration plays a major role in a variety of biological processes and a detailed understanding of associated mechanisms should lead to advances in the medical sciences, for example, in drug discovery for cancer therapy. However, the traditional methods used for analysis of cell migration cannot easily be scaled up for high-throughput screening. In this study, we have attempted to develop a novel simple method for high-throughput phenotypic screening for the identification of genes that are required for cell migration. As the appropriate cell line for the method, we found NBT-L2b cells that would be suitable for screening of migration-related genes in our method without influence by other cellular processes. Moreover, the idea for printing both the labeled fibronectin, for identification of the starting region of a cell, and the green fluorescent protein (GFP) expression vector, for identification of cells that had been transfected with siRNA and of the end point of migration, brings a rapid and efficient high-throughput screening procedure. Our new method will lead to an enhanced understanding of cell migration.

We developed a plasma recycling dialysis (PRD) system based on plasma exchange (PE). In this system, rapid reduction of toxic substances and restitution of deficient essential substances are performed by PE and subsequent blood purification is performed by dialysis between separated plasma recycled over a purification device and the patient's blood across the membrane of a plasma separator. To demonstrate the safety and efficacy of this system, we used a pig model of fulminant hepatic failure (FHF) and anion exchange resin, activated charcoal and hemodialysis for the purification device. FHF was induced by intraportal administration of alpha-amanitine (0.1 mg/kg) and lipopolysaccharides (1 microg/kg) in pigs. Three groups of animals were studied: group 1, diseased controls (N = 4); group 2, PE group (N = 4), 16 h after drug infusion the pigs underwent PE of approximately 1.2 L for 2 h; and group 3, PE + PRD group (N = 4), the pigs underwent PE followed by PRD for 6 h. The hemodynamic status of all animals was stable during the procedure. In group 3, the values of ammonia, total bile acid and total bilirubin continuously decreased and were significantly lower than those of the animals in group 2 24 h after the induction of FHF. The Fischer ratio was significantly higher than in group 2 after 24 h. Group 3 pigs maintained a higher level of consciousness and survived longer than group 2 pigs. Safety of this PE-based PRD system was demonstrated and the removal of toxic substances was significant. This study confirmed the clinical utility of this system as an artificial liver support.

Background/aims: The development of a bioartificial liver with xenogeneic hepatocytes requires sufficient hepatocyte mass for metabolic support, decreased immunogenicity, and prevention of viral transmission. Therefore, the authors developed a plasma dialysis system using a selective plasma filter, and combined this dialysis system with whole liver plasma perfusion (PDWLP). In this system, the patient's plasma was dialyzed with normal plasma recycled through the pig liver., Methodology: Female inbred Large White pigs were used for the hepatic failure model and for liver donation. Fulminant hepatic failure (FHF) was induced by the intraportal administration of alpha-amanitin and lipopolysaccharide. Nine hours after this injection, the FHF pigs were connected to the dialysis system for 6 h (n = 5). Control FHF pigs were not connected to the dialysis system (n = 10)., Results: Animal survival was lengthened, and cardiovascular stability was demonstrated in the PDWLP treated pigs. This system suppressed the production of substances that promote encephalopathy. The liver necrosis and neutrophil infiltration were significantly less in the experimental pigs., Conclusions: The experimental treatment attenuated the progression of FHF. Extracorporeal liver, perfused with oxygenated plasma, functioned for 6 h without hyperacute rejection. This type of hybrid bioartificial liver may be useful for treating FHF patients.

We herein report a case of a hilar tumor with extensive invasion to the proper hepatic artery, which was successfully treated with a radical resection in a 57-year-old female patient after a stepwise hepatic arterial embolization. She underwent right colectomy and partial hepatectomy for advanced colon cancer two years ago and radiofrequency ablation therapy for a liver metastasis one year ago, respectively. A recurrent tumor was noted around the proper hepatic artery with invasion to the left hepatic duct and right hepatic artery 7 mo previously. We planned a radical resection for the patient 5 mo after the absence of tumor progression was confirmed while he was undergoing chemotherapy. To avoid surgery-related liver failure, we tried to promote the formation of collateral hepatic arteries after stepwise arterial embolization of the posterior and anterior hepatic arteries two weeks apart. Finally, the proper hepatic artery was occluded after formation of collateral flow from the inferior phrenic and superior mesenteric arteries was confirmed. One month later, a left hepatectomy with hepatic arterial resection was successfully performed without any major complications.

Aim: To analyze gallbladder contractility in patients with black pigment stones (BPSs) and to compare this with patients with cholesterol stones (CSs) and healthy volunteers., Methods: The pattern of bile evacuation from the gallbladder was quantified by computer cholescintigraphy in 28 normal subjects, 22 patients with CSs and 14 with BPSs. The parameters of gallbladder contractility included ejection period (EP), ejection fraction (EF) and ejection rate (ER)., Results: A significantly shorter EP was observed in patients with BPSs in comparison to those with CSs (t = 2.4, P < 0.05). EF in BPS patients significantly decreased in comparison to that in CS and normal subjects (t = 6.4, P < 0.0001; t = 2.1, P < 0.05). EF in CS patients also significantly decreased in comparison to that in normal subjects (t = -3.0, P < 0.005). Consequently, ER in patients with BPSs and CSs was significantly smaller than that in normal subjects (t = 3.1, P < 0.005; t = -3.5, P < 0.001). Moreover, in cases where postprandial reflux of a radioisotope into the common hepatic duct from the gallbladder was observed, EF and ER of either CS or BPS patients showed a significant reduction., Conclusion: Bile evacuation from the gallbladder is reduced in patients with BPSs, in comparison to those with CSs and to healthy volunteers. Bile stagnation due to impaired gallbladder kinetics seems to be one of the predisposing factors for the development of BPSs.

Hemolymphangioma of the pancreas is a very rare benign tumor. There were only five reports of this disease until March 2008. Herein, we report a case of hemolymphangioma of the pancreas with gastrointestinal bleeding due to duodenal invasion. A 53-year-old man had been admitted a referral hospital because of severe anemia due to gastrointestinal bleeding in December 2005. He was then transferred to our institute with a diagnosis of a tumor of the head of the pancreas with duodenal invasion in January 2006. No abnormalities were revealed except for anemia in laboratory data including CEA and CA19-9. Gastrointestinal endoscopy revealed bleeding at the duodenum. Computed tomography also demonstrated a heterogenous mass at the pancreatic head and suspected invasion to the duodenal wall. Ultrasonography showed a huge mass at the pancreatic head with a mixture of high and low echoic areas. Pylorous-preserving pancreatoduodenectomy was performed. The pancreatic tumor was soft and had invaded to the duodenum. The pathological diagnosis was a hemolymphangioma of the pancreas invaded to the duodenum. His postoperative course was uneventful and he was discharged on the 26th d after surgery. Hemolymphangioma of the pancreas is a very rare benign tumor. In a literature review until March 2008, we found five case reports. Major symptoms are abdominal pain and distension due to the enlarged tumor. However, we experienced a case of hemolymphangioma of the pancreas with gastrointestinal bleeding due to invasion to the duodenum. This disease is a very rare entity, but should be considered when patients have gastrointestinal bleeding.

Hemosuccus pancreaticus is a rare cause of intermittent upper gastrointestinal bleeding. We report two cases of hemosuccus pancreaticus with multiple episodes of upper gastrointestinal bleeding. The causes of hemorrhage were rupture of pseudoaneurysm of the splenic artery and bleeding from the wall of pancreatic pseudocyst. Interventional radiology is the first modality for early diagnosis and possible treatment of hemosuccus pancreaticus. When angiography shows no abnormal findings or interventional radiological therapy can not be successful, surgery should be considered without delay. Our patients herein underwent surgery without recurrence or sequelae. Intraoperative ultrasonography and pancreatoscopy were helpful modalities for confirming the source of hemorrhage and determining the cutting line of the pancreas. When we encounter intermittent upper gastrointestinal bleeding with an obscure source, hemosuccus pancreaticus should be included in differential diagnoses especially in patients with chronic pancreatitis, which would lead to a prompt and proper treatment.

Aim: To analyze the efficacy of routine intraoperative ultrasound (IOUS) as a guide for understanding biliary tract anatomy, to avoid bile duct injury (BDI) after laparoscopic cholecystectomy (LC), as well as any burden during the learning period., Methods: A retrospective analysis was performed using 644 consecutive patients who underwent LC from 1991 to 2006. An educational program with the use of IOUS as an operative guide has been used in 276 cases since 1998., Results: IOUS was highly feasible even in patients with high-grade cholecystitis. No BDI was observed after the introduction of the educational program, despite 72% of operations being performed by inexperienced surgeons. Incidences of other morbidity, mortality, and late complications were comparable before and after the introduction of routine IOUS. However, the operation time was significantly extended after the educational program began (P < 0.001), and the grade of laparoscopic cholecystitis (P = 0.002), use of IOUS (P = 0.01), and the experience of the surgeons (P = 0.05) were significant factors for extending the length of operation., Conclusion: IOUS during LC was found to be a highly feasible modality, which provided accurate, real-time information about the biliary structures. The educational program using IOUS is expected to minimize the incidence of BDI following LC, especially when performed by less-skilled surgeons.

Claudins are transmembrane proteins and major constitutes of tight junctions, and participate in the paracellular barrior and cellular connecting functions. They have been shown to be differentially regulated in malignant tumors and play a role in carcinogenesis and tumor progression. Recent studies have shown changes in expression of claudins during tumorigenesis. However, a causal relationship between claudin expression and cancer recurrent status has not been established. In this study, we examined 54 esophageal cancer cases to assess immunohistochemical expression patterns of claudin-1. Eleven (20.4%) of 54 cases had negative immunostaining for claudin-1. Decreased expression of claudin-1 was statistically correlated with recurrence status (P = 0.018). The cases with lymphatic vessel permeation showed reduced expression patterns of claudin-1 (P = 0.033). Decreased expression of claudin-1 was also correlated with short diseasefree survival (P = 0.0003) and overall survival (P = 0.0045). The results indicated that claudin-1 expression was correlated with the recurrence status and poor prognosis in esophageal cancer and claudin-1 expression may be a good indicator of recurrence in esophageal cancer.

Aim: To clarify the effect of a high des-gamma-carboxy prothrombin (DCP) level on the invasiveness and prognosis of small hepatocellular carcinoma., Methods: Among 142 consecutive patients with known DCP levels, who underwent hepatectomy because of hepatocellular carcinoma, 85 patients met the criteria for small hepatocellular carcinoma, i.e. oneor=400 mAU/mL (P=0.02) were independent prognostic factors. In the group of patients who met the criteria for small hepatocellular carcinoma, DCP>or=400 mAU/mL was found to be an independent prognostic factor for recurrence-free (P=0.02) and overall survival (P=0.0005). In patients who did not meet the criteria, the presence of vascular invasion was an independent factor for recurrence-free (P=0.02) and overall survivals (P=0.01). In 75% of patients with small hepatocellular carcinoma and high DCP levels, recurrence occurred extrahepatically., Conclusion: For small hepatocellular carcinoma, a high preoperative DCP level appears indicative for tumor recurrence. Because many patients with a high preoperative DCP level develop extrahepatic recurrence, it is necessary to screen the whole body.

In clustering methods, the estimation of the optimal number of clusters is significant for subsequent analysis. Without detailed biological information on the genes involved, the evaluation of the number of clusters becomes difficult, and we have to rely on an internal measure that is based on the distribution of the data of the clustering result. The Gap statistic has been proposed as a superior method for estimating the number of clusters in crisp clustering. In this study, we proposed a modified Fuzzy Gap statistic (MFGS) and applied it to fuzzy k-means clustering. For estimating the number of clusters, fuzzy k-means clustering with the MFGS was applied to two artificial data sets with noise and to two experimentally observed gene expression data sets. For the artificial data sets, compared with other internal measures, the MFGS showed a higher performance in terms of robustness against noise for estimating the optimal number of clusters. Moreover, it could be used to estimate the optimal number of clusters in experimental data sets. It was confirmed that the proposed MFGS is a useful method for estimating the number of clusters for microarray data sets.

Aim: To clarify the usefulness of a new method for performing a pancreaticojejunostomy by using a fast-absorbable suture material irradiated polyglactin 910, and a temporary stent tube for a narrow pancreatic duct with a soft pancreatic texture., Methods: Among 63 consecutive patients with soft pancreas undergoing a pancreaticoduodenectomy from 2003 to 2006, 35 patients were treated with a new reconstructive method. Briefly, after the pancreatic transaction, a stent tube was inserted into the lumen of the pancreatic duct and ligated with it by a fast-absorbable suture. Another tip of the stent tube was introduced into the intestinal lumen at the jejunal limb, where a purse-string suture was made by another fast-absorbable suture to roughly fix the tube. The pancreaticojejunostomy was completed by ligating two fast-absorbable sutures to approximate the ductal end and the jejunal mucosa, and by adding a rough anastomosis between the pancreatic parenchyma and the seromuscular layer of the jejunum. The initial surgical results with this method were retrospectively compared with those of the 28 patients treated with conventional duct-to-mucosa anastomosis., Results: The incidences of postoperative morbidity including pancreatic fistula were comparable between the two groups (new; 3%-17% vs conventional; 7%-14% according to the definitions). There was no mortality and re-admission. Late complications were also rarely seen., Conclusion: A pancreaticojejunostomy using an irradiated polyglactin 910 suture material and a temporary stent is easy to perform and is feasible even in cases with a narrow pancreatic duct and a normal soft pancreas.

Oncogenesis of anaplastic carcinoma of the pancreas is a subject of controversy, because it shows sarcomatous nature with extremely poor prognosis. We herein report an unusual case of anaplastic carcinoma occurring with a recurrent mucinous cystic neoplasm in a 38-year-old female. A 10-cm retroperitoneal cystic mass was pointed out in the first pregnancy and a probable diagnosis of mucinous cystic neoplasm was made in October 2000. She refused surgery first and delivered her baby uneventfully. During her second pregnancy in 2002, however, she presented hematemesis and underwent urgent distal pancreatectomy, splenectomy and partial resection of the gastric wall where the tumor perforated. A diagnosis of borderline-type mucinous cystic neoplasm with ovarian-like stroma was made. Nine months later, CT visualized a recurrent cystic tumor near the pancreatic stump, which was subsequently resected. Pathology revealed that the tumor was composed of two different components of borderline-type mucinous cystic neoplasm and anaplastic carcinoma. The latter was intensely positive for vimentin, CD68, p53 and focally for cytokeratin, suggesting both sarcomatous and carcinomatous differentiation. She survived four years after the second surgery without tumor recurrence. Although the origin of anaplastic carcinoma has not been determined yet, it should be remembered that anaplastic carcinoma can occur in association with mucinous cystic neoplasm of more benign histology.

Background/purpose: Liver regeneration occurs through interactions between the receptors on hepatocytes, including proteoglycans (PGs) and glycosaminoglycans (GAGs), and various growth factors. We investigated serial changes in GAGs, particularly heparan sulfate (HS), in proliferating hepatocytes., Methods: We performed 70% hepatectomy in male Wistar rats, and we then isolated hepatocytes by a collagenase perfusion method after each surgery. DNA synthesis was evaluated by measuring proliferating cell nuclear antigen (PCNA). After we had treated the hepatocytes by delipidation and digestion with actinase E, endo-beta-xylosidase, and alpha-amylase, we quantified GAGs by a carbazole-sulfuric acid method. GAGs were analyzed by ion-exchange chromatography, and changes in molecular weight of the HS component were investigated by size-fractionation HPLC., Results: Hepatocyte mitosis peaked at 24 h after the amount of GAGs was increased at 24 and 72 h after surgery. The amount of HS was slightly increased at 3 to 12 h after surgery, and then peaked at 24 h. The molecular weight of the HS declined by 12 h, but had recovered to the preoperative level by 24 h., Conclusions: These results suggested that this HS molecule, which contained about ten disaccharide units during proliferation, may be an initiator of hepatocyte proliferation.

Claudins (CLDNs) constitute the major transmembrane proteins of tight junctions. It may be hypothesized that changes in or loss of expression of tight junctional proteins such as CLDNs can lead to cellular disorientation and detachment, which is commonly seen in neoplasia. Recent studies have suggested that claudin-1 (CLDN1) plays an important role in invasion and metastasis and claudin-4 (CLDN4) has a particular role in mammary glandular cell differentiation and carcinogenesis. In this study, we examined 83 breast cancer cases and demonstrated immunohistochemical expression patterns of CLDN1/CLDN4 in recurrent and non-recurrent groups. We found significant results between the recurrent and non-recurrent group for expression of CLDN1/CLDN4. The recurrent group (26 cases) showed decreased expression patterns of CLDN1 (p<0.001), compared to the non-recurrent group (57 cases). Decreased expression of CLDN1 (p<0.0001) correlated with short disease-free interval. The lymph node metastasis-positive group showed decreased expression patterns of CLDN1 (p=0.001). However, there was no significance between the recurrent group and non-recurrent group in CLDN4 expression. There was no significance between histological factors and CLDN4 expression. The results indicated that CLDN1 expression correlated with the recurrence status and malignant potential of breast cancer.

We developed a plasma recycling dialysis (PRD) system based on plasma exchange (PE). In this system, rapid reduction of toxic substances and restitution of deficient essential substances are performed by PE, and subsequent blood purification is performed by dialysis between separated plasma recycled over a purification device and the patient's blood across the membrane of the plasma separator. This study was performed to demonstrate the safety and efficacy of this system. Hyperbilirubinemia was induced by ligating the bile duct in pigs, and 7 days later, only PE for 2 h (group PE) or PE for 2 h followed by PRD for 6 h (group PE + PRD) was performed. The separated plasma was recycled over anion-exchange resin through the extra fiber space of the plasma separator. The safety and efficacy of this system were evaluated based on the values of hemodynamic and laboratory parameters. Transfer from PE to PRD was completed in a few minutes. The hemodynamic status and blood cells counts were stable and hemolysis was not observed during the procedure. In the PE + PRD group, the concentrations of total bile acids continuously decreased (pretreatment, 155.5 +/- 40.6 microM; 2 h [end of PE], 76.1 +/- 14.4 microM; 8 h [end of PRD], 25.8 +/- 9.1 microM) and the value was significantly lower than in the PE group after 6 h. The total bilirubin also continuously decreased during PRD (pretreatment, 55.3 +/- 11.5 microM; 2 h [end of PE], 33.8 +/- 8.4 microM; 8 h [end of PRD], 18.6 +/- 7.7 microM) and was significantly lower than in the PE group after 4 h. No significant change was observed in other laboratory values. This PE-based PRD system allowed a swift transfer from PE to sorbent-based blood purification. The safety of this system was demonstrated and the removal of toxic substances was significant. This study confirmed the clinical utility of this system as a platform for artificial liver support.

Motivation: Classifying genes into clusters depending on their expression profiles is one of the most important analysis techniques for microarray data. Because temporal gene expression profiles are indicative of the dynamic functional properties of genes, the application of clustering analysis to time-course data allows the more precise division of genes into functional classes. Conventional clustering methods treat the sampling data at each time point as data obtained under different experimental conditions without considering the continuity of time-course data between time periods t and t+1. Here, we propose a method designated mathematical model-based clustering (MMBC)., Results: The proposed method, designated MMBC, was applied to artificial data and time-course data obtained using Saccharomyces cerevisiae. Our method is able to divide data into clusters more accurately and coherently than conventional clustering methods. Furthermore, MMBC is more tolerant to noise than conventional clustering methods., Availability: Software is available upon request., Contact: taizo@brs.kyushu-u.ac.jp.

We believe that this is the first report of a retroperitoneal tumor consisting of heterotopic gastrointestinal mucosa and pancreatic tissue. The patient was a 19-year-old woman with the chief complaint being occasional back pain. Abdominal computerized tomography demonstrated a 3.1 x 2.5 x 3.2-cm low-density solid and cystic lesion adjoining the left renal vein between the aorta and inferior vena cava. Angiography revealed that the inferior vena cava was displaced by the hypovascular tumor. The retroperitoneal lesion was diagnosed preoperatively as a benign tumor such as a neurogenic neoplasm or lymphangioma. At laparotomy, a cystic tumor was found, which existed behind the inferior vena cava and renal vessels, and contained reddish-brown fluid, suggesting hemorrhage in the past. The cut surface of the tumor showed a unilocular cyst with partially hypertrophic wall. Histopathological examination revealed a cystic tumor lined with heterotopic gastric and duodenal mucosa, with pancreatic tissue in the muscularis propria. In addition, evidence of bleeding from the gastric mucosa was observed in the cystic tumor. External secretion from these tissues could have triggered the hemorrhage and expanded the tumor, possibly resulting in the back pain.

Background/aims: Orthotopic liver transplantation (OLTx) from non-heart beating donor (NHBD) often involves hepatic warm ischemia and reperfusion injury which is triggered by the inflammatory cytokines. This study was carried out to investigate whether a newly synthesized cytokine suppressive anti-inflammatory agent, FR167653, attenuates graft injury in OLTx from NHBD., Methodology: Porcine OLTx from NHBD was performed. No-heart beating time was scheduled to be 60 minutes. Animals were divided into two groups: no treatment control (CT) group (n=5), and FR167653 treated (FR) group (n=5), in which FR167653 was administered intravenously before the aortic cross clamp in the donor, and before and after the hepatic allograft reperfusion in the recipient continuously., Results: Four out of five pigs died within 24 hours and one on postoperative day 1 from graft liver failure in the CT group, while two pigs died on day 3, and three survived more than 7 days in the FR group (p<0.05). Microcirculatory disturbance was attenuated, liver injury was lessened, and ATP resynthesis was enhanced in the FR group. Additionally, FR167653 inhibited neutrophils infiltration in the liver tissue, and suppressed release of inflammatory cytokines after OLTx from NHBD., Conclusions: The treatments with FR167653 successfully prevented graft injury after OLTx from NHBD by means of improvement of liver microcirculation, and attenuation of neutrophils activation. The inhibitory effect of FR167653 on the release of inflammatory cytokines played an important role in the liver graft protection.

Anastomotic stricture sometimes causes hepatic congestion leading to decreased hepatic clearance of drugs. We herein describe a correlation between trough levels of tacrolimus and an anastomotic stricture of the hepatic vein. A 13-year-old boy underwent living donor liver transplant with a left lobe graft from his mother. Outflow blockage due to an anastomotic stricture of the hepatic vein developed 3 months after transplant. His anastomotic site had been repeatedly treated with percutaneous transvenous angioplasty (PTA) by balloon dilation. About 1 year after transplant, his trough level of tacrolimus promptly decreased after balloon dilation (from 15.7 to 5.6 ng/dL). Liver function tests showed abnormalities, which were diagnosed as acute cellular rejection, and he was treated with pulse steroid therapy. The calculated half-life of tacrolimus (T1/2) showed marked improvement after PTA (from 35 to 22 hours). A 45-year-old woman underwent living donor transplantation due to alcoholic liver cirrhosis with a left lobe graft from her brother. An anastomotic stricture of the hepatic vein developed 4 months after transplant. She was treated with balloon dilation, which caused an abrupt decrease in the trough level of tacrolimus (12 to 4 ng/dL). Her alkaline phosphatase was elevated and she was diagnosed with rejection, which was treated with an increase of dosage of tacrolimus. In outflow block, the T1/2 of tacrolimus increases probably due to decreased hepatic clearance by stagnation or congestion of the liver. However, hepatic clearance of drugs quickly recovers after PTA. Close monitoring of immunosuppressive agents is fundamental at the time of angioplasty to avoid acute cellular rejection as developed in our two cases.

Anastomotic stricture of the hepatic vein is an annoying complication, especially in living donor liver transplantation. Balloon dilation has been utilized but is sometimes associated with recurrences. Recently, a cutting balloon was invented for treatment of arteriosclerosis. Herein we report the results of application of this device for treatment of anastomotic strictures of the hepatic vein in two living donor liver transplant recipients who underwent percutaneous dilation of the hepatic vein with a cutting balloon (8 x 10 mm, Atherotome, Boston Scientific). Case 1, a 26-year-old woman transplanted for subacute fulminant hepatitis, had been treated for an anastomotic stricture by balloon dilation on 15 occasions over a 2- to 3-month interval. Case 2, a 13-year-old boy transplanted for cryptogenic liver cirrhosis, had been treated for an anastomotic stricture by balloon dilation biannually. The cutting balloon was applied safely without severe complications. The first case showed a recurrent anastomotic stricture at 6 months after dilation. Follow-up at 6 months in the second case revealed a mild recurrence of the stricture. Anastomotic stricture of the hepatic vein jeopardizes the graft and the recipient. The reported treatments involve venoplastic surgery and expandable metallic stents. Application of a cutting balloon seemed to be a safe, convenient modality. However, its effect was not indefinite, so a cutting balloon of greater diameter or application of an expandable metallic stent may be considered for patients with multiple recurrences of their anastomotic stricture.

Unlabelled: Donor and recipient factors are closely associated with graft survival after orthotopic liver transplantation (OLT). This study was performed to analyze clinical characteristics of recipients and donors, which affect 30-day graft loss after OLT., Materials and Methods: One hundred eighty-six livers from heart-beating donors were accepted between May 1997 and June 1998 at the University of Pittsburgh Medical Center. Donor variables that were analyzed included age, sex, cold ischemia time (CIT), warm ischemia time (WIT), imported versus local procurement, cardiopulmonary arrest, serum sodium level, and dopamine dose. The recipient characteristics included native liver disease and UNOS status. Two-sided Fisher exact test and stepwise logistic regression were used for univariate and multivariate analyses. P-values < .05 were considered statistically significant., Results: Twenty-eight grafts (15.1%) were lost within 30 days of OLT. The following factors were found to adversely affect graft survival: donor sodium > 155 mEq/L (P = .002); CIT > 12 hours (P = .002); WIT > 45 minutes (P = .002); and imported liver graft (P = .048). Logistic regression revealed that donor sodium (odds ratio, 3.03; 95% CI, 1.05 to 8.74), CIT (OR 1.20; 95% CI 1.05 to 1.38), WIT (OR 1.06; 95% CI 1.01 to 1.09) were independent predictors of early graft loss., Conclusion: Donor hypernatremia as well as warm and cold ischemia times independently affect graft outcomes in the early postoperative period after OLT. Avoidance of long preservation and correction of donor sodium level are recommended to optimize results and survival in OLT.

Aim: Corticosteroids have been considered the mainstay of immunosuppressive therapy after liver transplantation. However, the side effects of long-term steroid use such as diabetes, infections, and bone disease, including growth retardation in children, are serious problems. Our immunosuppression regimen includes FK506 and steroid withdrawal by 30 days after transplantation. The aim of this study was to determine the outcomes of liver transplant, using this immunosuppressive regimen., Patients: Fifteen primary liver transplant recipients were performed between January 1994 and May 2003 and data were reviewed retrospectively. Eight pediatric and four adult recipients, who had survived more than 3 months after transplantation, were included in this sample. The immunosuppressive regimen consisted of FK 506 (Prograf), initially at doses of 0.03 mg/kg, with dose adjustments to achieve daily trough levels of approximately 10 to 12 ng/mL, and predonisone, initially at 4 mg/kg/d, with a taper and cessation by 30 days when the graft was stable., Results: All recipients were successfully withdrawn by 30 days. Acute rejection episodes occurred in three patients, no patient was diagnosed with chronic rejection. The acute rejection-free rate at 5 year was 74.1%. No recipient had diabetes, serious infections or bone disease., Conclusion: Our primary immunosuppressive regimen of rapid steroid withdrawal is safe with regard to acute and chronic rejection with benefits upon steroid-related side effects.

Unlabelled: This study was performed to investigate whether intraoperative changes in blood lactate levels after hepatic allograft reperfusion reflect initial graft function in living donor liver transplantation (LDLT)., Patients and Methods: From 1994 to 2003, 15 of LDLT cases were divided into two groups based on the intraoperative blood lactate levels. Group A consisted of seven recipients whose new liver grafts started to consume lactate immediately after portal perfusion. Group B consisted of the remaining eight recipients whose intraoperative blood lactate values showed no change or an elevation for 2 hours after graft revascularization., Results: All Group A patients survived, whereas three out of eight patients in Group B died of infection and portal vein thrombosis within 3 months after LDLT. There was no significant difference in preoperative donor and recipient laboratory data. The recipient age and body size in Group B were significantly higher than those in Group A, indicating that Group B consisted of small-for-size liver transplant cases. Serum total bilirubin concentrations in Group B were significantly higher than Group A from postoperative day 5 to 23, whereas postoperative liver enzyme levels and prothrombin time were similar between the two groups., Conclusion: The change in intraoperative blood lactate after hepatic allograft reperfusion served as an accurate predictor of initial graft function which was associated with graft size in human LDLT.

This report discusses the pathophysiology of and therapeutic methods to address hepatic vein anastomotic stricture after living donor liver transplantation (LDLT). From 1994 to 2002, our 15 LDLTs using the lateral segments or left lobes included four recipients who experienced 28 occurrences of this complication after the operation. The period between LDLT and the first stricture was 4.0 +/- 1.2 months. The age of the affected recipients (31.0 +/- 8.2 years) was significantly higher than that of the nonaffected patients (7.0 +/- 4.1 years, P < .05). Graft liver/standard liver volume ratio was 39.1% +/- 3.8% in the former and 77.9% +/- 12.7% in the latter cases (P < .05). Initial symptoms of stricture were ascites (42.9%), abdominal distention (42.9%), liver enzyme elevation (10.7%), and gastrointestinal bleeding (3.6%). In addition, 14 of 28 stricture cases (50%) showed increased blood trough levels of tacrolimus. Doppler ultrasonography was used for diagnosis, and balloon dilatations performed in all stricture patients, thereby hepatic significantly reducing venous blood pressure from 33.5 +/- 1.7 to 20.3 +/- 1.5 cmH2O. All patients finally resolved the strictures after several treatments. The stricture after LDLT was associated with small-for-size grafts, suggesting that liver regeneration may lead to anatomical changes and strictures. Since tacrolimus is metabolized by the liver, its blood trough level is one initial symptoms of stricture. Balloon dilatation was useful and safe as the treatment, while problems have been reported after stent insertion in the hepatic vein.

Background: We previously demonstrated that even a low dose of bone marrow cells (BMCs) established donor-specific tolerance if mixed with splenocytes (SPLCs). In this study, T-cell subsets CD4 (CD4SP) and CD8 (CD8SP) of donor SPLCs were investigated for their contribution to the enhancement of BMC engraftment leading to donor-specific tolerance in sublethally irradiated mice., Methods: Sublethally irradiated C57BL/6 recipient mice were intravenously injected BMCs mixing with CD4SP or CD8SP harvested from BALB/c donor mice. The degree of chimerism in the peripheral blood lymphocytes (PBLs) and in the SPLCs was analyzed using FACS, mixed lymphocyte reaction, and skin graft transplantation 3 months after injection., Results: Recipients injected with 3 x 10(6) donor BMCs admixed with 10 x 10(6) donor CD8SP established chimerism. However, recipients injected with the same dose of BMCs admixed with 5 x 10(6) CD4SP, 10 x 10(6) CD4SP, and 5 x 10(6) CD8SP did not established chimerism. CD8SP contained 44% of Ly6A/E (Stem Cell Antigen-1 (Sca-1))-positive cells based on FACS analysis, whereas only 6% of CD4SP were positive for Ly6A/E. MLR supernates of donor SPLCs chimeric mice using admixture with CD8SP dominated by Th2 cytokines. In contrast, mixting with MLR supernates from failed chimera showed dominant Th1 cytokines., Conclusions: CD8SP seems to make a major contribution to enhance BMC engraftment and induce donor-specific tolerance. Ly6A/E (Sca-1)-positive cells need to be further investigated for their contribution to the establishment of chimerism.

Introduction: Many types of isolated hepatocytes-based bioartificial liver have been developed. However, to maintain hepatocyte-specific functions for a long period is still a significant challenge. The possibilities of rejection or viral transmission still remain as untackled obstacles. We developed a cross-circulation system, using a semipermeable membrane combined with whole liver perfusion. Detoxifying functions of the extracorporeal porcine liver and molecular movements across the membrane were evaluated in vitro., Methods: The hollow-fiber module has a molecular cutoff of 100 kD. A spiked solution containing 500 mL low molecular dextran solution spiked with 12 mg ammonium chloride, 500 mg D-galactose, and 300 mg lidocaine, which mimicked a patient, was recirculated through the inner fiber space. The extracorporeal liver perfusion circuit consisted of an extra-fiber spaces. A reservoir containing 1000 mL healthy pig plasma, a membrane oxygenator, and a porcine whole liver. Both circuits circulated in the opposite direction for 6 hours., Result: In 6 hours, 47.3% +/- 10.2% of ammonia, 89.5% +/- 1.7% of D-galactose, and 95.5% +/- 1.0% of lidocaine were eliminated from the circuits; 66.5 +/- 11.1 mg of urea were produced at the same time. Oxygen consumption was maintained between 0.248 and 0.259 mL/100 g liver/min for 6 hours. Movement of IgM was completely blocked by the 100-kD membrane, whereas albumin was freely transferred from the reservoir to the intrafiber space., Conclusion: The perfusion experiments showed the possibility of using a whole liver with oxygenated plasma perfusion in a bioartificial liver system in vitro.

Prolonged cold ischemia time (CIT) during graft preservation and warm ischemia time (WIT) during rewarming time have been reported to cause postoperative graft dysfunction after orthotopic liver transplantation (OLT). However, the effects of both CIT and WIT in combination on patient and graft survivals are not yet defined. The aim of this study was to determine whether simultaneously prolonged CIT and WIT were associated with early graft outcomes after clinical OLT. For analysis of liver graft survival within 90 days of OLT and postoperative graft function, 186 consecutive OLT cases were divided into four groups as follows: group A, CIT < 12 hours and WIT < 45 minutes; group B, CIT > 12 hours and WIT < 45 minutes; group C, CIT < 12 hours and WIT > 45 minutes; and group D, CIT > 12 hours and WIT > 45 minutes. The graft loss rates were 5.4% in group A, 9.8% in group B, 11.1% in group C, and 42.9% in group D. The mean highest aspartate aminotransferase (AST) value after OLT in group D (3352.3 +/- 569.4 U/L) was significantly greater than those in groups A (1411.7 +/- 169.2 U/L) and B (1931.3 +/- 362.6 U/L). The simultaneously prolonged cold and warm ischemia times significantly caused hepatic allograft injury and failure, suggesting some cumulative effects of CIT and WIT on postoperative graft function.

Background/aims: Prolonged cold ischemia time (CIT) during graft preservation and warm ischemia time (WIT) defined as a rewarming time have been reported to cause postoperative graft dysfunction after orthotopic liver transplantation (OLT). However, a synergistic effect of both CIT and WIT on patients of graft survival has not been confirmed. The aim of this study was to determine whether simultaneously prolonged CIT and WIT was associated with early graft outcome after clinical OLT., Methodology: Between May 1997 and July 1998, 186 consecutive OLT cases were divided into 4 groups as follows: group A, CIT < or =12 hrs and WIT < or =45 min; group B, CIT >12 hrs and WIT < or =45 min; group C, CIT < or =12 hrs and WIT >45 min; and group D, CIT > 12 hrs and WIT >45 min. Liver graft survival within 90 days of OLT and early postoperative graft function were analyzed., Results: The graft loss rates were 5.4% in group A, 9.8% in group B, 11.1% in group C, and 42.9% in group D. The mean highest aspartate aminotransferase (AST) values after OLT in group D (3352.3+/-569.4 U/L) was significantly higher than those in groups A (1411.7+/-169.2 U/L) and B (1931.3+/-362.6 U/L)., Conclusions: The simultaneously prolonged cold and warm ischemia time significantly caused hepatic allograft injury and failure, suggesting some synergistic effects of CIT and WIT on postoperative graft function.

Organ ischemia-reperfusion injury is caused by two consecutive steps, microcirculatory disturbance and neutrophil-endothelial cell interactions, which are caused by inflammatory cytokines. We examined the hypothesis that combination therapy with a donor (FK409) of nitric oxide, one of the potent mediators with diverse roles as a vosodilator and a platelet inhibitor, together with the cytokine suppressor agent (FR167653) attenuates warm ischemic injury in canine small bowel. Small bowel ischemia was initiated by clamping the superior mesenteric artery and vein. Animals were divided into two groups: a control group (n = 5) subjected to 2-hour small bowel ischemia only, and a combination therapy group (FK/FR group, n = 5) that received FK409 (300 mcg/kg/h) plus FR167653 (1 mg/kg/h) intravenously before and after the ischemic event. We evaluated animal survival, small bowel tissue blood flow, and enzyme release from the small bowel. All controls died from severe acidosis within 2 days and all the FK/FR animals survived 7 days (P < .05). The FK/FR group recovered more than 70% of blood flow immediately after the revascularization, while the flow was less than 40% among the controls. Serum creatine phosphokinase values in the control group after reperfusion were significantly higher than those in the FK/FR group. In conclusion improvement of the microcirculation by FK409 and inhibition of cytokine release by FR167653 together attenuated warm ischemic small bowel injury.

Implementation of the critical path in the healthcare system facilitates standardization of medical practices, which improves both quality and safety management, shortens the length of hospital stays, and economizes on medical resources. In a new payment system introduced to university hospitals in Japan, hospital fees are charged by the day according to the diagnosis-procedure combination (DPC). To prepare for DPC, standardization of medical care, shortening the average length of stay within all hospitals, cost reduction, establishment of hospital networks, and an increase in new patients are critical issues. Cost management can be achived effectively by using the critical path.

Background: Small-for-size grafts often cause persistent conjugated hyperbilirubinemia in the recipient after adult-to-adult living donor liver transplantation, but the cause has not yet been clarified. In physiologic status, bilirubin is excreted from hepatocytes to the bile canaliculus by means of multidrug resistance protein (MRP) 2 and, in particular circumstances, by means of MRP3 to the sinusoidal space. The aim of this study was to research whether there is any change in bilirubin excretion pattern during liver regeneration with reference to expression of MRP2 and MRP3., Methods: Sprague-Dawley rats underwent sham operation (n=37), 70% hepatectomy (n=38), or 90% hepatectomy (n=37). The degree of liver regeneration, total and direct bilirubin, protein synthesis, and interleukin (IL)-6 were serially assessed. Expression of MRP2 and MRP3 were semiquantified by Western blotting., Results: The proliferating cell nuclear antigen labeling index indicated rapid liver regeneration after 70% and 90% hepatectomy. Serum levels of total and direct bilirubin increased significantly (P<0.05), and conjugated hyperbilirubinemia was proved only in the 90% hepatectomy group. Coagulation factor VII dipped but increased as early as 12 to 24 hr postoperatively in both hepatectomy groups. Plasma IL-6 levels were significantly increased in the 90% hepatectomy group (P<0.05). Expression of MRP2 was decreased and MRP3 was expressed at 36 and 72 hr postoperatively in the 90% hepatectomy group, whereas no change was observed in MRP expression in the 70% hepatectomy group., Conclusions: During liver regeneration after critical hepatectomy such as 90% hepatectomy, decrease of MRP2 and expression of MRP3 may play an important role in postoperative hyperbilirubinemia.

Unknown genetic regulation mechanisms are expected to be discovered by information technology using large amount of biological data especially for gene expression data. In this study, we propose a novel inferring method for genetic interactions that combines our original preprocessing method and the Boolean algorithm. First, the performance of our method was evaluated using artificial data. The results showed that our method was able to infer genetic interactions with high specificity (specificity=0.629). Then, using our method, the genetic interaction was inferred from the experimental time course data collected using microarray on 69 genes of cell cycle for Saccharomyces cerevisiae. Our method estimated about 80% of all genetic interactions in Kyoto Encyclopedia Genes and Genomes (KEGG) for these genes. Furthermore, our method was able to infer several other genetic interactions that are not included in KEGG but whose existence is supported by other biological reports.

Background/aims: Inflammatory cytokines, such as interleukin-1 beta and tumor necrosis factor-alpha, which activate neutrophils, contribute to hepatic warm ischemia-reperfusion injury. However, the role of the cytokines in hepatic microcirculation immediately after reperfusion is still unclear. This study was carried out to investigate whether FR167653, a dual inhibitor of interleukin-1 beta and tumor necrosis factor-alpha, attenuates hepatic microcirculatory disturbance at the initial phase of reperfusion following liver ischemia., Methodology: Adult mongrel dogs were subjected to 90 minutes of liver ischemia by a Pringle's maneuver under portosystemic bypass. The animals were divided into two groups: a control group (n = 10), subjected to hepatic warm ischemia only, and a FR167653 administered group (n = 5), which received 1 mg/kg/h FR167653 for 4 hours since 30 minutes before the ischemia to 2 hours after the reperfusion continuously. Seven days animal survival, hepatic tissue blood flow, liver function test, hepatic venous blood concentration of endothelin-1 and plasminogen activator inhibitor-1, liver tissue biochemistry, and histopathology were analyzed., Results: The treatment with FR167653 attenuated microcirculatory disturbance, lessened liver injury, enhanced adenine nucleotides resynthesis, and improved animal survival after liver ischemia. In addition, FR167653 significantly inhibited release of both endothelin-1 and plasminogen activator inhibitor-1 from the liver cells., Conclusions: These results suggest that the inflammatory cytokines induce microcirculatory disturbance in the initial phase of reperfusion following liver ischemia.

Background: Allogeneic chimerism has been established in graft-accepting recipients and the donor cells in the host may act in a major way to facilitate the induction of tolerance. In this study, we examined the effects of allogeneic chimerism after injecting donor bone marrow cells (BMCs) mixed with splenocytes (SPLCs) to the sublethally conditioned recipients., Methods: In BALB/c(H-2(d)) to B6(H-2(b)) combination, B6 recipients were irradiated at 7.5 Gy and were injected a mixture of donor BMCs and SPLCs intravenously. On day 90 after injection, the degree of chimerism in peripheral blood lymphocytes (PBL) and in the splenocytes was checked by flow cytometry., Results: In groups which were injected varying BMCs, when > 45 x 10(6) BMCs were injected into B6, a large percentage of donor cells were detected in PBL and in the spleen. In contrast, when < 30 x 10(6) BMCs were injected into B6, only a small percentage of donor cells were detected. In the groups which were injected 3 x 10(6) BMCs with varying SPLCs, when > 10 x 10(6) SPLCs were added, a large percentage of donor cells were detected in PBL and SPLCs, but a small percentage of donor cells were detected with the addition of < 3 x 10(6) SPLCs. A high percentage of chimeric mice showed donor specific tolerance in vitro, mixed lymphocyte responses, and in vivo, skin grafting. In contrast, only a small percentage of chimeric mice showed no donor specific tolerance by skin grafting., Conclusion: These findings suggested that even a low dose of BMCs can establish a state of allogeneic chimerism and donor specific tolerance if combined with SPLCs.

Background: High dose of bone marrow cells (BMCs) has been reported to be essential to establish donor-specific tolerance. In clinical settings, a large quantity of BMCs is very difficult to be obtained. Our previous report demonstrated that even a low dose of BMCs could establish donor-specific tolerance if mixed with splenocytes (SPLCs). In the present study, various components of SPLCs were purified or removed and were investigated their contribution for enhancement of bone marrow engraftment leading to donor-specific tolerance in sublethally irradiated mice., Methods: Sublethally irradiated C57BL/6 recipient mice were intravenously injected 3 x 10(6) BMCs mixed with various components and various numbers of SPLCs harvested from BALB/c donor mice. One week after injection, skin grafting was performed. The degree of chimerism in peripheral blood lymphocytes (PBLs) and in SPLCs was analyzed by FACS 3 months after transplantation., Results: Recipients receiving 3 X 106 BMCs mixed with 10 x 10(6) T cell-enriched SPLCs established chimerism. Recipients receiving BMCs mixed with macrophage-depleted SPLCs also showed chimeirism and donor-specific tolerance. B cell-enriched SPLCs did not help small dose of BMCs to establish chimerism. Irradiated SPLCs were not effective to induce tolerance even with additional infusion to recipients., Conclusions: Active effects of splenic T cells were more important to help engraftment of small dose of BMCs than B cells, but the interaction between T and B cells might play some roles to enhance BMC engraftment. Splenic macrophages or dendritic cells might have some adverse effects against tolerance induction. Fatal graft-versus-host disease (GVHD) might be avoided by depleting adherent cells from SPLCs, so macrophages or dendritic cells were also considered as key components to induce donor-specific tolerance and prevent GVHD in this model.

Purpose: The association between hepatic allograft cold ischemia time (CIT) and graft transport distance (GTD) in human liver transplantation was examined by investigating whether extended graft transportation prolongs the CIT and adversely affects graft survival., Methods: We retrospectively analyzed 186 consecutive orthotopic liver transplants (OLTs) done between May 1997 and July 1998. The number of miles from the donor hospital to the University of Pittsburgh Medical Center in a straight line was measured in each case, and defined as the GTD. The OLTs were divided into two groups according to whether the GTD was 200 miles. The latter group was then subdivided into groups of GTD 200-400 miles, GTD 400-600 miles, and GTD >600 miles. The CIT and graft outcome within 90 days after OLT were assessed., Results: Extended GTD prolonged the CIT ( P < 0.001). The rate of hepatic allograft loss in the long GTD group was significantly higher than that in the short GTD group ( P = 0.018). When the OLTs were subdivided according to GTD, the CIT increased and graft survival decreased as the GTD extended. Hepatic allograft transportation for a long distance prolonged the CIT and decreased the graft survival rate., Conclusion: Since prolonged CIT is a major risk factor, avoiding long-distance graft transportation is recommended when the donor risk factors are high.

Background: Major obstacles to develop a bioartificial liver are xenogeneic immune reactions and viral infection from donor pigs. To solve these problems, we studied the effect of xenogeneic double filtration plasmapheretic cross-circulation (DFPCC) using a high performance semipermeable membrane on totally hepatectomized dogs., Methods: Mongrel dogs, weighing 12-15 kg, underwent total hepatectomy in one stage (n=18). One hr after total hepatectomy, the femoral vein and the jugular vein were cannulated in both dogs and pigs by using the blood access catheter tubes that were connected to the DFPCC system. In the DFPCC circuit, filtrated dog plasma and pig plasma counterflowed in a hollow fiber cartilage at a rate of 25 ml/min for 6 hr and met through a semipermeable membrane with 100 kd nominal molecular weight cut-off (n=5). In control dogs, the circuit was not connected to the pig (n=13)., Results: In vitro mass transfer study suggested that very little immunoglobulins crossed the semipermeable membrane. During and after 6 hr of DFPCC, anhepatic dogs had significantly lower blood ammonia and aromatic amino acid levels than did controls. DFPCC-treated dogs demonstrated decreased intracranial pressure and survived significantly longer than control dogs (20.75+/-3.80 hr vs. 14.75+/-1.30 hr, P<0.05). Histology showed no xenogeneic rejection in both dogs and pigs., Conclusions: Our DFPCC systems with a high permeability membrane demonstrated detoxification-function and contributed to intracranial decompression and longer animal survivals without adverse immune reaction and the possibility of zoonosis.

Background: We evaluated the effects of intraperitoneal transplantation of microencapsulated hepatocytes in a 3-stage total hepatectomy rat model., Methods: A new model of total hepatectomy was created as follows. First, the infrahepatic inferior vena cava was ligated just above the right renal vein. Seven days later, the portal vein was ligated and a portacaval shunt was established using a Teflon catheter over a venipuncture needle. Another 7 days later, total hepatectomy was completed by ligating and dividing the suprahepatic inferior vena cava, the hepatic artery, and the bile duct. Next, 4 x 10(7) hepatocytes (4% of the normal liver hepatocyte mass) isolated from male Wistar rats were microencapsulated within a collagen matrix enveloped by a 3-layer membrane of sodium alginate-poly-L-lysine-sodium alginate copolymer. Capsules containing hepatocytes (diameter, 500-800 microm) and empty capsules (control) were transplanted intraperitoneally 4 days before the total hepatectomy. Survival time and selected blood chemistry concentrations after the total hepatectomy were measured. The capsules were also examined histologically with hematoxylin and eosin staining and modified Gmelin's stain for bile pigments., Results: The survival time was greater in the rats given the microencapsulated hepatocytes than in the control rats (17.3 +/- 3 vs 3.7 +/- 0.1 hours; P <.01). The blood ammonia concentrations increased soon after total hepatectomy but remained significantly lower in the rats with microencapsulated hepatocytes (P <.05). The microcapsules contained numerous viable hepatocytes with abundant bile pigments and no lymphocytic infiltration., Conclusions: Microencapsulated hepatocytes with an ultrathin polymer layer that protects them from inflammatory and lymphocytic reactions may facilitate their ability to function. In this study, 4 x 10(7) hepatocytes significantly prolonged the survival of rats that underwent hepatectomy and supported ammonia metabolism. Further development of this technique may permit its use in patients with hepatic failure.