Bilirubin, a physiological antioxidant, can improve cryopreservation of human hepatocytes.

The availability of cryopreserved hepatocytes is required for a more widespread use of hepatocyte transplantation, but human hepatocytes are easily damaged during freezing-thawing. Here, preincubation with unconjugated bilirubin, a physiological antioxidant, resulted in increased viability and function of hepatocytes (as determined by trypan blue exclusion, mitochondrial succinate dehydrogenases activity, urea synthesis, and cytochrome P450 1A/2) compared with cells incubated without the pigment. These findings suggest that unconjugated bilirubin may be used as cryoprotectant in clinical hepatocyte transplantation.

Hepatocyte transplantation followed by auxiliary liver transplantation--a novel treatment for ornithine transcarbamylase deficiency.

We report the first successful use of hepatocyte transplantation as a bridge to subsequent auxiliary partial orthotopic liver transplantation (APOLT) in a child antenatally diagnosed with severe ornithine transcarbamylase (OTC) deficiency. A total of 1.74 x 10(9) fresh and cryopreserved hepatocytes were administered intraportally into the liver over a period of 6 months. Immunosuppression was with tacrolimus and prednisolone. A sustained decrease in ammonia levels and a gradual increase in serum urea were observed except during episodes of sepsis in the first 6 months of life. The patient was able to tolerate a normal protein intake and presented a normal growth and neurological development. APOLT was successfully performed at 7 months of age. We conclude that hepatocyte transplantation can be used in conjunction with APOLT as an effective treatment for severe OTC-deficient patients, improving neurodevelopmental outcomes.

Effects of serum from patients with acute liver failure due to paracetamol overdose on human hepatocytes in vitro.

OBJECTIVE: Our goal was to investigate the effects of serum from patients with acute liver failure due to paracetamol (acetaminophen) overdose on the function of human hepatocytes in vitro. METHODS: Freshly isolated human hepatocytes plated on collagen-coated culture plates were, incubated (24 hours 37 degrees C) in medium containing pooled human sera (0%-80%) obtained from normal individuals or from patients with acute liver failure due to paracetamol overdose. The effects of the sera on cell function were assessed using MTT, [14C]-leucine incorporation, and cytochrome P450 (CYP1A1/2) activity assays. RESULTS: The overall cellular metabolic activity was significantly greater at all concentrations after exposure to acute liver failure serum compared to normal serum. There were no significant differences in the decreases produced by pooled acute liver failure and normal sera at concentrations up to 80% on the [14C]-leucine incorporation or CYP1A1/2 activity. CONCLUSION: The overall cell function/activity of human hepatocytes was not impaired in vitro on exposure to serum from patients with acute liver failure due to paracetamol overdose.

Hepatocyte transplantation for metabolic disorders, experience at King's College hospital and review of literature.

Hepatocyte transplantation is emerging as a potential treatment for liver based metabolic disorders and acute liver failure. To date, clinical studies have shown that hepatocyte transplantation could be used as a "bridge" to liver transplantation in a few patients with acute liver failure and has changed the phenotype of metabolic patients in terms of reducing the severity of illness. For many years, research studies have been carried out to optimise conditions for a) hepatocyte isolation in order to obtain the highest possible number of viable hepatocytes isolated from various sources of unused donor liver tissues, and b) cryopreservation and storage of hepatocytes for immediate cell availability in emergency cases. In this review, we summarise the worldwide clinical experience in hepatocyte transplantation for liver based metabolic disorders including the experience of our centre at King's College Hospital, London (United Kingdom). We briefly comment on the possible future developments and improvements needed in this field.

Review of methods to remove protein-bound substances in liver failure.

A wide range of toxic substances accumulates in the circulation of patients with liver failure, including more lipid-soluble substances, which bind to plasma proteins. Serum albumin is the most important binding protein for ligands such as bilirubin and bile acids, which are potentially toxic and can cause apoptosis in astrocytes and hepatocytes respectively in vitro. Resin haemoperfusion was originally investigated to remove these compounds, as well as inflammatory cytokines. Current effective methods for removal of protein-bound compounds in patients with liver failure include high volume plasmapheresis and different forms of albumin dialysis. Bioartificial liver support systems need adsorbent and/or dialysis modules to replace the lack of excretory function.

Transforming growth factor-beta 1, activin and follistatin in patients with hepatocellular carcinoma and patients with alcoholic cirrhosis.

BACKGROUND: Transforming growth factor-beta 1 (TGF-beta1) exerts an inhibitory effect on DNA synthesis in hepatocytes. Activin, through different mechanisms, also exhibits an apoptotic effect on hepatocytes. Follistatin antagonizes the actions of activin. METHODS: Patients with hepatocellular carcinoma (HCC, n = 20), patients with alcoholic cirrhosis (n = 12), patients with cirrhosis due to other causes (n = 5) and normal controls (n = 19) were studied. TGF-beta1, activin and follistatin concentrations in blood and ascites were measured by ELISA. RESULTS: All three groups of patients had significantly higher serum levels of total TGF-beta1, activin and follistatin compared to those of controls. In patients with HCC, the total TGF-beta1 level correlated negatively with tumour size (r = -0.644, P = 0.001). The activin level correlated with alkaline phosphatase (ALP) level (r = 0.374, P = 0.046). The follistatin level correlated with the ALP level (r = 0.404, P = 0.026), and the glutamyl transpeptidase level (r = 0.457, P = 0.01). In patients with alcoholic cirrhosis, serum activin correlated with the Child-Pugh score (r = 0.601, P = 0.01). The levels of the cytokines in ascites (n = 16) did not correlate with the corresponding levels in serum. CONCLUSIONS: Serum levels of total TGF-beta1, activin and follistatin were elevated in patients with hepatocellular carcinoma and in patients with alcoholic cirrhosis. Apoptosis of tumour cells may be reduced by a subsequent decrease in serum TGF-beta1 levels when the tumours expand in size. Activin and follistatin were associated with tumour activity, as both correlated with ALP and/or GGT levels. Further studies are required to define the exact relationships between these cytokines, the dynamics of tumour growth and their significance in cirrhosis.

In vitro studies on the immunomodulatory effects of extracts of Osbeckia aspera.

Ayruvedic medical practitioners in Sri Lanka use aqueous extracts of the mature leaves of Osbeckia aspera to treat liver disease. The extract has been shown to have hepatoprotective effects in vitro and in vivo, and to have inhibitory effects on the complement system and on in vitro phagocytosis by polymorphonuclear cells. The aim of this study was to investigate the effect of an aqueous extract of Osbeckia on lymphocyte proliferation stimulated by mitogens and antigen. In control peripheral blood mononuclear cells (PBMC), high concentrations of the Osbeckia extract were inhibitory to proliferation stimulated by phytohaemagglutinin (PHA) and tuberculin purified protein derivative (PPD). On stimulation by phorbol myristate acetate and ionomycin (PMA+I) the extract showed stimulation of proliferation at low concentrations (<10 microg/ml) with inhibition at higher concentrations. A similar inhibitory pattern on mitogen/antigen stimulation was seen with PBMC from patients with chronic hepatitis C virus (HCV) infection. These results suggest that the inhibitory agent(s) in the aqueous extract of Osbeckia may have an effect on antigen-presenting cell function. The combined hepatoprotective and immunosuppressive effects of the extract are more likely to be beneficial in acute hepatitis rather than chronic hepatitis viral infection.

Expression of Fas antigen (CD95) in peripheral blood lymphocytes and in liver-infiltrating, cytotoxic lymphocytes in patients with hepatocellular carcinoma.

BACKGROUND: Fas-expressing cytotoxic T lymphocytes (CTLs) are important antitumor immune effector cells in patients with hepatocellular carcinoma (HCC). The role of transforming growth factor beta 1 (TGF-beta1) in modulating the expression of Fas by CTLs is not known in HCC. The objectives of this study were to characterize the expression of Fas by CTLs and natural killer (NK) cells among peripheral blood lymphocytes (PBLs) and tumor-infiltrating lymphocytes (TILs) in patients with HCC and to correlate the association, if any, with serum TGF-beta1 levels. METHODS: PBLs from 18 patients with HCC and TILs from 5 HCC liver specimens were isolated, and Fas expression was analyzed by three-color flow cytometry. The results were compared with results from normal control volunteers (n = 19 individuals). Serum TGF-beta1 levels in patients with HCC were measured by enzyme-linked immunosorbent assay. RESULTS: The median percentage of Fas expression by CD3 positive T cells was significantly higher in patients with HCC compared with normal controls (54.37% vs. 32.03%, respectively; P = 0.0036), and this was attributable solely to Fas expression by CD4 positive PBLs (54.46% vs. 34.90%, respectively; P = 0.0234). In contrast, Fas expression was significantly higher in all the subtypes of TILs (CD3 positive, CD4 positive, CD8 positive, NK cells, and natural T cells) compared with controls (all P values were < 0.001). Tumor size was inversely proportional to the TGF-beta1 levels (correlation coefficient [r] = -0.725; P < 0.0001), which were correlated inversely with Fas expression by CD4 positive PBLs (r = -0.516; P = 0.01). CONCLUSIONS: In patients with HCC, TILs exhibit significantly increased expression of Fas compared with PBLs that may enhance their susceptibility to apoptotic mechanisms. Larger tumors were associated with lower serum TGFbeta1 levels, and this was correlated with greater Fas expression by CD4 positive PBLs.

Assay to detect inhibitory substances in serum of patients with acute liver failure.

Patients with acute liver failure accumulate toxic substances in the circulation which may impair recovery of hepatic function. The aim of this study was to test an in vitro assay to detect inhibitory substances in the serum of patients with acute liver failure. Human liver-derived HepG2 cells were incubated for 24h in 96 well plates (30,000 cells/well) with sera (10%) from 24 patients with acute liver failure due to paracetamol overdose or NANB hepatitis and 11 normal controls. DNA synthesis was determined from the incorporation of 3H-thymidine and cell viability by the metabolism of the tetrazolium dye MTS. HepG2 cells exposed to acute liver failure sera incorporated significantly less 3H-thymidine (median 30% of control, range 0.2-169%) than normal sera (100%, 76-133%, p=0.002). Cell viability was also reduced (75%, 33-112% vs 100%, 96-105%, p<0.001). There was no correlation between these values and patient outcome or levels of plasma TNF-alpha or serum interferon-gamma. The assay detected inhibitory substances in sera of patients with acute liver failure and could be used to monitor the use of liver support systems.

Temporary extracorporeal liver support for severe acute alcoholic hepatitis using the BioLogic-DT.

Patients with severe acute alcoholic hepatitis develop multiple organ failure which is associated with production of inflammatory cytokines and a poor prognosis. The aim of the present pilot study was to evaluate the effects of the BioLogic-DT sorption-suspension dialyser in patients with severe acute alcoholic hepatitis. Ten patients with encephalopathy (grade II-IV) were entered into the study, 5 received treatment with the BioLogic-DT for 6 hours daily for 3 days and 5 received conventional treatment as controls. The system was biocompatible with no adverse effects on blood pressure or platelet counts, factor V, fibrinogen or antithrombin III. No bleeding episodes were observed even with the use of small doses of heparin. After 3 days, blood ammonia was lower in the BioLogic-DT treated patients than in the controls, although blood lactate was higher. There were slight increases in plasma TNF and IL-8 during treatment over and above the higher levels present initially, possibly as a result of activation of white cells in the extracorporeal circuit. The further development of the BioLogic-DT dialyser with the addition of a plasma treatment module capable of removing cytokines would be worth evaluating in acute alcoholic hepatitis.

Plasma cytokine levels and coagulation and complement activation during use of the extracorporeal liver assist device in acute liver failure.

Multiple organ failure frequently occurs in patients with acute liver failure, and this has been associated with increased cytokine production. Treatment by hemoperfusion with an extracorporeal liver assist device (ELAD) containing human liver-derived cells was performed in 12 patients with acute liver failure. Over the first 6 h, there were significant increases in plasma tumor necrosis factor alpha (TNFalpha; from 114+/-54 pg/ml [mean+/-SEM] to 236+/-161 pg/ml, p < 0.05) and interleukin (IL)-6 (260+/-121 pg/ml to 445+/-149 pg/ml, p < 0.05) but not in interferon gamma (IFNgamma). A similar pattern with a small peak increase was observed for complement C5b-9 complex. Plasma C-reactive protein (CRP) and thrombin antithrombin (TAT) III complex showed small peaks after 24 h of ELAD hemoperfusion. No such changes were seen in 12 control patients with acute liver failure who were treated with intensive care alone. These transitory effects, without changes in blood pressure, are likely to be due to the contact of the blood with the dialyzer membrane. There was no evidence of the clearance of cytokines by the ELAD.

Screening of hepatoprotective plant components using a HepG2 cell cytotoxicity assay.

Identification of the active components of plants with hepatoprotective properties requires screening of large numbers of samples during fractionation and purification. A screening assay has been developed based on protection of human liver-derived HepG2 cells against toxic damage. Various hepatotoxins were incubated with HepG2 cells in 96-well microtitre plates (30,000 cells well-1) for 1 h and viability was determined by metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS). Bromobenzene (10 mM) and 2,6-dimethyl-N-acetyl-p-quinoneimine (2,6-diMeNAPQI, 200 mM) had greater toxic effects than tert-butyl hydroperoxide (1.8 mM) or galactosamine (10 mM), reducing mean viability to 44.6 +/- 1.2% (s.e.m.) and 56.1 +/- 2.1% of control, respectively. Protection against toxic damage by these agents was tested using a crude extract of a known hepatoprotective Sri Lankan plant, Osbeckia aspera, and two pure established hepatoprotective plant compounds, (+)-catechin and silymarin (1 mg mL-1). Viability was significantly improved by Osbeckia (by 37.7 +/- 2.4%, P < 0.05, and 36.5 +/- 2.1%, P < 0.05, for bromobenzene and 2,6-diMeNAPQI toxicity, respectively). Comparable values for (+)-catechin were 68.6 +/- 2.9% and 63.5 +/- 1.1%, and for silymarin 24.9 +/- 1.4% and 25.0 +/- 1.6%. This rapid and reproducible assay should prove useful for the isolation and identification of active hepatoprotective compounds in crude plant extracts.

Use of extracorporeal liver assist device and auxiliary liver transplantation in fulminant hepatic failure.

The case history of a 14-year-old boy with fulminant hepatic failure secondary to non-A, non-B hepatitis who fulfilled selection criteria for orthotopic liver transplantation is described. Two forms of liver support were used (extracorporeal liver assist device and an auxiliary partial orthotopic liver transplantation) to provide additional time to allow spontaneous recovery to occur. During the 66 h of extracorporeal haemoperfusion through the device, haemodynamic stability was maintained along with improvements in serum bilirubin (555 to 381 mumol/l), and international normalized ratio (INR) (3.7 to 2.9). Deterioration in these parameters was observed following cessation of treatment and 10 h later, after a donor liver had become available, an auxiliary transplant was performed. Clinical recovery, though initially slow, was eventually complete, with histopathological and scintigraphic evidence of full liver regeneration at 3 months. Withdrawal of his immunosuppressive drugs began at 6 months and was complete by 14 months after auxiliary transplantation. He has since remained well with normal liver function tests. Temporary liver support may provide additional time for spontaneous recovery of the native liver to occur in selected cases of fulminant hepatic failure, even when criteria are fulfilled for orthotopic liver grafting.

Systemic and hepatic hemodynamic changes in acute liver injury.

Systemic and hepatic circulatory changes were studied in rats over the course of acute liver injury. Hepatic injury was induced by intraperitoneal injection of D-galactosamine (1.1 g/kg), and systemic and hepatic hemodynamics were measured over a 72-h period using a radioactive microsphere technique with direct measurement of arterial, portal venous, and hepatic venous blood oxygen content. Cardiac output increased to a maximum at 48 h, producing a marked increase (450%) in hepatic arterial blood flow so that it became the dominant supply of oxygen at the time of maximal hepatic injury. A subsequent increase in portal venous flow resulted in an overall increase in total hepatic blood flow of 500%. At this point the oxygen delivery by the hepatic arterial and portal venous systems was equal. These circulatory changes returned to control values by 72 h with recovery of liver function. These results demonstrate the development of a hyperdynamic circulation and a marked change in the normal relationship between portal venous and hepatic arterial blood flows that occur during hepatic injury.

Regional cerebral edema and chloride space in galactosamine-induced liver failure in rats.

The pathogenesis of cerebral edema, which is a major complication of fulminant hepatic failure, is poorly understood. In previous studies, increased regional brain water content was observed in rats at an early stage of acute liver failure caused by galactosamine. At a later stage when the animals had developed deep coma, brain water content was reduced, possibly as a result of generalized dehydration. In the present investigation, we have determined brain water content at a late stage of liver failure, 48 hours after galactosamine, in animals that had been maintained in fluid balance by continuous intraperitoneal infusion of glucose solution. In these animals, brain water content, determined from the ratio of wet to dry weight, showed a greater increase than that observed previously at the early stage (hindbrain region [cerebellum, pons, and brain stem] increased by 4.2%; forebrain region increased by 1.4% compared with controls). Regional analysis of brain water, using a tissue-specific gravity method, showed a significant increase in cerebellar gray matter water content. Analysis of chloride space showed the extra fluid to be mainly extracellular in the hindbrain region, but not in the forebrain region. Ultrastructural examination of capillaries in gray matter from cerebellum and cerebrum showed no evidence of gross disruption of the tight junctions. Swelling of the astroglial foot processes was observed in the cerebellar gray matter. These results suggest that both vasogenic and cytotoxic mechanisms of edema formation occur in the brain during liver failure.

Plasma levels and hepatic mRNA expression of transforming growth factor-beta1 in patients with fulminant hepatic failure.

BACKGROUND/AIMS: Transforming growth factor-beta1 is an important cytokine involved in cell growth and inflammation which has been shown to be inhibitory to hepatic DNA synthesis. The aim of this study was to investigate the plasma levels and hepatic mRNA expression of transforming growth factor-beta1 in patients with fulminant hepatic failure in whom liver regeneration may be impaired. METHODS: Plasma levels of transforming growth factor-beta1 and human hepatocyte growth factor were measured in 57 fulminant hepatic failure patients and 20 healthy volunteers by ELISA. Northern blot analysis of transforming growth factor-beta1 and H3 histone, a marker for liver proliferation, was performed in liver tissue of 14 fulminant hepatic failure patients. RESULTS: The plasma levels of total transforming growth factor-beta1 in fulminant hepatic failure patients on admission (median 38.8 ng/ml, range 8.4-108 ng/ml) were significantly higher than those in control subjects (23.0 ng/ml, 8.5-34.9 ng/ml, p<0.001). Significantly higher levels were observed in non-A, non-B hepatitis patients (57.9 ng/ml, 38.8-108 ng/ml, n=10, p<0.001) compared to patients with paracetamol overdose (37.1 ng/ml, 8.4-72.5 ng/ml, n=47). In contrast, the plasma levels of free transforming growth factor beta1 were greater in paracetamol overdose (623 pg/ml, 46.7-1241 pg/ml, n=21) than in non-A, non-B hepatitis (131 pg/ml, 77.2-254 pg/ml, n=9), with both being higher than control (72.3 pg/ml, 28.7-108, n=7, p<0.001). The plasma levels of human hepatocyte growth factor in patients with paracetamol overdose (7.04 ng/ml, 1.00-62.4 ng/ml) were significantly higher than those in patients with non-A, non-B hepatitis (4.48 ng/ml, 0.74-9.10 ng/ml, p<0.05). Northern blots showed increased mRNA expression of transforming growth factor-beta1 in paracetamol-overdose patients (n=8, p<0.05), but not in patients with non-A non-B hepatitis (n=6), compared to controls (n=4). CONCLUSIONS: The increased circulating plasma TGF-beta1 in FHF may be part of the tissue repair process in fulminant hepatic failure. In patients with non-A, non-B hepatitis, the increased total transforming growth factor-beta1 together with a less elevated hepatocyte growth factor could be related to impaired liver regeneration in this group.

Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure.

The objective of this pilot controlled study was to evaluate the extracorporeal liver assist device (ELAD) in patients with acute liver failure who were judged to still have a significant chance of survival (approximately 50%) and in those who had already fulfilled criteria for transplantation. Twenty-four patients were divided into two groups, 17 with a potentially recoverable lesion (group I) and 7 listed for transplantation (group II), and then randomly allocated to ELAD haemoperfusion or control. The median period of ELAD haemoperfusion was 72 hours (range 3-168 h). Biocompatibility of the device was good, with no acceleration in platelet consumption, and haemodynamic stability was maintained. Two patients were withdrawn from the study because of worsening of preexisting disseminated intravascular coagulation in one case and a hypersensitivity reaction in the other. Deterioration with respect to encephalopathy grade was more frequent in the control patients, 7 of 12 (58%), than in the ELAD-treated patients, 3 of 12 (25%). In group I where survival for the ELAD cases was 7 of 9 (78%), there was a higher than expected survival in the controls, 6 of 8 (75%). For group II cases, survival was 1 of 3 (33%) for the ELAD-treated patients, and 1 of 4 (25%) for the controls. Both of the survivors underwent transplantation. Assessment of additive function for the device revealed an improvement in galactose elimination capacity after 6 hours of haemoperfusion. Based on the results of this pilot-controlled trial, better indices of prognosis will be required, in addition to those used to select for transplantation, if patients at an earlier stage of clinical deterioration are to be included in future studies.