Mitotic centromere-associated kinesin is a novel marker for prognosis and lymph node metastasis in colorectal cancer.

Mitotic centromere-associated kinesin (MCAK) is a microtubule depolymerase that is essential for proper kinetochore-microtubule attachment during spindle formation. Overexpression of MCAK has been correlated with aggressive forms of carcinoma, resulting in poor prognosis of colorectal cancer. The purpose of this study was to quantify MCAK expression in malignant and benign colorectal tissues and to determine if MCAK expression levels correlate with clinicopathologic factors and prognosis in colorectal cancer patients. Paired colorectal tissue samples from tumours and the corresponding normal tissues were obtained from 120 patients with colorectal cancer who underwent surgical resection. The real-time reverse transcriptase-PCR and immunohistochemistry were used to analyse mRNA and protein expression status with respect to various clinicopathological factors. MCAK expression was higher in colorectal cancer tissue (P<0.01) than in corresponding normal tissue, and this elevated expression level was markedly associated with factors such as lymph node metastasis (P=0.0023), venous invasion (P=0.019), peritoneal dissemination (P=0.021) and Dukes' classification (P=0.0023). Patients with high MCAK mRNA expression also showed a far poorer survival rate than those with low MCAK mRNA expression (P<0.01). Elevated MCAK expression was an independent predictor of overall survival and lymph node metastasis. These data suggest that MCAK expression may serve as a good marker of prognosis and lymph node metastasis in colorectal cancer.

Influence of serum from rats with fulminant hepatic failure on hepatocytes in a bioartificial liver system.

Fulminant hepatic failure (FHF) is a life-threatening condition marked by many excessively increased unmetabolized toxins and growth factors. Recently developed bioartificial liver (BAL) systems containing hepatocytes can be used to treat patients with FHF However, the behavior of these hepatocytes on exposure to FHF serum in vitro remains unclear. In the present study, we used FHF rat models and the sera from these rats (i.e., FHF serum) contained elevated inflammatory cytokines (TNF-alpha, IL-1beta, and IL-6), HGF, and TGF-beta1. In addition, 1x10(8) hepatocytes were harvested from the livers of inbred rats and incubated with microcarrier beads. Four hours later, the hepatocyte-coated beads were inoculated into a hollow-fiber module (=BAL system). FHF serum or normal control serum circulated for 6 hours through the BAL system. Expressions of mRNA for albumin, GST A1, CYP 1A2, OTC and c-fos were investigated by RT-PCR, and PCNA staining was performed before and after perfusion. The expressions of albumin, GST A1, and CYP 1A2 mRNAs were markedly decreased, whereas those of OTC and c-fos were modestly decreased. PCNA positive cells were low and showed no difference between FHF and normal serum-exposed hepatocytes. In conclusion, the exposure of hepatocytes to hypercytokinemia, including inflammatory cytokines and positive and negative growth factors, caused a loss in liver specific functions. This environment also failed to facilitate hepatocyte regeneration.

Bioartificial liver treatment prolongs survival and lowers intracranial pressure in pigs with fulminant hepatic failure.

Intracranial hypertension leading to brainstem coning is a major cause of death in fulminant hepatic failure (FHF). We have developed a bioartificial liver (BAL) utilizing plasma perfusion through a bioreactor loaded with porcine hepatocytes and a column with activated charcoal. In a Phase I clinical trial, we observed a decrease in intracranial pressure (ICP) in FHF patients. However, these patients received BAL therapy together with other measures. We therefore examined whether BAL therapy alone could prevent development of intracranial hypertension in pigs with surgically induced FHF. Pigs (40-60 kg) underwent end-to-side portacaval shunt, transection of all hepatic ligaments, and placement of slings around the hepatic artery and bile duct. After 3 days, the slings were tightened to induce liver necrosis. After 4 h, Group 1 pigs (n = 6) underwent a 6 h treatment with the BAL utilizing 10 billion cryopreserved pig hepatocytes and a charcoal column, Group 2 pigs (n = 6) with the BAL containing charcoal but no cells, and Group 3 pigs (n = 6) with the BAL containing neither cells nor charcoal. Group 1 pigs maintained a normal ICP during BAL treatment and for 14 h afterward and because of this effect they survived longer than Groups 2 and 3 animals. In contrast, Groups 2 and 3 pigs showed an early (6-8 h) rise in ICP.

Regulation of c-met expression in rats with acute hepatic failure.

BACKGROUND: Earlier we described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobe necrosis. In FHF rats, lack of hepatocyte proliferation was associated with delayed expression of HGF and HGF receptor c-met. Since the c-met promoter region has Sp1 binding sites, we decided to examine whether in FHF rats down-regulation of c-met is associated with decreased Sp1 function and whether changes in blood HGF, IL-6, and TGFbeta1 levels might be responsible for these effects. MATERIALS AND METHODS: Induction of FHF, partial (2/3) hepatectomy (PH), and sham hepatectomy (SH) was performed in adult Sprague-Dawley rats. The levels of c-met mRNA and Sp1 DNA binding activity were studied in rat liver remnants at different time points after surgery. Blood levels of HGF, IL-6, and TGFbeta1 were also measured in these rats. Additionally, the effects of treatment with TGF-beta1, IL-6, or a combination of both on c-met expression and Sp1 DNA binding were studied in HGF-induced rat hepatocyte cultures. RESULTS: Compared to SH rats, in PH rat livers c-met was up-regulated after 6 h and Sp1 DNA binding was at or only slightly lower than levels at all time points studied. In FHF rat livers, c-met expression was markedly reduced after 2 and 6 h, moderate after 12 h, and undetectable after 24 h. At the same time, Sp1 DNA binding was detected at 2 h postinduction only. In FHF rats, blood levels of all three cytokines showed early and sustained elevation. In vitro, IL-6 had no effect on c-met expression, whereas TGFbeta1 up-regulated c-met. When used alone, none of the cytokines affected Sp1 DNA binding activity. In contrast, a combination of IL-6 and TGFbeta1 down-regulated c-met expression as well as Sp1 DNA binding activity. These effects were dependent on the IL-6 concentration used. This study suggests that following massive loss of hepatocyte mass in rats, early increase in blood IL-6 and TGFbeta1 levels may weaken the expression of HGF receptor c-met in surviving hepatocytes through suppression of Sp1 DNA binding.

Intraportal administration of low-dose recombinant human hepatocyte growth factor enhances effects of hepatocellular transplantation.

BACKGROUND/AIMS: Although recombinant human hepatocyte growth factor (rhHGF) is a potent mitogen, the dose used for patients is still not clear and must be low to avoid untoward effects. Firstly, the optimal strategy of the dose and route of rhHGF was investigated. Secondly, low-dose rhHGF, which would induce proliferation of transplanted hepatocytes, was explored using Nagase analbuminemic rats. METHODOLOGY: 1) Concentrations of rhHGF in the portal vein were measured after continuous administration of titrated rhHGF through the jugular vein or portal vein. 2) F344 rat hepatocytes (2 x 10(7) cells) were transplanted in the liver of Nagase analbuminemic rats. On the 7th day, the rats were subjected to a low-dose rhHGF treatment. RESULTS: When the rats were given rhHGF in a dose of 50 micrograms/kg/day, the mean concentration in the portal vein (0.8 +/- 0.1 ng/mL) was almost similar to the minimum concentration which stimulated hepatocyte proliferation in vitro. When low-dose rhHGF (50 micrograms/kg/day) was administered directly into the portal vein following hepatocyte transplantation in Nagase analbuminemic rats, the serum levels of albumin were significantly higher than in other groups. It was found that the concentration of rhHGF in the portal vein were 3.1 +/- 0.5 ng/mL with continuous intraportal infusion and 0.8 +/- 0.1 ng/mL with continuous systemic infusion. CONCLUSIONS: It was found that the minimal dose of rhHGF needed to stimulate hepatocyte proliferation was 50 micrograms/kg/day. With rhHGF (50 micrograms/kg/day), continuous intraportal infusion afforded a more favorable outcome in case of proliferation of hepatocytes.

Safe and efficient gene transfer into porcine hepatocytes using Sendai virus-cationic liposomes for bioartificial liver support.

Establishment of a bioartificial liver support system using genetically modified hepatocytes is a potential approach to improve the treatment of severe liver failure. We describe the development of an efficient ex vivo method of gene transfer into a large number of porcine hepatocytes using hemagglutinating virus of Japan (HVJ)-liposome. The transfection efficiency of HVJ-liposome into isolated porcine hepatocytes attached to microcarrier beads was evaluated by beta-galactosidase (beta-gal) staining, fluorescence activated cell sorting analysis for beta-gal and luciferase assay, respectively. To examine the function and cellular damage of transduced hepatocytes, we used enzyme-linked immunosorbent assay for porcine albumin synthesis, lidocaine clearance test (P-450 activity), aspartate aminotransferase, and lactic dehydrogenase release assays. The optimal conditions for gene transfer into the beads-attached hepatocytes using HVJ-liposome included 4 microg of deoxyribonucleic acid with 200 microg of lipid/2 x 105 cells and exposure duration of 90 min. Under these conditions, beta-gal and luciferase genes were transduced to 2.5 x 108 isolated porcine hepatocytes following attachment to the beads. Positive beta-gal staining was observed in more than 30% of the beads-attached hepatocytes. The gene transfer activity of HVJ-liposome method determined by luciferase activities was about 100-fold of that of the lipofection method. Transfected porcine hepatocytes remained functional without any significant cell damage. Our results demonstrated that HVJ-liposome mediated gene transfer into microcarrier-attached porcine hepatocytes is an efficient and nontoxic method suitable for a bioartificial liver support sytem.

[Treatment of liver cancer: current status and future prospectives].

The most common liver cancers are hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), and metastatic colorectal cancer. In HCC patients, the extent of the surgical resection is limited due to the functional status of the underlying cirrhotic liver. Limited resection, transarterial catheter embolization, ethanol injection, and microwave coagulation have been applied to treat the patients with liver hypofunction; however, the intrahepatic recurrence rate was relatively high in those patients. Therefore, liver transplantation is the only radical treatment to remove HCC and cirrhotic livers with viral infections. Recent advances in anti-viral agents promise to improve the outcome after liver transplantation in patients with HCC. On the other hand, CCC is outside the indications for liver transplantation because of the broad extension of lymph node and nerve plexus. In liver metastasis from colorectal cancer, overall survival is not greatly improved, although arterial chemotherapy reduces mortality related to liver metastasis. Surgical resection including repeated hepatectomy indicates better survival in patients with liver metastases. In the future, both CCC and metastatic liver cancer could be candidates for gene therapy. For the 21 century, a new therapeutic strategy incorporating clinical evidence, molecular biology, and organ replacement needs to be established for the treatment of liver cancer.

Inhibition of signal transducer and activator transcription factor 3 in rats with acute hepatic failure.

In fulminant hepatic failure, survival is not possible without recovery of sufficient hepatocyte mass. Remarkably, only a few studies exist that provide insight into the mechanisms that control proliferation of residual hepatocytes after extensive hepatocyte loss. In this regard, the role of growth-regulatory factors, including pro-inflammatory cytokines such as interleukin-6 (IL-6), is not well understood. In the present study we show that in rats with critically low (10%) hepatocyte mass, whether with or without ongoing liver cell necrosis, inhibition of liver regeneration is associated with early and sustained increase in blood IL-6 levels. Under these conditions, the signal transducer and activator of transcription (Stat3) DNA binding activity was lowered at the time of G1/S cell-cycle transition. We further demonstrate that the protein inhibitor of activated Stat3 (PIAS3) and the suppressor of cytokine signaling (SOCS-1) were up-regulated early after induction of liver failure (6-12 h). In vitro, IL-6 induced PIAS3 expression in HGF stimulated rat hepatocytes. These findings suggest that after massive hepatocyte loss, an early and rapid rise in blood IL-6 levels may weaken the hepatic regenerative response through up-regulation of Stat3 inhibitors PIAS3 and SOCS-1.

Comparative study of bioartificial liver support and plasma exchange for treatment of pigs with fulminant hepatic failure.

Recently, bioartificial liver (BAL) treatment was reported to provide beneficial effects for patients with fulminant hepatic failure (FHF). Some success in experimental or clinical trials has been reported; however, the evaluation of BAL efficacy remains unclear, especially in comparison with other treatments for FHF. The purpose of this study was to compare the efficacy between BAL and plasma exchange (PE) in experimentally induced FHF in pigs. Pigs undergoing hepatic devascularization (HD) were placed into the following groups: no treatment (control; n = 6), BAL treatment (BAL; n = 5), and plasma exchange (PE; n = 5). Each treatment was initiated 6 h after HD and lasted for 4 h. BAL treatment significantly improved liver functions in FHF pigs. The decrease in cerebral perfusion pressure was also significantly suppressed in the pigs with BAL, and their survival time was prolonged compared with the results in pigs with PE. The effects of BAL outperform those of PE in the treatment of experimental FHF model.

Transforming growth factor beta1 helps maintain differentiated functions in mitogen-treated primary rat hepatocyte cultures.

Mechanisms that control function and repair of the injured liver remain unclear. We hypothesized that after liver injury, elevated blood TGF-beta1 levels may reflect an adaptive response to help maintain differentiated functions in surviving hepatocytes affected by excessive amounts of HGF. We thus studied the effect of HGF, EGF, TGF-beta1, HGF + TGF-beta1, or EGF + TGF-beta1 on the expression of liver-enriched transcription factors and genes which remain under their regulatory activity. The peak [3H]thymidine uptake induced by 20 ng/ml of either HGF or EGF was seen after 72 h; however, DNA binding of C/EBP and HNF1 decreased already after 6 h (electrophoretic mobility shift assay). Addition of TGF-beta1 antagonized these effects. Also at the mRNA level, TGF-beta1 counteracted at one point or another the decrease in C/EBPalpha, C/EBPbeta, HNF1beta, and HNF4 expression; HNF1alpha and COUP-TF showed similar responses and, additionally, were downregulated by TGF-beta1 at 24 h (Northern blot). Albumin and apolipoprotein B mRNA levels were decreased after 24-h treatment with HGF, whereas addition of TGF-beta1 increased their levels. The same pattern was found with EGF, but not until 48 h. PEPCK mRNA was dramatically lowered with either EGF or HGF, and TGF-beta1 did not counteract these effects. Id-1 was expressed only in cultures treated for 24 and 48 h with both the mitogen (EGF, HGF) and TGF-beta1 and in those treated for 48 h with TGF-beta1 alone.

The significance of measuring the serum total bile acids levels during orthotopic liver transplantation.

BACKGROUND/AIMS: Bile acid is confined to the enterohepatic circulation and consists of intestinal absorption and hepatic elimination. We investigated whether measuring the serum total bile acids (TBA) levels was useful for both evaluating the function of the grafted liver and predicting the outcome in porcine orthotopic liver transplantation (OLT). METHODOLOGY: Twenty-two female Yorkshire pigs undergoing OLT were divided into 2 groups as follows: Group A consisted of 11 pigs which survived over 7 days with an uneventful early post-operative course, while Group B consisted of 11 pigs which died within 5 days due to hepatic failure. The serum TBA levels were measured before and after reperfusion in the recipients. RESULTS: Between Groups A and B, no significant difference was observed in the operative backgrounds including the operation time as well as the cold and warm ischemic time. In Group A, the levels of serum TBA rapidly increased during the anhepatic phase, and thereafter promptly decreased after the reperfusion of the grafted liver. A significant difference was observed in the levels of serum TBA before and after reperfusion (p < 0.01), whereas no significant difference was seen in Group B. The delta TBA, which represents the difference in the levels of serum TBA between just prior to reperfusion and 10 min after reperfusion, was 71.8 +/- 43.5 mumol/L in Group A and 20.1 +/- 34.5 mumol/L in Group B, and demonstrated a significant difference between these 2 groups (p < 0.01). On the other hand, no significant differences were seen in the levels of serum AST, ALT, ALP and TB at each time point between Groups A and B. CONCLUSIONS: The level of serum TBA was found to be a more sensitive parameter and also reflected the developing grafted liver function earlier than the conventional parameters for liver function. Moreover, delta TBA thus appeared to be a valuable predictor for the post-operative outcome.

Efficacy of Nafamostat Mesilate for improving the performance of a bioartificial liver using porcine hepatocytes.

Our bioartificial liver (BAL) consists of porcine hepatocytes attached to beads and plasma perfused through the system. The function of our BAL lasts for approximately 7 hours. The objective of the present study was to investigate the efficacy of Nafamostat Mesilate (NM), a protease inhibitor and potent complement inhibitor, for improving the performance of the BAL. The experimental groups were divided as follows; the NM group (n=7) where the BAL had porcine hepatocytes with 3.8x10(-4) M, of NM, and the control group where the BAL had no NM. Plasma obtained from patients suffering from hepatic failure was perfused through the BAL for 10 hours. The viability of the porcine hepatocytes and the levels of alanine aminotransferase (ALT) in the human plasma were measured during perfusion. After the 10-hour perfusion, another human hepatic failure plasma was perfused for an additional 1 hour and then the function of the BAL was evaluated. After the 10-hour perfusion, the viability of the hepatocytes in the NM group was 51 +/- 7%, whereas that in the control group was rapidly reduced by 35 +/- 5%. Although the levels of ALT in the human plasma in both groups increased with the perfusion time, those in the NM group were significantly lower than those in the control group (p < 0.05). These results suggest that NM prevented damage to the porcine hepatocytes in human hepatic failure plasma as compared to the control group. In the human hepatic failure plasma before perfusion, the partial thrombin time (PT) and the plasma ammonia (NH3) levels were 19.8 +/- 12% and 288 +/- 102 microg/dl, respectively. Fischer's ratios were 0.98 +/- 0.39. Even after the 10-hour perfusion, the BAL in the NM group significantly improved the levels of PT (38 +/- 10%; p < 0.05), NH3 (214 +/- 34 microg/dl; p < 0.05) and Fischer's ratios (1.4 +/- 0.3; p < 0.05). On the other hand, the BAL in the control group did not show any improvement in those parameters. In conclusion, NM was found to help in maintaining the viability of porcine hepatocytes in human hepatic failure plasma, thereby allowing the porcine hepatocyte-based BAL to function much better.

Bioartificial liver treatment in rats with fulminant hepatic failure: effect on DNA-binding activity of liver-enriched and growth-associated transcription factors.

BACKGROUND: We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobe necrosis. In FHF rats, lack of regeneration of the residual liver was associated with delayed expression of HGF and HGF receptor c-met and elevated blood HGF and TGF-beta1 levels. We then found that intrasplenic hepatocyte transplantation prolonged survival in FHF rats and triggered hepatocyte proliferation in the native liver. The latter effect was associated with accelerated expression of HGF and c-met mRNA in the liver and lowering of blood HGF and TGF-beta1 levels. In the present study we show that in FHF rats, treatment with a bioartificial liver (BAL) had similar effects. MATERIALS AND METHODS: FHF was induced in inbred Lewis rats and after 4 h, Group 1 rats were subjected to a 4-h whole blood perfusion through the BAL loaded with 3 x 10(8) microcarrier-attached syngeneic hepatocytes, whereas Group 2 control rats were treated with the BAL containing microcarriers only. RESULTS: Compared to sham-BAL-treated rats, the test rats lived longer (28 +/- 5 vs 17 +/- 2 h; P = 0.0005), had better coagulation parameters, maintained higher body core temperature, and showed decreased plasma TGF-beta1 levels. In addition, their liver remnants were HGF positive and showed increased DNA binding of transcription factors engaged in the modulation of hepatocyte proliferation (e.g., STAT3) and liver-specific gene expression (e.g., HNF1, HNF4, C/EBP). CONCLUSIONS: This study demonstrates that hepatocyte-based extracorporeal support not only can provide metabolic support by increasing the available functional liver mass but also is capable of modifying humoral and molecular mechanisms which are responsible for proliferation and organ-specific functions of residual hepatocytes.

Utility of technetium-99m-labeled-galactosyl human serum albumin scintigraphy for estimating the hepatic functional reserve.

Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (Tc-GSA) is a receptor binding agent, specific for asialoglycoprotein receptor, that resides exclusively on the plasma membrane of mammalian hepatocytes. The usefulness of Tc-GSA for estimating the hepatic functional reserve was retrospectively evaluated in patients undergoing a hepatic resection. Tc-GSA scintigraphy was performed in 35 patients before hepatectomy, and the hepatic uptake ratio (LHL15) was calculated. The LHL15 was then compared with the findings of conventional liver function tests, the indocyanine green retention rate in 15 minutes (ICG R15), and histologic activity index (HAI) score. Significant correlations were observed between the LHL15 and values of ICG R15, prothrombin time activity, serum levels of total bilirubin, hyaluronic acid, and values of HAI score. Ratios of LHL15 to preoperative liver volume (LHL-V) correlated well with the regenerative rates of the residual liver after major hepatectomy. In addition, patients with more than 0.76 of LHL-V value had no complications in postoperative course, whereas those with less than 0.73 had several complications due to hepatic dysfunction. Tc-GSA scintigraphy thus appears to be a useful diagnostic tool for evaluating functioning mass of the liver and the values of LHL-V seems to be able to demonstrate regenerative activity in the residual liver after hepatectomy.