Jacqueline Blanchard

Rapid clearance of transplanted hepatocytes from pulmonary capillaries in rats indicates a wide safety margin of liver repopulation and the potential of using surrogate albumin particles for safety analysis

BACKGROUND/AIMS Applications of liver repopulation by hepatocyte transplantation require analysis of cell biodistributions, particularly when portasystemic shunting coexists. The aims of this study were to determine the fate of hepatocytes transplanted into the pulmonary vascular bed and to examine whether cell biodistributions could be approximated by convenient surrogates. METHODS Rat hepatocytes and macroaggregated serum albumin particles of similar sizes were injected into the portal and pulmonary vascular beds of rats, followed by biodistribution, survival and function analyses. RESULTS Although functionally intact, virtually all hepatocytes were cleared from the pulmonary capillaries within 24 h. Serum albumin levels increased minimally in Nagase analbuminemic rats with or without portacaval shunting to enhance delivery of portal factors to transplanted cells in lungs. Despite intravenous injection of hepatocytes approaching >1x10(9) cells in humans, the hemodynamic changes were limited to transient increases in right atrial pressures. The hepatocyte distributions in specific vascular beds were largely reproduced by macroaggregated human serum albumin particles. CONCLUSIONS Incidental intrapulmonary cell translocations during liver repopulation will have a wide safety margin. Use of macroaggregated serum albumin particles as surrogates for initial short-term biodistribution and safety analysis will advance hepatocyte transplantation, as the cost of GLP-certified laboratories and consumption of scarce donor livers will be avoided.

Intrasplenic hepatocyte allotransplantation in dalmatian dogs with and without cyclosporine immunosuppression

Hepatocyte allotransplantation has been performed successfully in several small animal models for the amelioration of inborn metabolic errors. Before a human clinical trial of hepatocyte allotransplantation can be attempted, preliminary experience in a large animal model is needed. We transplanted isolated mongrel hepatocytes into the spleen of dalmatians in the attempt to cure their inborn error of uric acid metabolism. Of 10 dalmatian recipients, two that received 9-10 x 10(9) mongrel hepatocytes died early after surgery of acute portal hypertension and hemorrhage. The eight long-term survivors received 5-6 x 10(9) hepatocytes and were randomized either to no treatment or to oral cyclosporine (CsA). Levels of CsA were adjusted to maintain trough levels between 400 and 800 ng/ml. In the four nonimmunosuppressed dalmatians, a reproducible average reduction in urinary uric acid excretion (UUAEx) of 23.7% was achieved; values returned to baseline within 14 days. In the CsA-immunosuppressed dalmatians, the average decline in UUAEx was 30%. The partial correction of the metabolic defect persisted for an average of 25 days in three immunosuppressed dogs, whereas in one dog, the partial correction lasted for over 90 days. No change in UUAEx was observed in two dalmatians that underwent sham laparotomy and intrasplenic injection of saline solution; CsA given alone to dalmatians did not modify UUAEx. We conclude that the dalmatian dog is a valuable large animal model for studies of the role of hepatocyte transplantation in the cure of inborn hepatic metabolic errors.

Transplantation of Hepatocytes: An In-Vitro and In-Vivo Study in Canines

We isolated and transplanted hepatocytes in the canine, large animal model to evaluate hepatocyte yield and purity as well as the optimal site for hepatocyte engraftment (i.e., the spleen or the portal bed). We obtained viable, pure, single hepatocyte suspensions that were readily preserved at 4°C in University of Wisconsin (UW) solution for up to 3 days. Both intrasplenic and portal vein injection were well tolerated, with minimal recipient morbidity and mortality when 1-2 × 109 hepatocytes were injected into immunosuppressed allogeneic hosts. We noted the embolization of hepatocytes into the parenchyma of the native liver within 7 days of intrasplenic transplantation that produced a mild reversible derangement of liver function and histology. These results warrant consideration prior to clinical trials of hepatocyte transplantation in man.

Effectiveness of prostaglandin E1 and procarbazine hydrochloride in prolonging the survival of vascularized cardiac hamster-to-rat xenograft.

The immunosuppressive effects of prostaglandin E1 and procarbazine hydrochloride were compared in a vascularized organ xenograft model. Hamster-to-rat cardiac grafts were used. Both procarbazine hydrochloride and prostaglandin E1 prolonged survival of the xenograft. Prostaglandin E1 had the additional feature of coincident suppression of hemagglutination titer to hamster red cells. Prostaglandin E1 was somewhat less effective than procarbazine hydrochloride.

A new rat model to study the correlation of cardiac and skeletal muscle allograft rejection

As a first step to study the correlation of cardiac and skeletal muscle allograft rejection, we describe a new experimental rat model of simultaneous heterotopic heart and cutaneous maximus muscle flap allotransplant. Brown Norway rats were used as donors and Lewis rats as recipients. No immunosuppression was given. The grafts were revascularized with sequential end‐to‐side anastomosis of each vascular pedicle to the infrarenal aorta and vena cava. Syngeneic heart and cutaneous maximus muscle grafts remained functional and showed no sign of rejection at 7 days after the transplant. In contrast, both allografts developed severe rejection and functional compromise at 7 days after the transplant. Our experimental model is technically feasible and reproducible and may provide important information about the pattern of rejection of cardiac and skeletal muscle allografts.

Endothelial proliferation in experimental granulomatous colitis. Autoradiography and immunohistochemistry studies.

The time sequence and magnitude of endothelial cell proliferation was investigated in an experimental model of granulomatous colitis in rats, induced by intramural inoculations of mycobacterium Bacillus Calmette-Guerin. Colonic tissues were assessed by gross examination, histopathology, autoradiography, and immunohistochemistry. Gross examination of the colonic tissue showed thickening of the colonic wall, erythema, hemorrhage, and scattered ulcers. Histopathological findings were characterized by an acute transmural inflammation, progressing to chronic inflammation accompanied by regenerative changes in the glandular epithelium, goblet cell depletion, mucosal atrophy and fibrosis. Well-developed noncaseating granulomas were first observed at day 5 and were found to be a dominant feature up to day 17. Autoradiographic studies showed increased endothelial cell labeling up to 17% at 48 hr, compared to less than 1% labeling in control animals. Immunostaining for factor VIII-related antibody, an endothelial cell marker, showed increased numbers of microvessels and individual positive cells located in areas of inflammation as early as 24 hr. At day 5 these individual cells along with dilated neocapillaries were found surrounding the granulomas. This model of granulomatous colitis mimics many features of the human disease state. The early increase in endothelial cell proliferation that precedes granuloma formation during the course of the inflammatory response may suggest that the events leading to the expression of granulomatous colitis are dependent on endothelial proliferation.