Pig aortic endothelial cell antigens recognized by human IgM natural antibodies.

Human-to-pig xenoantibodies may constitute a major obstacle to the successful use of pigs as xenograft donors for human transplantation. Our studies demonstrate that normal human serum contains antibodies, primarily IgM, that are cytotoxic for pig aortic endothelial cells (PAECs). These antibodies bind to several antigens isolated from PAECs, lymphocytes, platelets, red blood cells, and the kidney. Absorption of human serum with pig lymphocytes removes the cytotoxic activity to PAECs and some, but not all, of the IgM antibodies capable of binding in an ELISA assay to the PAECs. The cytotoxic antibodies are inactivated by 2-mercaptoethanol, suggesting that they are primarily IgM. Whole cell extracts of PAEC, lymphocytes, platelets, red blood cells, and kidney were prepared and analyzed by Western blots to establish the cellular distribution of the xenoantigens that react with human IgM in pooled human serum. Results showed that several of the most intensely stained bands migrated between 24 and 66 kDa. High molecular weight bands (> 100 kDa) were observed only in kidney, platelet, and PAEC preparations. Human IgM xeniantibodies also reacted strongly in Western blots to endothelial cell membranes proteins with molecular weights of 62, 48, 42, 36, 34, 28, and 26 kDa. Absorption of human serum with pig lymphocytes removes IgM binding to all bands except for a 34-kDa Treatment of the PAEC membrane proteins with proteinase K disrupts the binding of the human IgM antibodies. Similar treatment with glycosidase F) resulted in a decrease in molecular weight of the 28- and 26-kDa bands, suggesting that these xenoantigens are glycoproteins and that antibody binding to some xenoantigens may not require glycosylation.

Treatment of hypercholesterolemia in the Watanabe rabbit using allogeneic hepatocellular transplantation under a regeneration stimulus.

Numerous studies have reported successful allotransplantation of hepatocytes. However, none have shown long-term correction of a liver-related metabolic defect. In this study, we used a method of regional hepatocyte transplantation and subsequent induction of transplanted cell proliferation by regeneration response in the transplant-bearing liver lobes. New Zealand White rabbits were used as cell donors and Watanabe heritable hyperlipidemic (WHHL) rabbits were used as cell recipients (2 x 10(8) cells/rabbit). All recipient rabbits were maintained on daily cyclosporine. Two weeks after baseline serum cholesterol determination, group I WHHL rabbits (n = 7) received an infusion of cells into the right lateral liver lobe, and a loose ligature was placed around the portal venous branch supplying the anterior lobe. After 1 week, to allow engraftment, the portal venous branch was ligated, which resulted in the atrophy of the affected liver parenchyma and induction of hyperplasia in the transplant-bearing liver tissue. Group II rabbits (n = 6) were transplanted with New Zealand White hepatocytes without portal branch ligation (PBL) and group III rabbits (n = 4) were subjected to sham transplantation (saline) and PBL. The experimental period extended to 150 days after transplantation. All WHHL rabbits transplanted with normal hepatocytes showed reduction in serum cholesterol and low-density lipoprotein (LDL) levels. Group I (PBL-stimulated) recipients demonstrated a more pronounced and sustained effect than group II animals (P < 0.05). Group III controls showed only a slight, typical for aging decrease in serum cholesterol. Group I recipient livers perfused with LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) showed much higher numbers of DiI-LDL-positive hepatocytes than those of group II recipients. In conclusion, a liver regeneration stimulus enhanced the population of transplanted hepatocytes and their functional effect in a large animal model of inborn error of liver metabolism.

Fetal rat hepatocytes: isolation, characterization, and transplantation in the Nagase analbuminemic rats.

BACKGROUND: In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative, less immunogenic, and resistant to cryopreservation and ischemic injury. These qualities could enhance FH engraftment, proliferation, and gene transfer requiring active DNA synthesis. METHODS: Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Free and cultured cells were studied using electron microscopy, fluorescence-activated cell sorting, and Northern analysis using alpha-fetoprotein and albumin as markers of hepatocyte lineage. DNA synthetic activity was measured in quiescent and mitogen-stimulated fetal and adult hepatocytes by [3H]thymidine incorporation. Susceptibility of cultured FH to retrovirally mediated gene transfer was studied using an amphotropic retroviral vector carrying the Escherichia coli lac-Z gene. Nagase analbuminemic rats were used as recipients to study the effects of intraportal FH transplantation. Analysis of serum albumin was carried out by enzyme-linked immunosorbent assay. RESULTS: In fetal liver, 87+/-2% of the cells showed morphological and molecular features of hepatocytes. DNA synthetic activity in nonstimulated cultured FH was 10 times greater than the maximal hepatocyte growth factor-driven response in adult rat hepatocytes. A total of 5-15% FH stained positive for X-gal; results of transduction in adult hepatocyte cultures were negative. In Nagase analbuminemic rat recipients, FH produced significant amounts of albumin only when a hepatic regenerative stimulus was applied. Immunohistochemistry confirmed presence of albumin-positive hepatocytes. CONCLUSIONS: Fetal rat liver from the late gestation period is highly enriched with hepatocyte progenitors. They are highly proliferative and susceptible to retroviral transduction and can engraft and function in the adult rat liver if transplanted under a hepatic regenerative stimulus.

Treatment of severe liver failure with a bioartificial liver.

Orthotopic liver transplantation (OLT) is the definitive therapy for severe liver failure. However, many patients die before an organ becomes available, mostly from cerebral edema. To provide temporary liver support, we developed a bioartificial liver (BAL) based on porcine hepatocytes and a charcoal column. Fifty-four consecutive BAL treatments were carried out in three groups of patients: Group I (n = 15) patients presented with FHF were listed for emergent OLT, Group II (n = 3) patients with primary non-function (PNF) of their liver grafts required urgent re-transplantation and Group III (n = 10) patients with acute exacerbation of chronic liver disease were not candidates for OLT. Patients were managed in a critical care unit receiving maximal standard support. Each BAL treatment was conducted for 6 hours. In Group I, all patients showed significant neurologic improvement, intracranial pressure (ICP) decreased and cerebral perfusion pressure (CPP) increased; other significant improvements, included lowered plasma ammonia and liver enzymes and increased glucose. One patient recovered spontaneously without OLT, all other patients were "bridged" to OLT, and recovered. Group II: PNF patients showed similar benefits. Group III: Chronic liver patients demonstrated transient beneficial effects after BAL treatment(s), however, most (n = 8) eventually succumbed to sepsis and multiple organ failure as they were not candidates for OLT; two patients, recovered, later were successfully transplanted and survived. Our clinical experience demonstrates that the BAL can serve as a bridge to OLT in patients with acute liver failure.

Early postoperative enteral feeding increases anastomotic strength in a peritonitis model.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) has been shown to decrease collagen synthesis and increase collagenase activity leading to impaired wound healing. Our hypothesis was that immediate postoperative feeding would decrease TNF-alpha, therefore increasing anastomotic healing in a peritonitis model. METHODS: Twelve Sprague-Dawley rats underwent cecal ligation and puncture to induce peritonitis. Six hours after induction of peritonitis an ileocecectomy and ileocolostomy was performed. Group 1 animals (n = 6) had immediate access to food and water, whereas group 2 (n = 6) had free access to water only. At 48 hours, weight loss, nitrogen loss, anastamotic bursting strength (ABS), TNF-alpha, interleukin-6 (IL-6), and IL-10 were measured. RESULTS: Weight loss was similar in the two groups. Group 1 rats had a significantly lower mean TNF-alpha level (17.3 +/- 10 versus 17.3 +/- 10 mcg/Dl, P = 0.05). ABS was also significantly higher in group 1 rats when compared with group 2 rats (81 +/- 34 versus 39 +/- 13 mm HG, P = 0.03). CONCLUSIONS: These data suggest that immediate postoperative feeding results in a beneficial change in the cytokine profile.

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

UNLABELLED: 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 (mumol/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 (mumol/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.

Loss and recovery of liver regeneration in rats with fulminant hepatic failure.

We earlier described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobes necrosis. After this procedure, lack of regenerative response in the residual viable liver tissue (omental lobes) was associated with elevated plasma hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta1) levels and delayed expression of HGF and c-met mRNA in the remnant liver. Here, we investigated whether syngeneic isolated hepatocytes transplanted in the spleen will prolong survival and facilitate liver regeneration in FHF rats. Inbred male Lewis rats were used. Group I rats (n = 46) received intrasplenic injection of 2 x 10(7) hepatocytes and 2 days later FHF was induced. Group II FHF rats (n = 46) received intrasplenic injection of saline. Rats undergoing partial hepatectomy of 68% (PH; n = 30) and a sham operation (SO; n = 30) served as controls. In 20 FHF rats (10 rats/group), survival time was determined. The remaining 72 FHF rats (36 rats/group) were used for physiologic studies (liver function and regeneration and plasma growth factor levels). In Group I rats survival was longer than that of Group II controls (73 +/- 22 hr vs. 33 +/- 9 hr; P < 0. 01). During the first 36 hr, Group I rats had lower blood ammonia, lactate, total bilirubin, PT, and PTT values, lower activity of liver enzymes, and higher monoethylglycinexylidide (MEGX) production than Group II rats. In Group I rats, livers increased in weight at a rate similar to that seen in PH controls and showed distinct mitotic and DNA synthetic activity (incorporation of bromodeoxyuridine and proliferation cell nuclear antigen expression). Plasma HGF and TGF-beta1 levels in these rats decreased and followed the pattern seen in PH rats; additionally, c-met expression in the remnant liver was accelerated. Hepatocyte transplantation prolonged survival in FHF rats and facilitated liver regeneration. Even though the remnant liver increased in weight four times reaching 30% of the original liver mass, the transplant-bearing rats expired due to inability of the regenerating liver to support the rat.

Differential patterns of reaction of human natural antibodies to pig hepatocytes and vascular endothelium.

We have recently conducted a series of experiments to characterize the pattern of reaction of human natural antibodies (NA) with individual pig liver cells. Pooled normal human serum (PHS) was incubated with cultured pig hepatocytes (HEP), aortic endothelial cells (AEC), and portal endothelial cells (PEC), and the reaction of NA to different cell types was measured by antibody-mediated cytotoxic (MTT assay), antibody binding (ELISA), and flow cytometric analysis. The human NA displayed a differential pattern of binding with hepatocytes exhibiting a more limited expression of xenoantigen expression than either aortic or portal endothelial cells. These differences in reaction patterns were also noted for Western blot analysis of individual cell membrane extracts. Preincubation of the pig cells with anti-pig MHC antibodies did not inhibit the binding of human IgM natural antibodies to the pig cells. Comparison of the pattern of NA absorption following the use of bioartificial liver support in patients with acute hepatic failure demonstrated limited ability of pig hepatocytes to absorb substantial amounts of NA. These studies indicate that pig hepatocytes are less vulnerable to NA cytotoxicity than pig vascular endothelial cells and that pig vascular endothelial cells express xenoantigens that are unique and not found on hepatocytes.

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

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

Nucleotide sequence analyses of the genes encoding the HN, M, NP, P, and L proteins of two host range mutants of Sendai virus.

Comparative nucleotide sequence analyses of the genome of Sendai virus (strain Z) and two host range mutants, ts-f1 and F1-R, previously described revealed that the ts defect of ts-f1 can be attributed to two nucleotide exchanges in the NP gene. These exchanges lead to a single amino acid substitution. A single base pair change was found in both the P and L genes of F1-R, but not of ts-f1. Both host range mutants have the two same exchanges in the M gene. These additional mutations are discussed concerning their significance in the pantropic properties of the host range mutants.