Fulminant Hepatic Failure Patients Research Articles

High prevalence of potentially hepatotoxic herbal supplement use in patients with fulminant hepatic failure

The use of potentially hepatotoxic herbal and dietary supplements is highly prevalent in the fulminant hepatic failure (FHF) patient population at our institution, and this subgroup of patients has a worse prognosis. Retrospective case series. Settings An adult tertiary care university hospital and a Veterans Affairs hospital in Oregon. All patients referred to the liver transplantation service for FHF from January 2001 through October 2002 (N = 20). We defined FHF as onset of encephalopathy within 8 weeks of onset of jaundice in the absence of preexisting liver disease. All patients underwent investigation for potential causes of liver injury. Potentially hepatotoxic supplements were defined as those with previously published reports of hepatic injury related to their use. Ten patients (50%) were recent or active users of potentially hepatotoxic supplements or herbs; 10 had no history of supplement use. In the supplement group, 7 patients (35%) had no other identified cause for hepatic failure. Six patients in the supplement group and 2 patients in the nonsupplement group underwent orthotopic liver transplantation. Five patients in each group died. There were no significant differences in transplantation rate (P =.07) or survival (P>.99) between groups. Supplement use alone accounted for the most cases of FHF during this period, exceeding acetaminophen toxicity and viral hepatitis. Herbal and dietary supplements were potential hepatotoxins in a high proportion of patients with FHF at our institution. Enhanced public awareness of the potential hepatotoxicity of these commonly used agents and increased regulatory oversight of their use is strongly urged.

Transcranial Doppler sonography in fulminant hepatic failure

The clinical course of patients with fulminant hepatic failure (FHF) is often worsened by the presence of cerebral edema and endocranial hypertension. In spite of the multiple studies using Transcranial Doppler Sonography (TCDS), few have shown the cerebral blood flow (CBF) pattern among patients with encephalopathy resulting from FHF. Objective Our objective was to characterize the CBF pattern in these patients through the use of TCDS to provide therapeutic strategies. Method The TCDS pattern was assessed in five patients diagnosed with FHF and compared with a control group who displayed critical neurologic conditions not associated with FHF. Pulsatile index, systolic, diastolic, and mean velocity of the middle cerebral artery were measured. Results The mean age of patients with FHF was 45.4 years. One hundred percent were women, with viral hepatitis as the predominant etiology. A cerebral hypoperfusion pattern was found in 80% of the FHF group and 40% of the control group. In the former group there was no evidence of hyperemia, as there was among 20% of the control group. The mean values of velocity and pulsatile index were 36.6 cm/sec and 2.4, respectively, in the FHF group and 47.8 cm/s and 1.8 in the control group ( P = 0.268, P = 0.402). Conclusions FHF patients show a predominance of cerebral hypoperfusion pattern with mean velocities lower than normal values and an increased pulsatile index. We recommend that clinicians take appropriate measures to improve cerebral perfusion and avoid hypoxia. Hyperventilation as a first level measure is contraindicated.

Lens culinaris agglutinin-A-reactive alpha-fetoprotein as a marker for liver atrophy in fulminant hepatic failure

Liver atrophy is frequently found at autopsy of patients with fulminant hepatic failure (FHF). Imaging studies in patients with FHF demonstrate that estimation of total liver volume correlates with prognosis. In this study, in order to determine serum markers for evaluating liver atrophy, we measured the weight of whole livers resected from 31 transplant recipients with FHF, and assessed the relation between the liver weights and several prognostic markers. The level of liver atrophy was evaluated by calculating the ratio of the removed whole liver weight to the body weight of each recipient, and 16 major variables including several serological markers were analyzed among those recipients. We found that the serum Lens culinaris agglutinin-A-reactive alpha-fetoprotein (AFP-L3) levels were significantly higher in patients with FHF than in those with other liver diseases. In FHF patients, the serum AFP-L3 level at the onset of encephalopathy was significantly higher in cases with mild atrophy than in those with severe atrophy ( P<0.05). Notably, the residual liver volume was significantly correlated with the serum AFP-L3 level (Pearson's correlation coefficient=0.63), but not with AFP. In conclusion, AFP-L3 is a possible serum marker for evaluating liver atrophy and/or liver regeneration in patients with FHF.

Early up-regulation of chemokine expression in fulminant hepatic failure.

CC-chemokines recruit and activate macrophages and T lymphocytes, the major components of inflammatory infiltrates in fulminant hepatic failure (FHF). To analyse the role of CC-chemokines in the pathogenesis of FHF, this study examined serum levels and intrahepatic expression of MCP-1, MIP-1alpha, MIP-1beta, and RANTES in the livers and sera of patients with FHF and controls by ELISA, immunohistochemistry, and competitive RT-PCR. Serum levels and intrahepatic expression of all chemokines studied in FHF exceeded the levels in chronic liver diseases and normal controls. Distinct patterns of expression of each chemokine were noted on Kupffer cells, sinusoidal endothelial cells, hepatocytes, lymphocytes, and bile ducts. Intrahepatic chemokine expression correlated closely with the extent of infiltration by macrophages and T lymphocytes (r = 0.65-0.95, p < 0.001). The functional relationship between intrahepatic chemokine release and infiltration was confirmed in chemotaxis assays by inhibiting chemotaxis induced by homogenates of liver tissue obtained from FHF patients with neutralizing MCP-1, MIP-1alpha, MIP-1beta, and RANTES antibodies. The time course of CC-chemokine release was studied in the concanavalin A and the galactosamine/LPS mouse models of FHF. In both models, intrahepatic chemokine up-regulation occurred as an early event prior to hepatic infiltration and liver damage. The data indicate that an abundant intrahepatic release of CC-chemokines is an early and pivotal step in the pathogenesis of FHF.

Hepatitis B virus genotypes and serotypes in western India: lack of clinical significance.

To determine hepatitis B virus genotype and subtype distribution among HBV infected individuals with different clinical manifestations in western India, serum samples from 19 asymptomatic hepatitis B surface antigen carriers, 30 chronic hepatitis B patients, 8 acute hepatitis B patients, 5 fulminant hepatitis B patients, and with circulating HBV DNA were genotyped and subtyped on the basis of the nucleotide sequence analysis of S region of the HBV genome. Genotype D was the predominant genotype circulating in western India (57/62; 91.93%). All 19 asymptomatic hepatitis B surface antigen carriers, 8 acute hepatitis B patients, 5 fulminant hepatic failure patients and 25/30 chronic hepatitis B patients were circulating genotype D and ayw3/ayw2 subtypes. HBV genotype A was prevalent in 8% (5/62) of the total number of patients and all belonged to chronic hepatitis B category. Subtyping analysis showed that all genotype A isolates were of subtype adw2. As most of the patients from different clinical categories were infected with HBV genotype D, it is concluded that this genotype did not influence the outcome of HBV infection.

High Metabolic Function of Primary Human and Porcine Hepatocytes in a Polyurethane Foam/Spheroid Culture System in Plasma from Patients with Fulminant Hepatic Failure

It has been demonstrated that plasma from patients with fulminant hepatic failure (FHF) interferes extensively with cellular function. We placed primary human and primary porcine hepatocytes in a polyurethane foam (PUF)/spheroid culture system and compared the metabolic functions in the plasma of patients with FHF in a 24-h stationary culture to those in a monolayer culture. The PUF/spheroid culture system using primary human and primary porcine hepatocytes significantly decreased ammonia content during 28-day culture. Fisher's ratio significantly increased at culture days 3 and 7. Tauroursodeoxycholic acid significantly increased and glycochenodeoxycholic acid and taurochenodeoxycholic acid decreased in the FHF patients' plasma at culture day 3. During at least a 24-h culture in the FHF patients' plasma, metabolic functions of primary human and primary porcine hepatocytes were almost identical. The present results indicate that the PUF/spheroid culture system using primary human or primary porcine hepatocytes demonstrated more advantageous metabolic functions in the plasma from patients with FHF than the monolayer culture.

Single vs Dual Vessel Porcine Extracorporeal Liver Perfusion

Background. The use of porcine extracorporeal liver perfusion (PECLP) to provide temporary hepatic support for patients in fulminant hepatic failure has been limited by the fact that individual perfusions can be sustained for only a few hours. Inadequate liver function and/or hemodynamic instability are the major contributing factors for early interruption of PECLP. Recent reports suggest that the choice of single (portal vein only) vs dual (portal vein and hepatic artery) vessel perfusion may influence the duration of perfusion. We hypothesize that PECLP with single vessel perfusion (SVP) is associated with worse liver function and greater hemodynamic instability than PECLP with dual vessel perfusion (DVP).Materials and methods. To eliminate the potentially confounding influences of liver failure and xenograft rejection, liver isografts procured from White-Landrace pig donors were perfused by either SVP or DVP via an extracorporeal circuit established with normal White-Landrace pig recipients. The function of perfused livers was evaluated by measuring production of bile and Factors V and VIII, clearance of ammonia and lactate, and extraction of O2 at baseline and at 0, 1, 3, 6, 12, and 24 h after initiation of PECLP. The impact of PECLP on recipient hemodynamic status was assessed by monitoring BP, heart rate, urine output, O2 saturation, etc. Among other parameters evaluated were serum albumin and total protein and hepatic release of IL-1β and nitric oxide to assess their possible contributions to hemodynamic instability.Results. DVP and SVP livers cleared ammonia and lactate similarly. Both approaches were associated with progressive hypoalbuminemia and hypoproteinemia. DVP livers produced more bile and Factor V and were associated with less recipient hypotension and IL-1β and NO release than SVP livers.Conclusions. Livers with DVP function better than livers with SVP. The duration of PECLP can be limited by recipient hypotension, although this complication is less severe with DVP than with SVP.

Prolonged unilateral vasodilatation and brain edema in fulminant hepatic failure, associated with symptomatic seizure

We report prolonged unilateral vasodilatation and hemispheric brain edema in a 49-year-old man with fulminant hepatic failure (FHF). The patient presented with a tonic–clonic seizure caused by a hypertensive subcortical hemorrhage in the left parietal lobe. Serial computed tomography (CT) scans showed progressive darkening of the ipsilateral hemisphere, suggesting hemispheric cerebral infarction, but the patient did not show clinical signs of deterioration. Brain magnetic resonance angiography showed dilation of the large arteries of the left hemisphere. Evaluation of cerebral blood flow 7 days postictus with single photon emission CT revealed marked ipsilateral hyperperfusion. The darkening of the hemisphere was brain edema elicited by hyperperfusion. Brain edema was reversible, disappearing 14 days postictus. Hemispheric brain edema was caused by unilateral cerebral vasodilatation and resultant hyperperfusion. Although brain edema is a major complication in FHF patients and cerebral hyperperfusion is responsible for edema formation, CT findings of these patients almost invariably show a bilateral lesion. Unilateral vasodilatation and subsequent hemispheric hyperperfusion may be due to overproduction of vasodilators, already abundant in the brains of patients with severe hepatic failure, by seizure activity.

Increased turnover of Gc-globulin in patients with hepatic encephalopathy.

A low serum level (< 100 mg/L) of the actin-scavenger Gc-globulin is a prognostic marker of non-survival in fulminant hepatic failure (FHF). It is unknown whether decreased production or increased consumption (or both) is responsible for the low Gc-globulin levels. Ten patients with FHF and four patients with acute or chronic liver disease (AOCLD) with hepatic encephalopathy (HE) grades II-IV were included. Eight patients with cirrhosis (chronic liver disease, CLD) without HE served as controls. Total, free, and actin-bound Gc-globulin were measured in samples from an artery, a central vein, and a hepatic vein. In 12 patients (9 FHF, 3 AOCLD), concentrations were measured before and after high volume plasmapheresis (HVP). Total Gc-globulin was reduced to 21%, 40%, and 43% of the normal level in the FHF, AOCLD, and CLD groups, respectively, whereas bound Gc-globulin was within normal range in all patients. The Gc:actin complex ratio was increased 3.8, 2.5, and 1.9-fold compared with normal levels. Total, free, and bound serum Gc-globulin levels did not differ among arterial, systemic venous, or hepatic venous blood. Total Gc-globulin rose to >100 mg/L in all patients after HVP, whereas bound Gc-globulin remained unchanged. The Gc-globulin production rate in FHF and AOCLD patients was increased to 4.1 +/- 1.3 mg/min compared to literature values of 0.6 mg/min in healthy individuals. The estimated half-life of total Gc-globulin was shorter in the patients compared to healthy individuals (127 +/- 56 min and 870 min, respectively). Gc-globulin levels were reduced in patients with FHF and AOCLD because a 7-fold increase of Gc-globulin production rate could not compensate for the accelerated clearance. Bound Gc-globulin was maintained within normal levels in all circumstances studied, indicating a possible regulatory role of this parameter in the clearance of actin.

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.

First clinical application of a second generation HepatAssist bioartificial liver support system without charcoal associated with neurological recovery in a fulminant hepatic failure patient

First clinical application of a second generation HepatAssist bioartificial liver support system without charcoal associated with neurological recovery in a fulminant hepatic failure patient

First clinical application of a second generation HepatAssist bioartificial liver support system without charcoal associated with neurological recovery in a fulminant hepatic failure patient

First clinical application of a second generation HepatAssist bioartificial liver support system without charcoal associated with neurological recovery in a fulminant hepatic failure patient

Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure.

The aim of this study was to elucidate the role of apoptosis in human fulminant hepatic failure. We studied the expression of Fas antigen on liver tissues, Fas ligand in lymphocytes, and soluble Fas ligand in patients' serum. On finding apoptotic cells in fulminant hepatic failure liver, we first examined them using the TUNEL method. Subsequently, the expression of Fas was studied by immunostaining. Simultaneously, Fas ligand presenting on both liver-infiltrated cells and peripheral lymphocytes was studied by reverse transcription-polymerase chain reaction, and soluble Fas ligand in sera was measured by ELISA. By using the TUNEL method, we first demonstrated that many apoptotic cells existed in fulminant hepatic failure but not in normal ones. Our immunohistochemistry study showed that many hepatocytes in fulminant hepatic failure strongly expressed Fas. In addition, Fas ligand on both liver-infiltrating lymphocytes and peripheral lymphocytes in fulminant hepatic failure patients was detected. The serum level of soluble Fas ligand was significantly increased in fulminant hepatic failure (mean value, 2.91 ng/ml in fulminant hepatic failure [n = 10], 1.62 ng/ml in acute hepatitis [n = 10], and 0.27 ng/ml in healthy controls [n = 10]). Furthermore, this serum level of sFas ligand was significantly associated with prothrombin time both in acute hepatitis and fulminant hepatic failure. The present results indicate that Fas-mediated apoptosis may be one of the triggers for the induction of fulminant hepatic failure.

Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure

OBJECTIVE: The aim of this study was to elucidate the role of apoptosis in human fulminant hepatic failure. We studied the expression of Fas antigen on liver tissues, Fas ligand in lymphocytes, and soluble Fas ligand in patients’ serum. METHODS: On finding apoptotic cells in fulminant hepatic failure liver, we first examined them using the TUNEL method. Subsequently, the expression of Fas was studied by immunostaining. Simultaneously, Fas ligand presenting on both liver-infiltrated cells and peripheral lymphocytes was studied by reverse transcription-polymerase chain reaction, and soluble Fas ligand in sera was measured by ELISA. RESULTS: By using the TUNEL method, we first demonstrated that many apoptotic cells existed in fulminant hepatic failure but not in normal ones. Our immunohistochemistry study showed that many hepatocytes in fulminant hepatic failure strongly expressed Fas. In addition, Fas ligand on both liver-infiltrating lymphocytes and peripheral lymphocytes in fulminant hepatic failure patients was detected. The serum level of soluble Fas ligand was significantly increased in fulminant hepatic failure (mean value, 2.91 ng/ml in fulminant hepatic failure [n = 10], 1.62 ng/ml in acute hepatitis [n = 10], and 0.27 ng/ml in healthy controls [n = 10]). Furthermore, this serum level of sFas ligand was significantly associated with prothrombin time both in acute hepatitis and fulminant hepatic failure. CONCLUSIONS: The present results indicate that Fas-mediated apoptosis may be one of the triggers for the induction of fulminant hepatic failure.

Bioartificial Liver: Hepatic Support and Liver Regeneration

Much like renal replacement therapy in the 1950’s and 1960’s, artificial hepatic support is entering a new era. Not to be minimized is the understanding that the liver serves complicated synthetic and detoxifying functions to maintain life. The liver contains over 600 key enzymes. There are currently many research and clinical efforts at providing a clinically relevant means to replace liver function. Acute liver dysfunction carries significant morbidity and mortality. Clinical consequences include hemodynamic instability, coagulopathy, kidney failure, and cerebral edema. The liver also is an organ that contains significant regenerative properties. The exact local or neural-humoral signals to regenerate are unknown. It is assumed that regeneration is a response initially stimulated by external factors that then leads to alterations in gene expression, growth factor modulation and subsequent morphological changes. Research efforts have focused on studying up and down regulated segments of cDNA fragments in a partial rat hepatectomy model. The translated amino acid sequences showed striking homology with KIM-1, a cell adhesion molecule and HAV cr-1, a hepatitis A virus cell receptor (1). Other significant efforts have focused on Cyclin E as the molecular target for coupling ras-dependent signals to the cell cycle machinery after partial hepatectomy (2). Using a fulminant hepatic failure model in rats, researchers have showed an increased DNA synthetic activity and corresponding decreases in plasma hepatocyte growth factor beta (TGF-beta 1). Other avenues of investigation have shown that tumor necrosis factor-1 (TNF-a) and IL-6 are important components to induce regeneration pathways (3). Whatever the mechanisms identified, the possibility that artificial liver support can be used until a liver can repair or regenerate is exciting. The need for a clinically important means to support liver function is obvious. Current clinical trials have focused on bridging acutely ill patients in fulminant hepatic failure to liver transplantation. In fulminant hepatic failure, mortality can be as high as 30–70% depending on the published study. The largest clinical segment that is in need for such therapy is the acute-on-chronic disease market. Many of these patients are not candidates for organ replacement and have no current medical or surgical therapies to support them during an acute exacerbation of their disease. Given the scarcity of organ donors, current surgical efforts have focused on either split liver or living–related donor operations to address this need. There will remain a need for temporary support of the small-for-size graft. Current clinical efforts have focused on two device concepts: mechanical detoxifying systems and bioartificial systems containing liver hepatocytes. Mechanical systems, such as the BioLogic DTTM (4) use a technique of hemodiabsortion and push-pull pheresis. The MARSTM system (5) utilizes charcoal absorption and albumin to effect bound toxin removal. There are two bioartificial liver assist device systems in clinical trials in the US. The HepatAssistTM Liver Support system (6) uses plasma separation and porcine hepatocytes in a hollow fiber cartridge. A random-

LIVER DIALYSIS IN TREATMENT OF HEPATIC FAILURE AND HEPATORENAL FAILURE

The Liver Dialysis Unit is a currently marketed artificial liver that employs hemodiabsorption (dialysis of blood against powdered sorbent) to remove the numerous small molecular weight toxins of hepatic failure. A randomized, prospectively controlled study of Liver Dialysis in treatment of hepatic failure was performed in 5 centers. Entry criteria were: decrease in hepatic function associated with Stage 2–4 encephalopathy, renal failure or respiratory failure allowed, but not already on dialysis or CVVH Another 28 patients were treated in crossover studies. Most patients had stage IV encephalopathy, renal insufficiency, and respiratory insufficiency or failure. Of the 75 patients, 32 had fulminant hepatic failure (FHF), and 43 had acute-on-chronic failure. Liver Dialysis (6 hrs, 3–5 days) significantly improved neurologic (70%) and physiologic status (72%) of all patients vs. control groups (27% and 10% respectively). Liver Dialysis significantly improved patient outcome (improvement of condition for transplant or recovery of liver function) for patients with A-on-C (57%) vs. control patients (36%), though not for FHF patients. In 32 patients with hepatic and renal failure on entry, Liver Dialysis significantly improved patient outcome (41%) vs. control patients (0%). Since market introduction, more than 50 patients, mostly A-on-C have been treated in many hospitals for hepatic failure, with 58% positive outcome, confirming the above studies. The suspension is easily modified by adding dry powdered chemicals to the sorbent bag. Branched chain amino acids added to the sorbent result in a greater increase in Fischer's ratio during treatment, and new ammonium sorbents increase the removal of ammonium. Clinical effects of Liver Dialysis in treatment of hepatic failure may be further hastened and augmented by these improvements.

A biodialysis system for liver support tested in a porcine hepatic failure model.

A practical liver support system for patients in fulminant hepatic failure (FHF) remains a needed therapeutic modality. A new method of bioartificial liver support, the liver biodialysis system (LBDS), is described. Porcine hepatocytes, removed from direct contact with the treated subject's circulation, are in culture in a bioreactor which is combined in a dialysis circuit for patient treatment. The LBDS was tested in a porcine ischaemic hepatic failure model. The viable hepatocyte content of the bioreactor was 2.49 +/- 0.72 x 10(10). Cells remained viable in culture throughout the experiments (30 +/- 3 h) without evidence of immunological damage. A decrease in the degree of accumulation in the blood of ammonia (P < 0.02) and of 14 amino acids (P < 0.001) was achieved by the LBDS. Cerebral perfusion pressure was maintained at significantly higher levels in LBDS-treated animals (P < 0.05). In the LBDS, hepatocytes in large numbers and satisfactory culture conditions in a bioreactor have sustained viability and function. When combined in a dialysis circuit for the treatment of FHF pigs, immune reactions between the blood and hepatocytes were prevented and beneficial metabolic effects were observed.

Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes.

A porcine endogenous retrovirus (PERV) has been shown to infect human embryonic kidney 293 (HEK293) cells in vitro. The PERV proviral sequence exists in the genome of all porcine cells, including hepatocytes used in a bioartificial liver (BAL). We examined the possibility of PERV infection in HEK293 cells during exposure to supernatant from cultured pig hepatocytes. Pig hepatocytes were cultured in media supplemented with serum from patients in fulminant hepatic failure (FHF) to simulate conditions of an extracorporeal BAL. Pig hepatocytes were cultured in serum-free media for 24 hours and then exposed to fresh medium containing serum from a patient with FHF (22 patients tested). Twenty-four hours later, supernatant was collected and analyzed by polymerase chain reaction (PCR), with and without reverse transcriptase. Primers targeting the pol gene of PERV were used for PCR. Products of amplification were detected by an enzyme-linked immunosorbent assay-based technique using an internal capture probe also targeting the pol gene. Levels of PERV sequences were estimated by serial dilution. All positive samples were tested for infectivity in HEK293 cells. Porcine kidney 15 cell supernatant and fresh culture media were studied as positive and negative controls, respectively. Pig hepatocytes were also studied in the absence of FHF sera and in the presence of mitogenic stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate-13-acetate (PMA). PERV DNA and PERV RNA were detected in all supernatants of cultured pig hepatocytes. The level of PERV RNA in the supernatant of pig hepatocytes was not altered by exposure to human FHF serum or stimulation with PHA and PMA. In addition, PERV RNA was undetectable in the supernatant of HEK293 cells for up to 50 days after exposure to pig hepatocyte supernatant (with or without FHF sera). These findings show that production of PERV by cultured pig hepatocytes was unaffected by exposure to growth factors and cytokines present in human FHF sera.

Bile acids in xenogeneic ex-vivo liver perfusion: function of xenoperfused livers and compatibility with human bile salts and porcine livers.

In recent years, hepatic support systems using xenogeneic cells have been developed to support patients in fulminant hepatic failure. The extent to which xenogeneic hepatocytes metabolize and excrete human organic anions is unclear. In these studies we examined the ability of the ex vivo porcine liver to clear human bile acids during extracorporeal liver perfusion (ELP). Four patients with fulminant hepatic failure underwent extracorporeal liver perfusion with 9 porcine livers. The venovenous circuit was designed as previously described (NEJM,1994,331:234) as were the immunologic features (Transplantation 1994,58:1162). Bile from the porcine liver and serum samples were collected hourly during perfusion. Three bile acids (glycocholic, glycodeoxycholic, taurodeoxycholic acid) were selected as markers for human bile and three (glycohyocholic, glycohyodeoxycholic, and glyco-3alpha-hydroxy-6-oxo-5beta-cholanoic acid) for markers of pig bile. Bile acids from both serum and bile were processed and analyzed through high performance liquid chromatography. The Students' t test was used for statistical analysis. The mean duration of perfusions was 4.1+/-1.5 hr. The mean total bile acid clearance from serum (243+/-44 micromol/h) was similar to the total bile acid biliary excretion (286+/-84 micromol/hr, P = 0.06). After 1 hr of perfusion, bile samples demonstrated a predominance of pig bile salts (65%). After 3 hr of perfusion, human bile acids made up 85% of total biliary bile acids. Pig bile acids appeared in patients' sera after 1 hr of perfusion, and after 3 hr, 35% of serum bile salts were pig-specific. Porcine livers perfused with human blood can clear the serum of potentially toxic human bile acids and excrete them into bile. Simultaneously, the percentage of pig-specific bile acids in patient serum increases during xenogeneic perfusion for unknown reasons. The relative hepatic uptake of bile acid from serum is similar to bile acid excretion in bile. Further development of systems using porcine livers or hepatocytes is warranted.

Influence of human fulminant hepatic failure sera on endogenous retroviral expression in pig hepatocytes

A porcine endogenous retrovirus (PERV) has been shown to infect human embryonic kidney 293 (HEK293) cells in vitro. The DERV proviral sequence exists in the genome of all porcine cells, including hepatocytes used in a bioartificial liver (BAL). We examined the possibility of PERV infection in HEK293 cells during exposure to supernatant from cultured pig hepatocytes. Pig hepatocytes were cultured in media supplemented with serum from patients in fulminant hepatic failure (FHF) to simulate conditions of an extracorporeal BAL. Pig hepatocytes were cultured in serum-free media for 24 hours and then exposed to fresh medium containing serum from a patient with FHF (22 patients tested). Twenty-four hours later, supernatant was collected and analyzed by polymerase chain reaction (PCR), with and without reverse transcriptase. Primers targeting the pol gene of PERV were used for PCR. Products of amplification were detected by an enzyme-linked immunosorbent assay-based technique using an internal capture probe also targeting the pol gene. Levels of PERV sequences were estimated by serial dilution. All positive samples were tested for infectivity in HEK293 cells. Porcine kidney 15 cell supernatant and fresh culture media were studied as positive and negative controls, respectively. Pig hepatocytes were also studied in the absence of FHF sera and in the presence of mitogenic stimulation with phytohemagglutinin (PHA) and phorbol 12-myristate-13-acetate (PMA). PERV DNA and PERV RNA were detected in all supernatants of cultured pig hepatocytes. The level of PERV RNA in the supernatant of pig hepatocytes was not altered by exposure to human FHF serum or stimulation with PHA and PMA. In addition, PERV RNA was undetectable in the supernatant of HEK293 cells for up to 50 days after exposure to pig hepatocyte supernatant (with or without FHF sera). These findings show that production of PERV by cultured pig hepatocytes was unaffected by exposure to growth factors and cytokines present in human FHF sera.