CURRENT STATUS OF LIVER SUPPORT SYSTEMS

ADVANCES IN LIVER TRANSPLANTATION

1089-3261/00 $15.00

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CURRENT STATUS OF LIVER SUPPORT SYSTEMS Levent Kaptanoglu, MD, and Andres T. Blei, MD

The efforts to develop an ”artificial liver” span more than four decades of work. The persistence of this search illustrates the difficulties in merging the myriad of hepatic functions into one structure. The liver is critical in multiple processes, including the clearance of endogenous compounds, in the synthesis of macromolecules, and in organ-specific biotransformation processes. The reproduction of this complexity in a device that will allow patients with liver failure to survive the acute insult (in the case of fulminant hepatic failure, FHF) or an episode of decompensation (in the case of cirrhosis) has been a daunting challenge. Survival in these critical clinical situations may be influenced by additional factors that are beyond the control of any support system, such as the nature of the liver injury, the presence of multiorgan failure, or the underlying health status of the individual. The outlook for the artificial liver has been radically changed by the success of liver transplantation. The traditional objective for these devices; demonstration of an increased survival, has been transformed to a more manageable goal; survival to the transplant procedure. Their effectiveness can now focus on intermediate end-points (e.g., intracranial pressure) whose control is critical for the performance of a successful liver transplant. The term “bridge to transplantation” has been coined for this new clinical end-point. Under this aegis, potential new tools and approaches are being tested. Clinical trials are being performed whose design and scope far exceed the experience of the past. These are exciting times in this area of research. Conceptually, liver support systems can be divided into three main groups: artificial (nonbiologic) support, biologic approaches, and hybrid devices. Recent

Supported by a Merit Review from the Veterans Administration Research Service

From the Departments of Medicine and Surgery, Northwestern University Medical School, Chicago, Illinois

CLINICS IN LIVER DISEASE VOLUME 4 NUMBER 3 * AUGUST 2000

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reviews of this topic have been published.M* 68* 79 A general word of caution is due before the experience with each group is examined. Promising results in an animal model set the stage for clinical testing; however, in many studies in models of acute liver failure the treatment is begun at the time of onset of liver injury or shortly thereafter, a very different scenario from the clinical arena where treatment is begun with the syndrome fully developed. In the latter situation, a support system is involved in the reversal of injury already present. Timing of the initiation of therapy may be a critical factor in the effectiveness of a liver support system. The critical functions that need to be replaced by the support system are not well defined. It is still not known what the exact substances are that need to be cleared, or which synthetized compounds are critical for survival, or which biotransformation processes are a priority. Without this knowledge evaluation is based on an assessment of clinical success rather than on pathophysiology. Clinical trials for liver support systems are difficult to perform. Few modalities have been rigorously tested using well-designed prospective studies. The incidence of fulminant hepatic failure, the condition most frequently evaluated for liver support, is estimated at only 2000 individuals in the United States affected per year.4O Multicenter trials are needed to acquire enough patients to accurately test the efficacy of a support system, trials that are onerous and complex. Patients need to be stratified according to disease cause and severity, factors that are known to affect ~urviva1.7~ Also, results of any controlled evaluation depend on the quality of care in the control group. Most studies have been performed in patients with FHF. Acute-on-chronic liver failure, that is, the patient with decompensated cirrhosis, has received less attention. The clinical problems are not exactly similar to those of FHF, because symptomatology from portal hypertension can predominate in cirrhosis but is rarely a problem in FHF. Still, acute-on-chronic liver failure offers a broader population base to test the effectiveness of a support system. ARTIFICIAL (NONBIOLOGICAL) LIVER SUPPORT

Conceptually, the focus of extracorporeal artificial support systems is the efficient removal of circulating toxins. These toxins may arise from other organs (such as ammonia arising from the gut) or be released from the necrotic liver itself. Toxins differ in their molecular weight, in their protein binding, and in their ability to be removed by artificial systems. These substances can affect mental state, can participate in diverse components of the multiorgan failure syndrome, and can impair the liver's regenerating capacity. Toxins: Effects on Neurologic Function

Hepatic encephalopathy is a major complication of liver failure, including the development of brain edema in FHF, a major cause of death in this syndrome." Improvement in mental state has been the usual criteria to determine the effectiveness of a liver support system. Several candidate toxins can be removed by artificial means. Ammonia is the classic example of a small molecular weight, water soluble compound that can be effectively dialyzed. Classically the concept of synergism of ammonia with other toxins emphasized other products arising from the gut.lo5The notion of synergism has been conceptually transformed with the observation that the astrocyte is the main cellular element

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affected in hepatic encephalopathy and that multiple factors (hyponatremia, cytokines, benzodiazepines) may interact with ammonia in causing astrocyte dy~function.3~ Toxins: Multiorgan Failure

The multiorgan failure syndrome seen in patients with FHF may arise from the injured liver itself. Toxic species are generated in the context of hepatocyte necrosis and apoptosis or infiltration of the liver with inflammatory cells. Performance of a hepatectomy can improve the cardiovascular instability that characterizes the critical stages of liver failure." Cytokines released by the inflammatory process within the liver are candidates for such systemic effects. Elevated levels of tumor necrosis factor (TNF)a, interleukin (1L)-1, IL-lRa, and IL-6 have been reported in FHF,70,89 with bacterial endotoxin being an initiating factor in the signaling cascade that leads to cytokine release from mononuclear cells? Support for the subsequent generation of nitric oxide within the endothelium, well described in cirrhosis,1oois suggested by the elevated circulating levels of cGMP seen in FHF?7 Increased arteriolar vasodilatation can result in the opening of arteriovenous shunts, a factor involved in the decreased oxygen use seen in FHF? Toxins: Hepatic Regeneration

Multiple pathways are activated in the complex process that leads to hepatic regeneration." Substances are present in the serum of patients with FHF that inhibit hepatic Interferon-gamma may contribute to this effect, with inhibitory effects on liver regeneration in the classic partial hepatectomy rat model.%The activity of transforming growth factor (TGF)-P, the main endogenous inhibitor of hepatic regeneration, has been shown to be increased in the livers of patients with FHF.69Alternatively, increased levels of substances that promote hepatic regeneration are seen in the plasma of patients with FHF, such as hepatocyte growth factor (HGF).37Although the necrotic liver tissue may not effectively respond to promoters of regeneration, the depletion of such factors could potentially become a detrimental effect of extracorporeal systems. The effects of such toxins may be exerted at different levels. Ammonia, in conjunction with other synergistic compounds arising from the gut (mercaptans, phenols, short-chain fatty acids), may also inhibit hepatic regeneration.'" Interleukin-2 may exert neurologic effects without requiring entry into the brain, because binding to cerebral endothelial receptors can result in the generation of nitric oxide and prostacyclin within brain tissue.61It would seem then, that both small and large molecular weight ( M W ) compounds as well as water soluble and protein bound compounds could be targets for removal by a liver support system. The clinical experience with different modalities is reviewed later. Hemodialysis

Children with urea-cycle enzyme deficiencies and life-threatening hyperammonemia (with brain edema and intracranial hypertension) are treated with hem~dialysis,'~ an example of the ability of artificial systems to remove watersoluble toxins implicated in the pathogenesis of brain edema. Recently, our

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group has shown in an in vitro system that elimination of ammonia by dialysis depends on blood flow (the extracorporeal circuit’s flow), the dialysate flow (elimination of ammonia is optimal with a 2 : 1 ratio to blood flow) and the permeability surface.*O In animal studies, hemodialysis with semipermeable membranes has been shown to almost double the survival time of pigs with liver failure secondary In the late 1950s, hemodialysis was shown to reduce ammonia to i~chemia.2~ levels in cirrhosis, with some improvement of neurologic status.SoA decade later, one patient with acute liver failure was shown to improve transiently after dialytic The introduction of polyacrilonitrile (PAN) membranes in the mid-1970s focused on the possible elimination of molecules of higher molecular weight, the so-called middle molecules. French investigators carried out a large (but uncontrolled) clinical study in 41 patients with FHF, with an average Neurologic improveof 4.5 treatments per patient, using the PAN ment was seen in 24/41 patients (58%), with survival in 9/17 subjects with full neurologic recovery and 25% survival overall, a rate that approaches values of spontaneous survival in FHF in the late 1 9 9 0 Similar ~~ results (40%neurologic improvement, 33% survival) were noted in a series of 24 patients with FHF from London.% Patients with liver failure often exhibit renal failure and artificial kidney support is often indicated. When dialysis is instituted, continuous methods are preferable (see hernofiltration below). Standard dialytic procedures result in volume shifts that may aggravate cerebral edema.= The inability to remove protein-bound substances has been a major theoretical drawback of dialysis. Over the last years, albumin dialysis has received increasing attention. Placement of albumin in the dialysate allows the transfer of protein-bound substances from the intravascular space into the dialysis fluid; impregnation of the polymers that constitute the polysulphone membrane allows for binding of substances otherwise avidly bound to protein. Bile acids (with potential toxic effects on hepatocytes) and fatty acids (with potential effects on the brain) can be removed.92A single case report of fulminant Wilson’s disease treated with albumin dialysis illustrates the capacity of such therapy to remove albumin-bound copper.” The clinical experience in decompensated cirrhosis51is too preliminary for conclusions to be reached. In summary, hemodialysis has not been shown to improve survival in the pretransplant era, but studies were uncontrolled and animal studies showed effectiveness when begun at the time of onset of liver injury. Currently, removal of small molecular weight compounds, such as ammonia, and protein-bound substances, is possible with technical refinements. Hernofiltration, Alone and Combined

In hemofiltration, elimination of solutes of M W less than 10,000 (middle molecules of FHF), including immunoglobulins and cytokines, occurs by continuous convection without large shifts in fluid and electrolytes. Net fluid removal is achieved with a lower rate of fluid replacement (for review, see ref. 30). Continuous venovenous hemofiltration is the preferred approach to support the kidneys in patients with combined renal and hepatic failure, although attention to anticoagulation is required in the presence of defects in the synthesis of coagulation factors and platelet abnormalities. The uncontrolled clinical studies with hemofiltration have been inconclusive. In an uncontrolled series of 10 patients with FHF, a 50% survival was seen

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when treatment was applied for an average of 92 hours.= An experience with five patients with FHF in the United States showed survival in three, one of them transplanted.80Thrombocytopenia was severe, including the development of bleeding, a complication that may be ameliorated with the addition of dipyridamoleZ5or prostacyclin." A Japanese experience in eight patients with FHF using hemofiltration and plasma exchange= showed no discernible benefits on encephalopathy or survival. When combined with dialysis, a process termed hemodiafiltration, molecules removed by diffusion can also be eliminated. Hemodiafiltration, using a high performance membrane with large pores, has been used in combination with plasma exchange in patients with FHF (Fig. l).lo4The Japanese series was expanded to 67 subjects,103with the best results ever reported for liver support: 97% regained consciousness (including 33/35 patients in stage 111-IV coma); however, survival was 55% overall (38% in patients with FHF of indeterminate cause) at a time when liver transplantation was unavailable. Uncertainty surrounds these results in the absence of a controlled evaluation. Blood and Plasma Exchange Exchange transfusions were used in the mid 1960s to manage hepatic coma in FHF. Trey treated 11 patients and reported a 50% ~urvival.9~ Trey's experience, however, was not replicated in a clinical trial; the results were worse in the exchange group (Table 1):' Plasmapheresis with plasma exchange was initially evaluated in a small series of 5 patients with FHF,6O with recovery of conscious-

*\

I

Membrane

Centrifugal ator

+r

Exchange Filtration Sorption

I- Physicochemical alterations

Figure 1. Concept of plasma separation and plasma treatment. (From Takahashi T, Malchesky PS, Nose Y: Artificial liver: State of the art. Dig Dis Sci, 36:1327-1340,1991; with permission.)

A 62 h (3-72 h)

A: 17 with potentially recoverable lesions 9 ELAD, 8 Control B: 7 listed for transplant 3 ELAD, 4 Control

B: 90 h (80-129 h)

A 7/9 (78%) ELAD

8: None

B: 5 patients with FHF, grade 4 coma 24 patients with ALF

ELAD Ellis 199626

A: 115 (20%) survival B: 3/5 (60%) survival

A 6 h / d x 5 days

A 5 patients with FHF, grade 4 encephalopathy

6/ 8 (75%)control B: 1 / 3 (33%) ELAD 1/4 (25%) control

2. Survival rate 34.5%

Biologic DT-Sorbent dialysis Hughes 299Z4*

A 2-4 perfusions for 5 h 8: 2-4 perfusions for 10 h

A: 75 patients with FHF, grade 2 coma; 5 vs. 10 h hemoperfusionl day B: 62 patients with FHF, grade 4 encephalopathy, Nonperfusion vs. 10 h perfusion/day

B: 4 survival A 1. Survival rate 51.3% 2. Survival rate 50.0% B: 1. Survival rate 39.3%

B: None

A No survival

A 1-3 h daily

once and one twice vs. B: No treatment

Results

A 8 patients received exchange transfusion, 7

Length of Therapy

Charcoal O'Grady 1988'j

Exchange transfusion Redehzr 197381

Patient Characteristics

Table 1. CONTROLLED TRIALS OF LIVER SUPPORT

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ness after many days of multiple exchanges. More recently, high volume plasmapheresis has been evaluated in Denmark, based on the premise that the removal of toxins should include the volume of the extracellular space and not solely the plasma compartment. In a series of 11 patientss3in stages I11 and IV encephalopathy, an average of 2.6 exchanges were performed over 3 days with volumes equivalent to 16% of body weight (approximate volume of the extracellular space). Five of 11 patients survived, all with acetaminophen-induced injury. The nonsurvivors were able to maintain a stable hemodynamic and neurologic status for a period of 7 days before death. The same group reported a follow-up of this uncontrolled experience, noting a 54% survival, including 6/11 patients in grade IV c0ma.5~ Hemodynamic studies were performed, indicating an improvement in systemic vascular resistance and peripheral oxygen consumption.56Cerebral oxygenation, as studied by Doppler flowmetry and jugular vein oxygen saturation, seemed to improve with high volume plasmapheresis.58A recent report notes the ability of plasma exchange to reduce the level of circulating endotoxin and cytokines, such as tumor necrosis factor and interleukin-6, that are elevated in patients with FHF?5 These uncontrolled clinical observations and additional pathophysiologic observations suggest that plasma exchange and high-volume plasmapheresis, or both, may be of benefit in FHF. A controlled evaluation of the latter approach is currently under way in Scandinavian countries. Hemoperfusion

Removal of toxins could also occur if blood is circulated through adsorbent systems. Of these, hemoperfusion through charcoal was extensively tested in the 1970s and 1980s. Based on animal studies and an encouraging result in one patient with FHF,l6the effects of charcoal hemoperfusion were initially evaluated in a series of 22 patients with FHF, noting a 46% survival.33Problems with biocompatibility of blood with charcoal included leukocyte and platelet loss. Charcoal was microencapsulated and prostacyclin was added to prevent platelet activation and improve bi~compatibility.~~ An uncontrolled series reported a 65% survival rate in patients in whom charcoal hemoperfusion was begun at stage I11 coma in contrast to 20% survival and almost 80% brain edema in those treated in stage IV coma.35 In the late 1980s, a large controlled trial of charcoal hemoperfusion was published.73When applied to 75 subjects in stage I11 coma, 10 hours of perfusion did not confer benefits over 5 hours of perfusion, with survival noted to be 51% and 50% respectively. In 62 patients in stage IV coma, charcoal hemoperfusion did not confer a survival benefit when compared with conventional treatment, with mortalities of 61% and 65%, respectively. A thoughtful editorial accompanied this paper, noting the importance of controlled trials in FHF, which in the case of charcoal hemoperfusion disproved the tenet of its original proponents7 In fact, most controlled studies in this disease have not shown differences between groups. Another approach to avoid the problems of sorbent compatibility with blood is the use of hemodiabsorption. In this system, blood is dialysed across a cellulose membrane whereas the dialysate contains powdered charcoal and a cation ion exchange resin. The exchange surface is increased, blood elements are not in contact with the sorbent, and the dialysate is continuously renewed. One such system, the Biologic-DT, has undergone clinical testing. Ten patients with

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FHF and stage IV coma were randomized; of the 5 on hemodiabsorption, one survived. In contrast, 3 of 5 subjects in the control group survived." It was concluded that technical developments were needed to improve the biocompatibility and efficiency of the system. The manufacturers have added a push-pull sorbent-based pheresis step, which essentially represents plasma filtration with powdered sorbent surrounding the membranes. A recent review suggests that the Biologic-DT system has benefits in the control of encephalopathy in patients with decompensated cirrhosis? The device is available commercially in the US. Its role in the management of patients with liver failure remains uncertain until careful and commercially unbiased controlled evaluations are performed (see Table 1).

BIOLOGICAL SYSTEMS Extracorporeal Liver Perfusion

It could be argued that the best liver support system is a liver itself. Perfusion of the patient's blood through an extracorporeal liver has been reported since the mid-1960s. Livers from several species have been used, including cow, dog, baboon, and pig, and in one occasion, multiple species were used in the same patient.' In the case of baboon livers,@the duration of perfusion in 14 patients with stage IV encephalopathy lasted from 5 to 27 hours, with one to four perfusions per patient. A patient survival of 50% has not been replicated in other (similarly) uncontrolled series. There are important technical considerations in the perfusion of an extracorporeal liver. The optimal rate of perfusion has to approximate in vivo blood flow (approximately lmL/ g tissue). Recent studies suggest that perfusion through both the hepatic artery and portal vein provides better function than perfusion through the portal vein alone, especially as it relates to bile formatiomz9Recent technologic advances have improved the ability to withdraw blood from a venous access, provide adequate oxygenation of the perfused liver, and optimize local anticoagulation. While awaiting emergency liver transplantation, four patients were perfused through extracorporeal pig livers with one subject successfully "bridged to tran~p1ant.l~ The complexity of the perfusion and the logistical needs for repeated organ perfusions in the same subject require a major commitment for this approach to be successful. A significant problem in the use of cross-species livers is biocompatibility. Preexisting antibodies activate complement in the endothelial cell of the extracorporeal liver, resulting in vascular thrombosis and eventual malfunction of the organ. The development of transgenic pig livers that express human complement regulatory proteins in the hepatic endothelial cell has been one approach to prevent this pr0b1em.l~No clinical reports of its application in humans are available at the time of this writing. Concerns have risen regarding the potential for transmission of zoonoses (see next section). Human livers have been used for extracorporeal perfusion. Three patients were perfused through livers that after harvesting had been deemed unfit for Perfusions were maintained for up to 40 hours, with a reduction of intracranial pressure and concomitant increase in cerebral perfusion pressure, correction of encephalopathy, and improvement in prothrombin time. Two patients survived. Generalization of this approach is quite limited because

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it requires livers nonsuitable for transplantation at the exact time the patient is in need of therapy. Hepatocyte Transplantation

The use of hepatocytes to support an acutely failing liver has received recent clinical scrutiny. An animal experience had yielded encouraging results. When implanted into the spleen of rats that underwent total hepatectomy, survival could be prolonged two to three fold.98 A volume of conditionally immortalized hepatocytes equivalent to 3% of liver mass improved hepatic encephalopathy in rats after portacaval anastomosis receiving an ammonia infusion.88 The limited clinical experience can be described as a phase I experience. Varying volume of hepatocytes isolated from human livers that were unusable for transplantation have been injected into the spleen or liver. In a recent series of 5 patients with FHF of viral and toxic causes who were deemed nontransplant candidates,1° hepatocytes were injected into the spleen via the splenic artery. In two subjects, hepatocytes were also injected into the liver via the portal vein, with a catheter placed in the latter via a transjugular access. The volume of injected hepatocytes was 1%to 10% of hepatic mass. After 2 days, coma improved while survival was extended for 2 to 7 weeks in 3 of the subjects, maintained on immunosuppressive drugs. At autopsy, engraftment of the hepatocytes in the liver and spleen was demonstrated. In another series of five patients with both acute (n=2) and chronic liver disease, injection of hepatocytes into the spleen resulted in evidence of eng r a f t m e ~ ~One t . ~ ~of the cases had alpha-1-antitrypsin deficiency and an increase in the circulating levels of this protein was demonstrated after transplantation. Three of five subjects underwent successful orthotopic transplantation. One patient with Criggler-Najjar syndrome showed an improvement in bilirubin levels after the injection of hepatocytes into the portal vein.3I There are still major technical issues to be resolved in this area. Hepatocytes have to be available on demand, and the optimal preservation of hepatocytes requires attention to freezing and thawing conditions. Immortalization of hepatocytes may be one solution to this problem (see next section). The critical mass that needs to be injected is still unknown. It is possible that a relatively small mass of hepatocytes could exert beneficial effects on the regenerative activity of the injured liver. Also, there are limits to the ability of the liver to receive repeated injections. In the series from Colorado,*olung infiltrates developed within the first 24 hours after injection into the portal vein, suggesting embolization of hepatocytes into the lung. Although the performance of a randomized clinical trial has been some of these technical issues need to be resolved before a multicenter study can be considered. HYBRID SYSTEMS

An extracorporeal device that combines features of both artificial and biologic support systems was first proposed by Wolf, who in the 1970s constructed the first experimental hollow-fiber liver bioreactor seeded with rat hepatoma cells.101A large number of studies have been published on the technical aspects of such an approach (for review see ref. 82). Two such hybrid systems have undergone phase I clinical evaluation; the ELAD (extracorporeal liver assist

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device) and the BAL (bioartificial liver) and are now being evaluated in phase I1 and I11 trials. In a recent commentary, the technology of biologic extracorporeal liver assist devices has been described as moving from infancy to adole~cence.4~ The complexity of this approach has resulted in three distinct areas of technologic development, which are described below. Extracorporeal perfusion

Although the ELAD system uses direct perfusion of blood through the hepatocyte-containing device,&the BAL system separates plasma and perfuses it through the device in an independent circuit." The former method decreases the complexity of the extracorporeal circuit but carries risks of consumptive coagulopathy or platelet consumption, requiring prophylaxis with heparin with its attendant risks. The clinical experience, however, has not shown major problems with biocompatibility using the ELAD with a median perfusion time of 72 hours in one study. In the BAL system, the extracorporeal circuit of the separated plasma is complex, involving the use of charcoal hemoperfusion to remove potential cytotoxic elements to hepatocytes present in the plasma of these individuals?l The Bioreactor

The effectiveness of the hepatocyte-containing device can be improved in an environment that favors hepatocyte function and that maximizes the exchange between blood and plasma and the liver cells. For the most part, hepatocytes are separated from the flowing compartment by a semipermeable membrane, whose molecular weight cut-off excludes passage of proteins greater than 100 kilodalton (kD) and allows immunoprotection of the xenogeneic cells. New innovations include (1) a system that allows decentralized cell perfusion by having four interwoven hollow fibres arranged at planes of 90" to each other,% (2) a spirally nonwoven polyester fabric in which liver cells are cultured as small or (3) a system whose aim is to incorporate nonparenchymal cells for maximal hepatocyte function? Choice of Hepatocytes

Recommendations of the Workshop on Fulminant Hepatic Failure held in 1994 provide guidance to this choice?O Liver cells used in extracorporeal devices should ideally be human in origin, of a normal phenotype, rapidly and easily grown in cell culture to a high mass, stable for prolonged periods in a welldifferentiated state, and able to perform the entire spectrum of liver functions. It is useful to examine the current experience under the light of these guidelines. Species

The choice of human hepatocytes is limited by the lack of general availability of such cells and the poor proliferation of adult hepatocytes in culture2l One alternative approach has been the use of porcine hepatocytes, because the yield of hepatocytes is plentiful and the pig shares physiologic features of liver function with humans. Pig hepatocytes have a tendency to form cell aggregates,=

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a three-dimensional arrangement that favors their function within extracorporeal cartridges and that can be further enhanced by the addition of collagen-coated microcarriers. Concerns of infection with a porcine endogenous retroviral agentn have not been borne out in the clinical experience?8'Still, repetitive exposure to pig hepatocytes results in the formation of human xenoreactive antibodies of the IgG and IgM types directed at pig tissues5; the possible deleterious effect of such sensitization with subsequent exposure to pig hepatocytes has been

Phenotype

Another approach for hepatocyte use is differentiated cell lines. The ELAD system uses the C3A hepatoblastoma cell line, cells that maintain hepatocyte differentiated function and exhibit contact inhibition.* This cell line, however, is a tumoral cell line and concerns have risen about the escape of such cells into the patient, with an undefined malignant potential. Filters in the outflow system of the ELAD reportedly prevent the release of the C3A cells. Mass of Hepatocytes

What is the residual liver mass after liver resection that is compatible with postsurgical survival? In man, 20% or 300 g seems to be the minimal viable mass after such Hepatocyte seeding of the BAL provides approximately 2% of liver mass while this value is considerably higher with ELAD, approximately 20%. As discussed in the section on hepatocyte transplantation, these calculations may not reflect the effects of liver cells in liver failure, where the mass requirements for patient survival are also dependent on the ability of such cells to divide, to detoxify toxins, and to improve hepatic regeneration. Stability for Prolonged Periods

This technologic aspect has not been fully resolved for the use of procine hepatocytes, which in culture lose differentiated function after several passages. In the case of BAL, the hepatocytes for clinical use need to be replaced on a daily basis. Novel approaches to increase the stability of cultured cells are being explored. This includes immortalization of hepatocytes after transfection with a variant of the simian virus 40 (SV40) large-T antigen gene (a temperaturesensitive control of cell cycle proteins).'02 Alternatively, the performance of hepatocytes can improve after coculture with extracellular matrix and nonparenchyma1 cells.14 Cryopreservation of freshly isolated hepatocytes, critical for the potential use of this therapeutic approach, is improved after their encapsulation with alginate or cellulose acetate or their attachment to polystyrene or glass microcarriers.52 Performance

The two systems discussed in detail have undergone pilot clinical evaluations. In the case of ELAD, an initial experience in 11 patients with ALF was not very promising, with 6/11 patients dying with cerebral edema.%A subsequent study in 24 patients with acute liver failurez6(ALF) divided them into two strata: those with greater likelihood of spontaneous survival and those listed for transplantation (see Table 1). Subjects were randomized to ELAD or control therapy. The results did not reveal a major impact of ELAD on survival. Deterioration with respect to the grade of encephalopathy was more frequent in the

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control group. No hemodynamic deterioration was noted with the treatment though evidence of metabolic function was modest, as assessed by galactose elimination capacity. More conventional parameters, such as blood ammonia, lactate, albumin or fibrinogen were not affected by treatment. Animal studies indicate efficacy of the BAL system in maintaining survival in anhepatic pigs.ls Bioartificial liver has undergone a phase I study in 31 patients, 18 with FHF, 10 with acute-on-chronicliver failure, and 3 with primary graft n0nfunction.9~ The main positive impact in FHF has been the reduction in intracranial pressure coupled with improvements in a neurologic score that indicate an increased responsiveness to external stimuli. The magnitude of these changes is modest and the values of intracranial pressure were only mildly elevated at baseline. The reduction in ammonia values was also small (from a mean of 155 kmol/L to 130 pmol/L) with no indications that metabolic functions were being contributed by the device. On a positive note, in patients with FHF in this series and in a smaller series of 8 subjects in France,% appropriate candidates were able to be successfully transplanted. A large phase I1 trial of BAL is currently in progress in the US and Europe. In summary, the last decade has witnessed an explosion of interest in hybrid support systems. Conceptual and technologic advances have propelled the bioartificial approach as one offering a promising outlook. Neither the ELAD nor the BAL system however, have been shown to result in an improvement of parameters of hepatic synthetic function or liver regeneration. One case report suggested that duration of perfusion may be important to promote metabolic function.66 The main focus for most of the current systems is still an improvement of the hepatic clearance function, that is, the ability to remove toxins from the bloodstream. Any system that solely substitutes the clearance function of the liver may be insufficient to provide a survival advantage for the complexity of medical problems present in fulminant hepatic failure. The choice of intermediate end points, such as a bridge to transplantation has been an advance; still, this end point is fraught with unpredictable variables such as timing of organ availability, a major problem throughout the United States, and financial considerations, which cloud its universality. BRAIN EDEMA: INSIGHT INTO LIVER SUPPORT

Brain edema is a unique and fatal complication of FHF because patients with other conditions associated with multiorgan failure do not succumb to intracranial hypertension. One group has used the improvement in intracranial pressure as an indicator of effectiveness of treatment.” Elucidation of the pathogenesis of brain edema should offer a guide to the design of systems that optimize the clearance of purported toxins.12From the authors’ current work in this area, several principles of treatment with current liver support systems can be proposed. Removal of Ammonia is an Important End-point for Any Support System

The development of cerebral herniation in FHF is related to arterial ammonia levels.19Formation of glutamine within astrocytes is a critical first step in the development of brain edema (Fig. 2) and can be demonstrated in humans in v ~ v oThe .~~ elimination of ammonia in the normal livern and in an extracorporeal

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Figure 2. Swollen foot processes of astrocytes surround a capillary of the cerebral cortex in a rabbit with galactosamine-induced fulminant hepatic failure. (From Traber PG, Dal Canto M, Ganger DR, et al: Electron microscopic evaluation of brain edema in rabbits with galactosamine-induced fulminant hepatic failure: Ultrastructure and integrity of the bloodbrain barrier. Hepatology, 7:1272, 1987;with permission.)

systemz0depends on blood flow: ammonia is a substance that undergoes h g h first-pass elimination and its hepatic or extracorporeal clearance, therefore, depends on the rate of perfusion. Most extracorporeal systems use rates of 100 mL/min to 300 mL/min, which is considerably lower than the normal hepatic blood flow of approximately 1500 mL/min. Removal of urea and glutamine, ammonia precursors, is also important to reduce ammonia levels. Improvement of ammonia elimination should be sought by all systems. Removal of Cytokines May Be Important to Prevent Additional Brain Swelling

Elevated levels of TNFa are present in FHF, a cytokine that can affect astrocyte volume when applied to glial cultures? Tumor necrosis factora particia pates in the development of a hyperdynamic circulatory state in cirrhosi~,~~ hemodynamic disturbance also present in FHF. An increase in cerebral blood flow in excess of brain metabolic needs is seen in cases of FHF with brain edema.2 57 As a result, cerebral blood volume increases and expansion of this compartment contributes to the rise in intracranial pressure. The increase in cerebral blood flow seems to arise from the swollen brain itself,@but vasodilatory stimuli related to cytokine activation may contribute to this cerebral vasodilatation.

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Treatment Should be Started Early

The rise in intracranial pressure is a critical event in the course of hepatic encephalopathy in FHF. Once the brain is swollen and intracranial hypertension is present, the therapeutic window is clearly reduced. Small changes in blood volume may affect intracranial pressure independently of any beneficial effect from a liver support system. In fact, a recent study has shown moderate hypothermia (32" to 33°C) to be very effective in reducing intracranial hypertension in FHF:6 an effect that is mediated through a reduction in cerebral blood flow and hence cerebral blood volume?* The true effectiveness of a liver support system may lie in the prevention rather than the treatment of brain edema.

CONCLUSION

An effective liver support system is still a long-cherished goal in hepatology. None of the current systems can provide a complete solution to the complexity of liver failure, which requires clearance of toxins, hepatic synthetic function, and improved hepatic regeneration in varying degrees. For artificial systems, improvement in the ability to remove ammonia and cytokines may optimize their clearance function. For biologic systems, the availability of extracorporeal livers on demand is a major hurdle to overcome; for transplanted hepatocytes, the spleen seems to be the best site for implantation but technical aspects that surround its use have not been yet resolved. Hybrid systems have garnered most of the recent attention, but the promise of an improved synthetic function is still unfulfilled. Still, each of these three areas is undergoing fast technologic and conceptual developments. The long quest for a liver support system has acquired a new energy that hopefully will yield a safe and accessible tool to assist patients with severe liver failure.

References 1. Abouna GM, Serrou B, Beohmig HG, et al: Long-term hepatic support by intermittent

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