Extensive Adhesions in Living Donor Liver Transplantation: A Retrospective Analysis.

BACKGROUND: Adhesions are abnormal fibrous bands of scar tissue between internal organs and tissues. With respect to recipient hepatectomy in living donor liver transplantation (LDLT), we defined extensive adhesions as adhesions in at least two separate locations that required more than 5 % of the total surgical time to lyse. We aimed to identify the etiology and consequences of this preventable burden. METHODS: A simple retrospective case-control study of all cases with extensive adhesions from August 2011 to September 2014 matched by age, sex, and diagnosis at surgery. RESULTS: A total of 380 cases were studied. Thirty-eight and five patients had extensive adhesions from surgical and non-surgical causes, respectively. The incidence and complications in pediatric patients were far less than in adults. In the adult group, the mean operative time was increased by 75 min (12.3 %) and blood loss by 2.5 L.The incidence of bowel perforation and biliary infections were increased in adults, while there was no significant difference in the rate of ascitic or wound infections. The 1-year survival was slightly less (92 %) than the control group (100 %). CONCLUSIONS: The most common cause of extensive adhesions at LDLT was prior liver resection. Extensive adhesions caused increased morbidity by increased blood loss, transfusion requirements, and increased cold ischemia time. There is also a higher risk of bowel perforation during enterolysis. The use of commercially available barrier techniques is advisable in adults at high risk of developing adhesions with a possibility of liver transplantation, such as liver resection for HCC.

Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis.

This study investigated the pro-fibrogenic role of high mobility group box 1 (HMGB1) peptides in liver fibrogenesis. An animal model of carbon tetrachloride (CCl4)-induced liver fibrosis was used to examine the serum HMGB1 levels and its intrahepatic distribution. The increased serum HMGB1 levels were positively correlated with elevation of transforming growth factor-ß1 (TGF-ß1) and collagen deposition during fibrogenesis. The cytoplasmic distribution of HMGB1 was noted in the parenchymal hepatocytes of fibrotic livers. In vitro studies confirmed that exposure to hydrogen peroxide and CCl4 induced an intracellular mobilization and extracellular release of nuclear HMGB1 peptides in clone-9 and primary hepatocytes, respectively. An uptake of exogenous HMGB1 by hepatic stellate cells (HSCs) T6 cells indicated a possible paracrine action of hepatocytes on HSCs. Moreover, HMGB1 dose-dependently stimulated HSC proliferation, up-regulated de novo synthesis of collagen type I and α-smooth muscle actin (α-SMA), and triggered Smad2 phosphorylation and its nuclear translocation through a TGF-ß1-independent mechanism. Blockade with neutralizing antibodies and gene silencing demonstrated the involvement of the receptor for advanced glycation end-products (RAGE), but not toll-like receptor 4, in cellular uptake of HMGB1 and the HMGB1-mediated Smad2 and ERK1/2 phosphorylation as well as α-SMA up-regulation in HSC-T6 cells. Furthermore, anti-RAGE treatment significantly ameliorated CCl4-induced liver fibrosis. In conclusion, the nuclear HMGB1 peptides released from parenchymal hepatocytes during liver injuries may directly activate HSCs through stimulating HSC proliferation and transformation, eventually leading to the fibrotic changes of livers. Blockade of HMGB1/RAGE signaling cascade may constitute a therapeutic strategy for treatment of liver fibrosis.

Outcomes of long storage times for cryopreserved vascular grafts in outflow reconstruction in living donor liver transplantation.

The outflow reconstruction of the right anterior sector in a right liver graft (RLG) with cryopreserved vascular grafts (CVGs) is crucial for preventing graft congestion in living donor liver transplantation (LDLT). The impact of the duration of cryopreservation has not been evaluated so far. From 2006 to 2009, 250 LDLT were performed: 47 of these patients (group 1) received CVGs stored for ≦1 year, and 33 patients (group 2) received CVGs stored for >1 year. Single or multiple segment 8 hepatic veins were reconstructed. The number of anastomoses did not affect vascular graft patency (P = 0.21). The length of the cryopreservation time did not affect the histological findings for CVGs. The preoperative and postoperative liver graft volumes were 783.8 ± 129.7 and 1102 ± 194.7 cc, respectively, for group 1 and 753.7 ± 158.5 and 1097.2 ± 178.7 cc, respectively, for group 2. The regeneration indices for liver grafts in the whole patient group, group 1, and group 2 were 48.9%, 47.4%, and 51.05%, respectively. In conclusion, the storage duration has no impact on the patency of CVGs in outflow reconstruction or on the regeneration of RLGs in LDLT. CVGs stored for >1 year can be safely used for the outflow reconstruction of RLGs in LDLT.

Homogenous phenomenon of graft liver CYP2C19 genotypes after living donor liver transplantation.

BACKGROUND: The donor liver grafts with different allelic patterns do not affect CYP2C19 genotypes in the peripheral blood of living donor liver transplantation (LDLT) recipients. AIM: This study investigated the influence of graft liver CYP2C19 genotypes on recipients who received the same or different CYP2C19 genotypes from donors after LDLT. METHODS: There were 30 donors and 30 recipients with the same CYP2C19 genotypes and 47 donors and 47 recipients with different CYP2C19 genotypes. Genomic DNA was isolated from the liver tissue of recipients. The CYP2C19 haplotypes were determined by polymerase chain reaction. RESULTS: A homogenous phenomenon in the sequences of graft liver CYP2C19 genotypes was indicated because the recipients showed mixed patterns that were similar to that of the original donor after LDLT. A significant decrease in homozygous extensive metabolizer (HomEM) and an increase in poor metabolizer (PM) distribution were observed in recipients with different CYP2C19 genotypes from their donors compared with recipients with the same CYP2C19 genotype as their donors (P < 0·05). CONCLUSIONS: Homogenous phenomenon of sequence changes in graft liver CYP2C19 from the different genotypes between the donors and the recipients may play a role in graft stability by causing decreased HomEM and increased PM after LDLT.

Intra-operative management of low portal vein flow in pediatric living donor liver transplantation.

For pediatric living donor liver transplantation, portal vein complications cause significant morbidity and graft failure. Routine intra-operative Doppler ultrasound is performed after graft reperfusion to evaluate the flow of portal vein. This retrospective study reviewed 65 children who had undergone living donor liver transplantation. Seven patients were detected with suboptimal portal vein flow velocity following vascular reconstruction and abdominal closure. They underwent immediate on-table interventions to improve the portal vein flow. Both surgical and endovascular modalities were employed, namely, graft re-positioning, collateral shunt ligation, thrombectomy, revision of anastomosis, inferior mesenteric vein cannulation, and endovascular stenting. The ultrasonographic follow-up assessment for all seven patients demonstrated patent portal vein and satisfactory flow. We reviewed our experience on the different modalities and proposed an approach for our future intra-operative management to improve portal vein flow at the time of liver transplantation.

Western blotting analysis for quantitative detection of CYP2C19 expression in liver tissues in the setting of living donor liver transplantation.

BACKGROUND: The cytochrome P450 (CYP) drug-metabolizing enzymes play an important role in cellular metabolism. Therapeutic failure or drug toxicity in the period after liver transplantation (LT) is influenced by the drug metabolizing capacity of the graft. The expression levels of CYP2C19 enzyme are often used as an indicator of the functioning of the CYP system. The aim of the present study was to assess the CYP2C19 protein expression in the setting of living donor liver transplantation (LDLT) by using western blotting analysis. METHODOLOGY: We performed CYP2C19 genotyping of liver biopsy samples obtained from 24 donors and 8 recipients each in the pre- and post-LT periods, after which we analyzed the CYP enzyme activity by using western blotting analysis. RESULTS: The CYP2C19/ß-actin ratio, which was an indicator of CYP expression, was 61.75% (23-100%) in donors, 59.13% (15-100%) in pre-LT recipients and 46.71% (12-67%) in post-LT recipients (p>0.05). The CYP2C19 expression levels associated with different genotypes were as follows: homozygous extensive metabolizers (HomEMs; n=24), 56.63±24.74%; heterozygous extensive metabolizers (HetEMs; n=15), 63.0±25.14% and poor metabolizers (PMs; n=1), 82.0% (p>0.05). CONCLUSIONS: Western blotting analysis showed low CYP2C19 protein expression not only in samples from the pre- and post-LT recipients but also from the donors.

Impact of artificial sunlight therapy on the progress of non-alcoholic fatty liver disease in rats.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is recognized as the most severe form of non-alcoholic fatty liver disease, with likely progression to liver cirrhosis and hepatocellular carcinoma. However, there is no unified standard for diagnosis and therapeutics. This study aimed to characterize lipid transfer/metabolic proteins as non-invasive diagnostic markers, and to evaluate the therapeutic effects of phototherapy on the progression of NASH in rats. METHODS: Lewis rats given a choline-deficient and iron-supplemented l-amino acid-defined (CDAA) diet and Zucker fa/fa rats were used as a diet-induced and an obesity-related NASH models, respectively, with or without phototherapy. RESULTS: Serum apolipoprotein E and low molecular weight-adiponectin levels were gradually reduced and reached the lowest level at fatty liver/NASH stage both in CDAA diet-induced NASH model and in genetically obese model. Total-adiponectin levels were dramatically elevated after NASH was established in CDAA diet-induced NASH model. Phototherapy ameliorated hepatocyte apoptosis, inflammation, fibrosis, and insulin/leptin resistance caused by CDAA diet with alteration of the levels of lipid transfer/metabolic proteins and elevation of the circulating active form of vitamin D(3). Vitamin D(3) supplementation ameliorated NASH progression in CDAA diet-induced NASH model. However, phototherapy failed to ameliorate the obesity and steatosis, suggesting that phototherapy may possess anti-inflammatory/fibrotic activity rather than anti-obesity/steatotic activity. CONCLUSIONS: These results suggest that serum lipid transfer/metabolic proteins and vitamin D(3) status may be effective biomarkers for non-invasive diagnosis of NASH progression, and that phototherapy may be a good complementary therapy for NASH because of its regulation of lipid transfer/metabolic proteins and vitamin D(3).

Two cases of massive pleural effusion noted only after induction of anesthesia in living donor liver transplantation.

Two adult patients who underwent living donor liver transplantation with acute accumulation of right-side pleural effusion are reported. The chest X-ray of patient 1 showed no specific finding 3 days before the operation, and patient 2 was known to have pleural effusion and underwent pigtail drainage before transplant. After anesthesia induction and insertion of central venous catheters, a portable chest radiograph was taken to confirm the positions of the central venous catheters and endotracheal tube. A massive right-side pleural effusion was noted unexpectedly in both patients. Approximately 2,000 ml transudative fluid was surgically drained through the right diaphragm in patient 1 upon opening of the abdominal cavity. The acute accumulation of massive pleural fluid in patient 2 was caused by clamping of the pigtail drainage tube during patient transfer to the operating room; upon unclamping of the tube, 2,000 ml fluid was drained. The intraoperative and postoperative transplant courses of both patients were uneventful. Both were discharged from the hospital in stable condition. Our cases suggest that chest X-ray after induction of the anesthesia and before liver transplantation surgery is recommended. In addition to documenting the positions of the central venous catheters and endotracheal tube, a potential life-threatening pleural effusion requiring appropriate management may be detected.

Upregulation of hepatoma-derived growth factor is involved in murine hepatic fibrogenesis.

BACKGROUND & AIMS: Hepatoma-derived growth factor (HDGF) expression is correlated with progression of hepatocellular carcinoma. Since liver fibrosis frequently occurs before hepatoma development, this study investigated the expression profile of HDGF and its relationship with transforming growth factor-beta (TGF-beta) signaling in experimental models of hepatofibrogenesis. METHODS: Liver fibrosis was induced in mice receiving bile duct ligation (BDL) or carbon tetrachloride (CCl(4)) administration. The expression levels of HDGF and other fibrosis-related markers were measured using quantitative RT-PCR, Western blotting, and enzyme-linked immunosorbent assays. Hepatic HDGF overexpression was achieved by adenovirus gene delivery. Rat hepatocytes were used to study the interplay between HDGF and TGF-beta1. RESULTS: In both liver fibrosis models, HDGF de novo synthesis significantly increased during the progression of fibrosis. The HDGF upregulation was observed mainly in hepatocytes and correlated with the expression of TGF-beta1 and collagen COL1A1 and COL1A2 proteins. Hepatic HDGF overexpression itself deteriorated hepatocellular structure and integrity, and aggravated the extents of BDL- and CCl(4)-induced liver fibrosis with concomitant upregulation of TGF-beta1 and COL1A1. Exogenous TGF-beta1 stimulated HDGF expression only in cultured primary hepatocytes grown on collagen matrix, whereas exogenous HDGF also increased TGF-beta1 production in hepatocytes in a collagen-dependent manner. Moreover, HDGF enhanced Smad2 phosphorylation dose-dependently and the TGF-beta1-driven luciferase activities. CONCLUSION: HDGF plays a pro-fibrogenic role during liver fibrosis in mice through activation of TGF-beta pathway. The mutual regulation between TGF-beta1 and HDGF may facilitate a vicious cycle to promote the progression of hepatic fibrogenesis.

Solitary pulmonary nodule in the liver transplant candidate: importance of diagnosis and treatment.

Our objectives were to define the incidence and etiology of solitary pulmonary nodules (SPNs) in patients undergoing living donor liver transplantation (LDLT), describe a diagnostic approach to the management of SPNs in LDLT, and define the impact of SPNs on the overall survival of adult LDLT recipients. Nine patients (9/152, 5.9%) were diagnosed with an SPN on the basis of chest radiography findings during the pretransplant survey. All were male. The mean age was 52 years. All the patients had hepatitis B virus-related cirrhosis with hepatocellular carcinoma. All were asymptomatic for the lung lesion. All underwent contrast-enhanced chest computed tomography (CT) to verify the presence and possible etiology of the SPNs. In 3 cases, CT was used to definitely determine that there was no pulmonary nodule; in 2, CT led to a definite diagnosis of pulmonary tuberculosis. In 4, CT led to a definite identification of an SPN but not to an etiological diagnosis. Two patients underwent outright thoracoscopy and biopsy of their SPNs. Biopsy showed cryptococcosis in both patients. One received a therapeutic trial of an antituberculosis treatment, and repeat CT after 1 month showed a regression in the size of the SPN. A diagnosis of tuberculosis was made. One patient had an inconclusive whole body positron emission tomography scan and subsequently underwent thoracoscopy where biopsy showed tuberculosis. A concomitant malignancy, either primary lung cancer or metastasis from the liver tumor, was not identified. All patients were surviving with their original grafts and were lung infection-free. The overall mean posttransplant follow-up was 54 months (range = 33-96 months).

Donor graft does not affect the P450 2C19 genotype expressed in peripheral blood in recipients of living donor liver transplantation.

The function of cytochrome P450 2C19 (CYP2C19) is altered in patients with end-stage liver disease (ESLD) that require liver transplantation (LT). The status of CYP2C19 is of considerable interest because the transplanted healthy donor livers are perfused with the blood of the recipient with ESLD. This study aims to clarify the changes in CYP2C19 in the peripheral blood before and after LT. Thirty pairs of living donors and recipients were enrolled in this study. The CYP2C19 genotype in peripheral blood mononuclear cells (PBMCs) was studied immediately before operation in donors, on the day preceding the operation in the unstable recipients, and one month after LT in stable recipients. Limited data suggest that the post-LT genotype in liver biopsy is the same as donor's original genotype in most cases (80.0%) and that only 2 patients in the study cohort had the same liver tissue genotype as the respective recipient PBMCs. However, expression of the CYP2C19 genotype after living donor LT (LDLT) was identical to pre-transplant expression in 100% (30/30) of recipients, i.e., CYP2C19 genotypes in recipient PBMCs did not change after LDLT, suggesting that the donor liver did not render any mutations to the CYP2C19 genotypes after LT.

Comparison of Glucose Loads and Blood Glucose Changes in Patients With and Without Diabetes Mellitus Undergoing Living Donor Liver Transplantation.

BACKGROUND AND PURPOSE: The purpose of this study was to identify the quantitative amount of glucose load, which maintained the blood glucose levels between 100 and 180 mg/dL in patients with and without diabetes mellitus (DM) undergoing living donor liver transplantation (LDLT). METHODS AND PATIENTS: The anesthesia records of 477 adult LDLT patients were reviewed retrospectively. The total amount of glucose loads and the changes in blood glucose between groups were compared by using Mann-Whitney U test. One-year patient survival between groups was compared with Pearson's χ2 test. A P value of <.05 was considered statistically significant. RESULTS: Eighty patients diagnosed with DM, who were all type II except one, were placed in group 1 (G1); and 397 patients without DM were placed in group 2 (G2). Table 1 shows that G1 received significantly less glucose loads in comparison to G2, but all the measured blood glucose levels, except in the reperfusion phase, were significantly higher in G1 than in G2. Both groups received glucose loads of 0.342 ± 0.191 and 0.774 ± 0.191 mg/kg/min for G1 and G2, respectively. No difference in 1-year survival between groups was observed. CONCLUSION: Patients with DM required significantly lower glucose loads compared to patients without DM.

Anesthetic Fluid Management of Patients With Hyponatremia Undergoing Living Donor Liver Transplantation.

OBJECTIVE: The objective of this study is to evaluate the changes in serum sodium levels in adult recipients with and without hyponatremia undergoing living donor liver transplantation (LDLT) without using hypertonic solution. METHODS: Patients were divided into 2 groups according to serum sodium level higher (GI) or lower (GII) than 130 mEq/L. The changes of serum sodium levels during an LDLT procedure and total sodium loads were compared between groups by using the Mann-Whitney U test, while the changes in the same group were paired by using the Student t test. A P value <.005 was considered significant. RESULTS: The total sodium load for GI (n = 438) and GII (n = 28) were 2737 ± 2159 mEq and 4017 ± 2830 mEq, respectively. Although GI received a significantly lower sodium load than GII, the serum sodium levels during the procedure were always within a normal range and higher than GII at all the measured time points; however, the changes of serum sodium level in GI from one point to the next measured point in the same group were unremarkable, while that of GII increased significantly between the 2 measured time points during the procedure. The mean total increase of serum sodium in GII was 5.57 ± 4.9 mEq/L in 14 hours of the LDLT procedure. None of the patients developed central pontine myelinosis (CPM) postoperatively. CONCLUSION: Patients with hyponatremia can be managed safely without using a hypertonic solution during liver transplantation. The mean increase of serum sodium of GII was of 5.57 ± 4.9 mEq/L, which was still within the acceptable and safe level. No postoperative CPM was observed in our GII patients.

Acute Kidney Injury in Adult Living Donor Liver Transplantation Performed with Different Methods of Inferior Vena Cava Clamping Maneuvers.

OBJECTIVE: The aim of this retrospective study is to evaluate and compare the incidence of acute kidney injury (AKI), defined as increase serum creatinine (SCr) of 0.3 mg/dl or increase in SCr to ≥1.5 times from baseline within 48 hour, in adult living donor liver transplantation patients performed with total cross clamp vs side clamp of the inferior vena cava (IVC). METHODS AND PATIENTS: Sixty adult living donor liver transplantation (LDLT) patients were divided into 2 groups: 30 patients in total IVC clamping (G1) and 30 in IVC side clamping (G2) during the anhepatic phase. Patients' characteristic, hemodynamic changes in percentage (%) as a result of different methods of IVC clamping, urine output during anhepatic phase were compared by using the Student t test, and the incidence of AKI were compared by using the χ2 test between groups. P value <.05 was regarded as significant. RESULTS: The negative impact of the 2 different ways of IVC clamping was significantly more severe in G1 compared to G2; consequently, the urine output of G1 was significantly less than G2. Although there was significantly more urine output of G2 during the anhepatic phase, the incidence of the postoperative AKI between groups was similar. CONCLUSION: The side clamp of the IVC had a significantly less negative impact on the hemodynamic parameters and provided sufficient urine output during the anhepatic phase (2.24 ± 3.17 vs 0.39 ± 0.33 mL/kg/h) compared to the total clamp of the IVC. But this favorable data did not protect the patient suffering from postoperative AKI in LDLT.

Routine microsurgical biliary reconstruction decreases early anastomotic complications in living donor liver transplantation.

Biliary reconstruction using a microsurgical technique in living donor liver transplantation was routinely performed on 88 grafts primarily transplanted into 85 patients. All procedures were performed under a microscope by a single microsurgeon. Except for biliary atresia and Alagille syndrome, duct-to-duct reconstruction was performed. Stents were not used. The outcomes with microsurgical biliary reconstruction (MB) were compared with the outcomes of a cohort of 86 grafts in 85 patients that underwent conventional biliary reconstruction (CB). The identification of complications included only up to 12 months of follow-up for each recipient in both groups. The average graft duct sizes were 2.8 mm for MB and 3.4 mm for CB. Most complications occurred in the first 15 cases with MB, and these cases were considered to constitute the learning curve phase. The MB complication rate was 46.7% in the first 15 cases, 20.0% in the next 15 cases, and 5.4% in the last 55 cases. When the learning curve phase was excluded, the overall complication rate over time with MB (8.9%) was significantly lower than that with CB (21.9%). CB increased the risk of biliary complications by 2.5 times (relative risk: 2.5; attributable risk: 128; odds ratio: 2.9). In conclusion, routine MB is a technical innovation that leads to decreased early anastomotic complications in living donor liver transplantation.

Serum factors potentiate hypoxia-induced hepatotoxicity in vitro through increasing transforming growth factor-beta1 activation and release.

BACKGROUND: Although transforming growth factor-beta (TGF-beta), a growth regulator of hepatocytes, induces cell death under pathological conditions, responsiveness of hepatocytes to hypoxic stimulus has not been fully defined. This study aimed at investigating the role of TGF-beta1 in hypoxia-induced hepatotoxicity using cultured clone-9 hepatocytes with or without serum supplementation. METHODS/RESULTS: Presence of serum significantly potentiated hypoxia-induced hepatotoxicity after 72h of exposure, as evidenced by fluorescent viability stain and LDH cytotoxicity assay. Quantitative PCR showed that TGF-beta1 gene expression decreased, while ELISA revealed that latent TGF-beta1 in conditioned media prominently increased in serum-treated groups under hypoxia. Western blotting indicated that both type I and II receptors of TGF-beta were up-regulated in serum-free groups, but down-regulated in serum-treated groups under hypoxia. Smad2 phosphorylation was only detectable in cells supplemented with serum, and hypoxia potentiated the extent of Smad2 phosphorylation, implicating that the activated TGF-beta1 induces hepatotoxicity in an autocrine manner. Addition of exogenous TGF-beta1 deteriorated, while TGF-beta1 blockade by neutralizing antibody ameliorated hypoxia-induced hepatotoxicity with serum supplementation. Gelatine zymography and immunofluorescent stain evidenced that elevated MMP-2 and MMP-9 activity and serum-dependent CD44 expression and its membranous localization may contribute to TGF-beta1 activation. CONCLUSION: The results suggest that the mechanism governing TGF-beta activation plays a crucial role in hypoxia-induced hepatotoxicity. Thus, interventions on TGF-beta1 bioavailability and/or its cognate signaling may be of benefit in preventing hypoxia-related liver injuries.

Living donor liver transplantation for hepatocellular carcinoma: a single-center experience in Taiwan.

BACKGROUND: Living donor liver transplantation (LDLT) demonstrates certain survival benefits over deceased donor liver transplantation for hepatocellular carcinoma (HCC) but there is no consensus on criteria for the use of LDLT for HCC for hepatocellular carcinoma (HCC) taking into account strategies to improve survival. METHODS: Thirty-five patients (89% men) underwent LDLT for HCC. The mean age was 51 years (range, 22-61). The median disease severity scores were B, 11-20, and 2B for Child-Turcotte-Pugh, Model for End-stage Liver Disease, and United Network for Organ Sharing, respectively. The transplant records were retrospectively analyzed. RESULTS: All were within Milan criteria at time of transplantation. A novel approach to downstaging tumors initially beyond the Milan criteria was evaluated using transarterial embolization or percutaneous ethanol injection. Our initial results were encouraging as recipients whose tumors had been downstaged had not had recurrence to date. Seven (20%) patients underwent hepatectomy for HCC before undergoing transplant. The overall mean posttransplant follow-up in this series was 40.3 months (range, 23-75). The overall posttransplant complication rate requiring intervention was 11%. There was only one malignancy recurrence for an overall recurrence rate of 3%. Vascular invasion and small- for-size transplants did not seem to influence tumor recurrence. The nonestimated recipient 1-year, 3-year, and 5-year survivals were 98%, 96%, and 90%, respectively. CONCLUSION: This review emphasizes the need for early disease recognition and prompt intervention when Milan criteria are met to improve survival from HCC after LDLT.

Heat preconditioning ameliorates hepatocyte viability after cold preservation and rewarming, and modulates its immunoactivity.

BACKGROUND: Heat preconditioning significantly preserved liver graft function after cold preservation in animal experimental model. The elevation of heat shock protein 70 (HSP70) was claimed to play a critical role in protecting grafts against cold preservation-induced hepatocyte apoptosis. However, little is known about whether HSP70 also plays an immunomodulatory role in cold preserved cells. This study aimed at investigating the relationship between HSP70 protein and the immunoactivity in response to lipopolysaccharide (LPS) stimulation. METHODS AND RESULTS: A normal rat hepatocyte cell line was preserved with University of Wisconsin (UW) solution, Ringer's lactate solution (RL), and phosphate-buffered saline (PBS) at 4 degrees C. No significant morphological alteration was noted in UW-preserved cells after 24 h through phase-contrast microscopic observation and fluorescent viability stain. Western blotting showed a two-fold increase in the ratio of HSP70/Bax proteins in cells after 24 h of UW preservation. Heat preconditioning significantly enhanced the recovery of lactate dehydrogenase (LDH) activity in both RL- and UW-preserved cells that were stored for a period of 12 h or less. Moreover, heat preconditioning promoted HSP70 and NF-kappaB p50 nuclear translocation and suppressed the LPS-induced nuclear p50 accumulation in cells before UW preservation. Immunofluorescent stain revealed that the LPS-induced p50 protein redistribution to nuclear membrane might contribute to NF-kappaB activation, while heat preconditioning and UW cold preservation completely abrogated the p50 intranuclear redistribution. Thus NF-kappaB p50 might be responsible for the endotoxin tolerance induction. CONCLUSIONS: These findings strongly suggest that heat preconditioning not only preserves hepatocyte viability after cold preservation and rewarming, but also ameliorates its immunoactivity.

Involvement of autoimmunity against nuclear histone H1 in liver transplantation tolerance.

BACKGROUND: Our recent studies suggested that anti-histone H1 autoantibody (auto-Ab) plays an important role in experimental and clinical liver allograft tolerance as a natural immunosuppressive factor. The present study aimed to explore how the autoimmune response against histone H1 is involved in tolerance induction. METHODS: The measurement of anti-histone H1 auto-Ab and immunohistochemical analysis were performed in serum and liver allografts after orthotopic liver transplantation (OLT). To compare the auto-Ab response against histone H1 between the recipients of rejector (DA-LEW) and tolerogenic (DA-PVG) OLT models, naïve recipients were immunized with calf thymus histone H1. The immunosuppressive state of histone H1-immunized rats was assayed by mixed lymphocyte reaction (MLR). RESULTS: Anti-histone H1 Ab titer was transiently increased during the rejection phase after OLT (days 7-21) in the DA-PVG combination, while no such response was confirmed in the DA-LEW acute rejection model. Nuclear histone H1 antigens were found in the cytoplasm and the extracellular environment in liver allografts at the rejection phase in the tolerogenic model but not in the rejector model, resulting from the transient induction of anti-histone H1 auto-Ab in recipient PVG rats after OLT. Low dose and short-term immunization with histone H1 upregulated the anti-histone H1 Ab titer in naïve PVG rats, which exhibited a low allogeneic immune response, while no such response was found in naïve LEW rats. CONCLUSIONS: These results suggest that the sensitivity to nuclear antigens such as histone H1 may be a key factor determining the acceptance or rejection of donor liver grafts, at least in DA-PVG and DA-LEW combinations.

Immunological role of indoleamine 2,3-dioxygenase in rat liver allograft rejection and tolerance.

BACKGROUND AND AIM: Indoleamine 2,3-dioxygenase (IDO) is expressed in the placenta and plays an essential role in maternal tolerance. Recent data showed that giving IDO inhibitor blocked liver allograft tolerance. However, the immunological role of IDO in rat liver allograft models has not been characterized. In the present study, the time-course of IDO expression and the localization of IDO were analyzed to address the role of IDO in the induction of tolerance. METHODS: Rat orthotopic liver transplantations (OLT) were performed and IDO gene expression of OLT livers was analyzed. Immunohistochemistry was used to evaluate the localization of IDO-expressed cells in the liver. RESULTS: The IDO gene was detected in the allogeneic liver graft at the acute phase but the signal could not be detected when these OLT rats were treated with cyclosporinee A. The time-course of IDO gene expression in liver grafts of the spontaneous tolerant OLT model revealed that the IDO mRNA was expressed in both the rejection phase and the induction phase of tolerance, but the signal was gradually lowered during the maintenance phase of tolerance. Immunohistochemistry confirmed that the IDO protein was detected in antigen-presenting cells but not in hepatocytes. CONCLUSION: Our results demonstrated that IDO is induced in antigen-presenting cells of rat liver allografts under drug-free status, suggesting that indirect or direct recognition of donor antigen and further T-cell activation may be inhibited. IDO may act as a local immunosuppressive molecule to protect transplanted cells, tissues and organs from immune attack.