Steve C. Chen

Clinical experience with a porcine hepatocyte-based liver support system.

The only clinically proven effective treatment of fulminant hepatic failure (FHF) is orthotopic liver transplant (OLT). However, many patients die before an organ becomes available. Thus, there is a need for development of an extracorporeal liver support system to “bridge” these patients either to OLT or spontaneous recovery. We developed a bioartificial liver (BAL) based on plasma perfusion through a circuit of a hollow-fiber cartridge seeded with matrix-anchored porcine hepatocytes to treat patients with severe acute liver failure. Two groups of patients were studied. Group 1 (n = 12): patients with FHF. All patients were successfully “bridged” to OLT. “Bridge” time to OLT was 21-96 hr (mean: 39.3 hr). All patients were discharged neurologically intact. Reversal of decerebration was noted in all 11 deep stage 4 coma patients. There was reduction in intracranial pressure (ICP mmHg, 18.2 ± 2.2 to 8.5 ± 1.2; p<0.004) and increase in cerebral perfusion pressure (CPP mmHg, 71.1 ± 4.0 to 84.7 ± 2.6; p<0.006). Laboratory values pre- and post- BAL treatment: glucose (mg/dl) 122 ± 11 to 183 ± 21, p<0.002; ammonia (μmol/l) 155.6 ± 13.2 to 121.6 ± 9.5, p<0.02; total bilirubin (mg/dl) 21.6 ± 2.8 to 18.2 ± 2.2, p<0.001; PT (sec) 23.2 ± 1.7 to 21.9 ± 1.0, p<0.3. Group II (n=8): patients with chronic liver failure experiencing acute exacerbation. Two patients survived and later underwent OLT. Six patients (not OLT candidates) died 1-14 days after last BAL treatment. Laboratory values pre- and post-treatment: ammonia (μmol/l) 201 ± 47 to 143 ± 25, p<0.06; total bilirubin (mg/dl) 22.8 ± 5.2 to 19.5 ± 4.4, p<0.01; PT (sec) 22.5 ± 2.0 to 21.8 ± 1.1, p<0.6. Conclusion: our clinical experience with the BAL suggests that it may serve as “bridge” to OLT in patients with FHF primarily by reversing intracranial hypertension, but it is not a substitute for OLT in patients with end-stage liver disease who are non-transplant candidates.

Long-term correction of albumin levels in the Nagase analbuminemic rat : repopulation of the liver by transplanted normal hepatocytes under a regeneration response

Numerous studies have reported successful transplantation of hepatocytes with demonstration of function. However, none have shown long-term correction of a liver-related metabolic defect. Male Nagase analbuminemic rats, immunosuppressed with cyclosporin-A, were transplanted with normal hepatocytes (2 x 10(7) cells/rat) isolated from allogeneic male Sprague-Dawley rat donors. Hepatocytes were selectively transplanted via the portal vein tributary into the posterior liver lobes of Nagase analbuminemic rats. Following 2 wk, to allow engraftment, selected transplanted rats (Group I) were reoperated and the portal venous branch supplying the anterior liver lobes was permanently ligated, resulting in their atrophy and induction of regeneration in the residual transplant-bearing lobes. Control rats consisted of: Group II-transplanted with normal hepatocytes without portal branch ligation; Group III-transplanted with analbuminemic hepatocytes with portal branch ligation; and Group IV-nontransplanted analbuminemic rats with portal branch ligation. The experimental period extended to 3 mo posttransplantation. All rats transplanted with normal hepatocytes demonstrated a significant elevation in serum albumin levels (ELISA). Group I rats had dramatic elevations in serum albumin to near normal levels (1.78 +/- 0.20 g/dl), and maintained these levels until the end of the experiment. Albumin levels in Group II rats reached 0.26 +/- 0.07 g/dl (p < 0.001), whereas Group III and IV rats showed no changes in serum albumin levels throughout the experiment. Immunohistology of liver tissue obtained from Group I rats, demonstrated large numbers (22.6 +/- 7.5%) of albumin-positive hepatocytes populating the recipient liver. This is the first report of near-total and sustained correction of a genetic defect in liver function in an experimental animal model following allogeneic hepatocyte transplantation.

Transplantation of hepatocytes for prevention of intracranial hypertension in pigs with ischemic liver failure.

Intracranial hypertension leading to brain stem herniation is a major cause of death in fulminant hepatic failure (FHF). Mannitol, barbiturates, and hyperventilation have been used to treat brain swelling, but most patients are either refractory to medical management or cannot be treated because of concurrent medical problems or side effects. In this study, we examined whether allogeneic hepatocellular transplantation may prevent development of intracranial hypertension in pigs with experimentally induced liver failure. Of the two preparations tested--total hepatectomy (n = 47), and liver devascularization (n = 16)--only pigs with liver ischemia developed brain edema provided, however, that animals were maintained normothermic throughout the postoperative period. This model was then used in transplantation studies, in which six pigs received intrasplenic injection of allogeneic hepatocytes (2.5 x 10(9) cells/pig) and 3 days later acute liver failure was induced. In both models (anhepatic state, liver devascularization), pigs allowed to become hypothermic had significantly longer survival compared to those maintained normothermic. Normothermic pigs with liver ischemia had, at all time points studied, ICP greater than 20 mmHg. Pigs that received hepatocellular transplants had ICP below 15 mmHg until death; at the same time, cerebral perfusion pressure (CPP) in transplanted pigs was consistently higher than in controls (45 +/- 11 mmHg vs. 16 +/- 18 mmHg; p < 0.05). Spleens of transplanted pigs contained clusters of viable hepatocytes (hematoxylin-eosin, CAM 5.2). It was concluded that removal of the liver does not result in intracranial hypertension; hypothermia prolongs survival time in both anhepatic pigs and pigs with liver devascularization, and intrasplenic transplantation of allogeneic hepatocytes prevents development of intracranial hypertension in pigs with acute ischemic liver failure.

MATRIX-INDUCED LIVER CELL AGGREGATES (MILCA) FOR BIOARTIFICIAL LIVER USE

Ex vivo reproduction of liver microstructure using isolated hepatocytes is critical for bioartificial liver use. We have developed a method of producing matrix-induced liver cell aggregates (MILCA) using a small number of collagen-coated beads as a nidus for formation of hepatocyte aggregates. Porcine hepatocytes were obtained by EDTA/collagenase digestion. Cell viability was assessed by trypan blue exclusion and LDH release. Cytochrome P-450 activity was determined at 4 and 24 hours by measuring the formation of 7-hydroxycoumarine (7-HC) from 7-ethoxycoumarine (7-EC). At 4 hours, the viability of MILCA was 92±2%, LDH release was 100+22 U/L and 7-HC formation was 140±34 nM/g cells. At 24 hours, MILCA viability remained greater than 90%, but 7-HC formation was lower than that of parallel control monolayer hepatocyte cultures (194±43 vs 481±78 nM/g cells; p<0.002). On transmission electron microscopy, MILCA ultrastructure resembled that of a normal liver (maintenance of cell polarity, gap junctions, bile canaliculi, intact organellae, glycogen granules). MILCA were subsequently inoculated into hollow-fiber bioreactors which were perfused for 6 hours with plasma recovered from patients with fulminant hepatic failure (n=6; 5x109 cells/cartridge, recirculation of 350 ml of plasma at 400 ml/min). In these studies, lidocaine (20 μg/ml) was cleared in less than 3 hours and 7-HC production at 6 hours was 71+8 nM/g cells. Other MILCA effects noted in this system included lowering of plasma lactate, bilirubin and ammonia and increase in the level of several non-essential amino acids.

A previously unreported mutation in a Currarino syndrome kindred.

Currarino syndrome consists of autosomal dominant hereditary sacral dysgenesis that is caused by mutations of the HOX gene, HLXB9. Sacral malformation, presacral mass, and anorectal malformations comprise the classic triad, but other common symptoms and malformations include neonatal‐onset bowel obstruction, chronic constipation, recurrent perianal sepsis, renal/urinary tract anomalies, female internal genital anomalies, tethered spinal cord, and anterior meningocele. Up to 33% of patients are asymptomatic. There is marked inter‐ and intrafamilial variability in expression, and no genotype/phenotype correlations have been identified. To date, 32 different mutations have been identified in HLXB9: all nine missense mutations were found in the homeodomain, while the others were nonsense, frameshift, splice site mutations, or heterozygous whole‐gene deletions. We report a four‐generation family with Currarino syndrome varying in severity from very mild to full expression of the Currarino triad. They were found to carry a previously unreported nonsense mutation, E283X, absent in tested asymptomatic first‐degree relatives. This family provides additional information on the degree of intrafamilial variability associated with HLXB9 mutations.

Tension pyopneumothorax in a child: a case report

Pneumonia is an infection of the lung parenchyma that may result in pleural thickening, effusion, or an empyema. When there is air or gas in association with purulent exudate in the pleural cavity, a pyopneumothorax exists. The progression to pyopneumothorax under tension is extremely rare. We present a case of tension pyopneumothorax in a child.

Tissue engineering: Liver

Various attempts have been made to support animals and patients with liver insufficiency, utilizing various extracorporeal support systems including cross-circulation, whole liver blood perfusion, hemadsorption, hemodialysis, plasma exchange, total body washout, use of microsomal enzymes bound to artificial carriers and others [1]. However, none of these therapeutic modalities succeeded in gaining wide clinical acceptance.