Yvette Middleton

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.

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.

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.

Intrasplenic transplantation of encapsulated cells: a novel approach to cell therapy.

Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained 1 × 107 rat hepatocytes. Human HepG2 cells were encapsulated using alginate/poly-l-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6–10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.

Identification and characterization of monoclonal antibodies that partially block human natural antibody binding to pig endothelial cell xenoantigens

Abstract: The shortage of human donors for clinical transplantation has led to a serious consideration of the use of non‐human species as organ donors. The major barrier to the clinical use of xenografts from species such as the pig in human transplantation has been the aggressive nature of the immune‐mediated rejection of the graft. We have recently identified the molecular weights of several endothelial cell surface proteins that may be targets of human antibody‐mediated responses to pig aortic endothelial cells (PAEC). In this series of experiments, we produced a panel of rat monoclonal antibodies (Mabs) to PAEC in an effort to identify Mabs that detect pig xenoantigens. Mabs were selected based on flow cytometric binding to PAEC, pig platelets, and various pig cell lines, including a pig kidney cell line (LLC‐PK1) reported to react with human natural antibodies (HNA). Eleven of the eighty‐three antibodies produced were cytotoxic for PAEC. Six of the cytotoxic clones recognized a 44 kDa protein and two of the clones recognized a 115 kDa protein expressed on the surface of PAEC. Since PAEC target antigens recognized by human natural antibodies include both 115 and 44 kDa antigens, these Mab clones were selected for further study. Several distinct patterns of tissue reactivity were demonstrated within this group of antibodies by immunohistochemical analysis; however all monoclonal antibodies were highly reactive with endothelial cells in all tissues examined. Two monoclonal antibodies recognizing antigens that are highly expressed on pig endothelial cells (92–98%) and pig platelets (74–92%), but moderately expressed on pig splenocytes (33–38%), were capable of reproducibly blocking 48–53% of human IgM binding to pig endothelial cells when analyzed with flow cytometry. This data suggests that these Mabs may recognize epitopes of potential significance in the human‐to‐pig xenograft reaction.