Can clamping of splenic vessels prevent abrupt increase of portal vein pressure and migration of transplanted hepatocytes to the liver after intrasplenic hepatocyte transplantation?

BACKGROUND/AIMS: It is well known that hepatocyte transplantation can retain some proper functions, significantly improve the survival rate of rats with different models of acute fulminant hepatic failure, correct some congenital genetic disorders, and improve liver function in cirrhosis. Portal hypertension and hepatic embolization have been described following intrasplenic hepatocyte transplantation. We evaluated the effect of temporary occlusion of splenic vessels on changes in portal vein pressure and on distribution of transplanted hepatocytes after hepatocyte transplantation into the spleen in normal rats. METHODOLOGY: Liver cirrhosis has been induced in rats by 1% dimethylnitrosamine (Sigma, St. Louis, Mo) dissolved in normal saline at the dose of 10 ml of DMN/Kg, i.p., 3 consecutive days a week for 4 weeks. Donor hepatocytes were harvested by in situ ethylenediaminetetraacetic acid (EDTA) perfusion. Changes in portal vein pressures were monitored by a pressure monitor and distribution of transplanted hepatocytes was assayed by measurement of radioactivity of 51Cr-labeled transplanted hepatocytes according to clamping or non-clamping during intrasplenic hepatocyte transplantation. RESULTS: The changes in portal pressure remained significantly high 10 min after hepatocyte transplantation in the nonocclusion groups compared to the occlusion groups. However, the changes in portal vein pressures in cirrhotic rats returned to normal faster than in normal rats after cell transplantation in the nonocclusion groups. The distribution of 51Cr-labeled transplanted hepatocytes into the spleen significantly diminished radioactivity of the liver at 10 min, 2 hours, and 24 hours in the occlusion groups compared to the nonocclusion groups. Also, duration of clamping time of splenic vessels did not influence the initial distribution of transplanted hepatocytes at the time of intrasplenic hepatocyte injection. CONCLUSIONS: These results suggested that temporary occlusion of splenic vessels should be routinely used during intrasplenic hepatocyte transplantation.     

Entry and integration of transplanted hepatocytes in rat liver plates occur by disruption of hepatic sinusoidal endothelium

To establish the process by which transplanted cells integrate into the liver parenchyma, we used dipeptidyl peptidase IV-deficient F344 rats as hosts. On intrasplenic injection, transplanted hepatocytes immediately entered liver sinusoids, along with attenuation of portal vein radicles on angiography. However, a large fraction of transplanted cells (>70%) was rapidly cleared from portal spaces by phagocyte/macrophage responses. On the other hand, transplanted hepatocytes entering the hepatic sinusoids showed superior survival. These cells translocated from sinusoids into liver plates between 16 and 20 hours after transplantation, during which electron microscopy showed disruption of the sinusoidal endothelium. Interestingly, production of vascular endothelial growth factor was observed in hepatocytes before endothelial disruptions. Portal hypertension and angiographic changes resulting from cell transplantation resolved promptly. Integration of transplanted hepatocytes in the liver parenchyma required cell membrane regenesis, with hybrid gap junctions and bile canaliculi forming over 3 to 7 days after cell transplantation. We propose that strategies to deposit cells into distal hepatic sinusoids, to disrupt sinusoidal endothelium for facilitating cell entry into liver plates, and to accelerate cell integrations into liver parenchyma will advance applications of hepatocyte transplantation.     

A single intravenous dose of murine megakaryocyte growth and development factor potently stimulates platelet production, challenging the necessity for daily administration

Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) have been implicated in the pathogenesis of various inflammatory liver disease states, including viral and autoimmune hepatitis as well as liver allograft rejection. Tumor necrosis factor alpha (TNF-alpha) is an inflammatory cytokine known to up-regulate adhesion molecules as well as major histocompatibility complex (MHC) class I expression, and has been demonstrated to be important in the rejection of vascularized organ allografts. The current studies address the effect of TNF-alpha and the role of ICAM-1 expression on liver cell immunogenicity in vitro in mixed lymphocyte hepatocyte culture (MLHC), in vitro in mixed lymphocyte liver nonparenchymal cell culture (MLNPC), in vivo in hepatocyte sponge matrix allografts (HC-SMA), and in vivo in liver nonparenchymal cell sponge matrix allografts (NPC-SMA). Purified allogeneic hepatocytes (HC) and liver nonparenchymal cells (NPC) under naive, unstimulated conditions demonstrated different profiles of MHC antigen and adhesion molecule expression, but both liver cell populations stimulated the proliferation and development of allospecific cytotoxic effectors in vitro and in vivo. Despite significant up-regulation of MHC class I and ICAM-1 on both HC and liver NPCs by in vivo treatment with TNF-alpha, the immunogenicity of TNF-alpha-stimulated liver cells was not appreciably different from naive, unstimulated liver cells. In contrast, ICAM-1-negative HC and NPCs were significantly less immunogenic both in terms of lymphocyte proliferative responses and the generation of allospecific cytolytic effectors. These results suggest that constitutive expression of ICAM-1 enhances the immunogenicity of "donor" liver cells but is not absolutely required to elicit immune responses to allogeneic liver cells. Further studies to determine the role of adhesion molecule expression on trafficking of host immune cells to the liver and the role of adhesion molecule expression by host cells are required to clarify their role in immune responses to liver cells.     

Immunosuppressive effects of human CTLA4Ig in a non-human primate model of allogeneic pancreatic islet transplantation

Ag-specific T cell activation requires a CD28-mediated costimulatory interaction. This observation has suggested novel approaches to suppress donor-specific immunity, including the use of soluble CD28 antagonists, such as CTLA4Ig, which suppresses transplant rejection in small animal models. In this study, CTLA4Ig therapy was examined in a non-human primate model of allogeneic pancreatic islet transplantation. Two of five CTLA4Ig-treated monkeys showed prolonged graft survival, which correlated with donor-specific hyporesponsiveness in vitro. Humoral responses to the transplanted tissue were suppressed in all treated animals. These results suggest that CTLA4Ig is effective in suppressing both humoral and cellular immune responses in a non-human primate model of allogeneic transplantation.     

Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats

To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P < .01). Additionally, at 12 hours post-hepatectomy, transplanted rats had significantly lower blood ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.     

Amyloid precursor protein fragment and acetylcholinesterase increase with cell confluence and differentiation in a neuronal cell line

BACKGROUND: Physiopathology of hepatic encephalopathy remains unclear. Recent studies have suggested that ammonia would not act by itself but through an increase in glutamine in the brain. We have previously demonstrated that transplantation of syngeneic hepatocytes into the spleen was able to correct both behavioral deficits and plasma amino acid changes observed in portacaval shunted rats. The aim of the present work was to show a correlation between the correction of chronic hepatic encephalopathy by means of intrasplenic hepatocyte transplantation and two parameters, brain glutamine concentration and ultrastructural aspects of astrocytes. METHODS: Inbred male Wistar Furth rats were divided into three groups: sham-operated rats (n = 10), rats subjected to portacaval shunt (n = 10), and rats subjected to portacaval shunt and intrasplenic hepatocellular transplantation of 10(7) hepatocytes isolated from livers of syngeneic rats (n = 10). Chronic hepatic encephalopathy was quantified 30 and 60 days after operation by means of nose-poke exploration and spontaneous activity. Pathologic examination and measurement of glutamine concentrations in the corpus striatus and in the cerebral cortex were performed 60 days after operation. RESULTS: Portacaval shunt rats showed reduced spontaneous activity and nose-poke exploration scores. After portacaval shunt a significant glutamine increase occurred in the corpus striatus and in the cerebral cortex when compared with sham rats (p < 0.05). Ultrastructural examination showed modification of astrocytes named Alzheimer type II after portacaval shunt. Correction of behavioral abnormalities by means of intrasplenic hepatocyte transplantation was associated with partial correction of striatal glutamine increase and with decrease in astrocyte alterations. Cortex glutamine concentration in portacaval shunt-intrasplenic hepatocyte transplantation group and in portacaval shunt rats did not differ significantly. CONCLUSIONS: These data show that intrasplenic hepatocyte transplantation not only prevents neurologic disorders of hepatic encephalopathy but can also decrease glutamine and ultrastructural alterations in the corpus striatus in an experimental model of chronic liver failure. These data are in favor of the involvement of glutamine in chronic hepatic encephalopathy. These results suggest that intrasplenic hepatocyte transplantation might be of therapeutic interest in chronic liver failure.     

Costimulation pathways in host immune responses to allogeneic hepatocytes

BACKGROUND: This study investigated the role of the CD28/B7 (blocked by CTLA4Ig) and CD40/CD40L (blocked by MR1) costimulation pathways on in vivo host T-cell- mediated immune responses to allogeneic hepatocytes. METHODS: Survival of allogeneic hepatocytes (H-2q) in C57BL/6 (H-2b) mice untreated or treated with MR1, CTLA4Ig, L6 (control fusion protein), or a combination of MR1 and CTLA4Ig fusion protein was determined. RESULTS: Median survival time for hepatocellular allografts was 10, 84, 10, 10, and 84 days in untreated (n= 10), MR1-treated (n=7) (P<.0001), CTLA4Ig-treated (n=7) (P=0.02), L6-treated (n=3) (P, not significant), and the combination of MR1- and CTLA4Ig-treated (n=6) (P=0.0003) groups, respectively. CONCLUSIONS: Host treatment with MR1, but not CTLA4Ig, prolonged hepatocellular allograft survival. These data suggest that CD28/B7 interactions appear relatively unimportant, whereas CD40/CD40L interactions provide critical costimulator signals for T-cell-dependent immune responses to allogeneic hepatocytes.     

The lack of long-term detrimental effects on liver allografts caused by donor-specific anti-HLA antibodies

Recent reports indicate a higher incidence of both acute and chronic liver allograft rejection when, at the time of transplantation, the recipients serum contains donor-specific anti-HLA antibodies. From 9/89 to 5/91, 133 liver allografts were performed at our institution. Thirteen liver recipients had donor-specific IgG anti-HLA antibodies (complement-fixing) at the time of transplantation. In eleven patients, antibodies reacted to donor class I antigens while in 1 patient the donor-specific antibody had class II reactivity. Twelve patients have been followed for a minimum of 12 months (median 18 months, range 28-12 months). No hyperacute rejection was seen in any of the cases and four patients had acute rejections. Thus far only one of the twelve patients has biopsy evidence suggestive of chronic liver injury. The remaining have normal liver enzymes and bilirubin. Three of these twelve patients died (one from a myocardial infarction and the others from sepsis) accounting for a one-year graft survival of 75%. There was no significant statistical difference in the one-year graft survival in those recipients without donor-specific antibodies (i.e., 80.5%). In eight of the twelve patients, pretransplant preformed antibody level (PRA) was > 50%. In six of the thirteen patients donor-specific antibody was present at dilutions greater than 1:64. As previously reported, the donor-specific antibody disappeared from the serum posttransplant within hours and did not reappear. In vitro studies demonstrated no factor in portal or hepatic artery blood that could inhibit rabbit complement mediated lysis of anti-HLA antibodies. We conclude that it is not a contraindication to do liver transplants in the presence of donor-specific anti-HLA antibodies.     

Evidence for immune regulation in the induction of transplantation tolerance: a conditional but limited role for IL-4

Most experimental models of allograft tolerance depend on manipulation of immune responses at the time of transplant. In such systems, the graft itself probably plays an important role in the induction of unresponsiveness but as a consequence may suffer immune mediated damage. Ideally, recipients would be made specifically unresponsive before transplant such that the graft is protected from the outset. In this report, we demonstrate that CBA mice pretreated with donor-specific transfusion plus anti-CD4 Ab 28 days before transplant accept cardiac allografts indefinitely without further intervention. Adoptive transfer of spleen cells from mice with long term surviving grafts results in donor-specific graft acceptance in naive secondary recipients, indicating that tolerance in this system involves immuneregulation. Regulation develops as a result of the pretreatment protocol alone, since transfer of cells from pretreated but untransplanted mice to naive recipients also leads to prolonged allograft survival without additional therapy. Neutralizing IL-4 at the time of tolerance induction had no effect on graft outcome in primary recipients. However, removal of IL-4 from the adoptive transfer donors at the time of tolerance induction prevented long term engraftment in the majority of secondary recipients. Our data demonstrate that pretreatment of transplant recipients can establish immune regulation powerful enough to override the responses of an intact immune repertoire and that under stringent conditions at least, development of this regulatory population may in part be dependent on IL-4.     

Liver tumor targeting of drugs: Spherex, a vascular occlusive agent

In order to determine whether the donor-specific T cell allorepertoire evaluated in patients before transplantation can be predictive for kidney graft survival, a study has been set up in which the number and activation state of donor-specific T lymphocytes before transplantation were correlated to transplant survival time. Limiting dilution analysis assays were carried out to determine precursor frequencies of both T helper and cytotoxic T lymphocytes. The activation state of these cells was studied by evaluating the inhibitory capacity of cyclosporine on helper and cytotoxic T cells and/or a monoclonal antibody directed against CD8 on cytotoxic T cells. This study shows that neither a significant difference in the number nor activation state of donor-directed helper and cytotoxic T cells before transplantation could be detected when patients who acutely rejected their grafts were compared with patients who still had a well-functioning kidney graft after more than 10 years. Moreover, no significant differences in the donor-specific T cell repertoire were found when patients who had been subject to multiple rejection episodes were compared with patients who experienced few complications after transplantation. Therefore, we conclude that in individuals who have not been sensitized to HLA antigens of the donor, the donor-specific peripheral T cell allorepertoire prior to transplantation is not predictive of kidney graft survival.     

Tolerance to experimental cardiac allografts produced by neonatal intrathymic injection of donor cells

Intrathymic inoculation of allogeneic cells after systemic administration of antilymphocyte serum in adult experimental animals has produced donor-specific tolerance to cardiac allografts. We investigated whether thymic injection of allogeneic cells without antilymphocyte serum in neonatal Lewis rats (day 1 of life) with immature immune systems also produced tolerance to cardiac grafts. Intrathymic or intraperitoneal injection of 5 x 10(7) Lewis (control) or Lewis-Brown Norway (allogeneic) spleen cells in Lewis neonates was followed by heterotopic cardiac transplantation using Lewis, Lewis-Brown Norway, or Wistar Furth (third-party allograft) hearts at 6 to 8 weeks of age. Graft survival was prolonged with both intraperitoneal and intrathymic allogeneic cells. Recipients of cells by the intrathymic route had longer graft survival, and 2 of 5 animals achieved permanent graft acceptance (longer than 100 days). As expected, Lewis isografts survived indefinitely, whereas third-party Wistar Furth allografts were rejected in the usual time frame. Intrathymic introduction of allogeneic cells in a neonatal recipient with an immature immune system can produce donor-specific tolerance to a subsequent graft without the need for a systemic immunosuppression regimen, even transiently.     

Evaluation of in vitro precipitation methods

As CD44 is believed to be a homing receptor involved in lymphoid trafficking and inflammatory responses, it is expected to be closely linked to transplant rejection. In this study, the expression of CD44 during liver transplant rejection was compared with the expression of lymphocyte-function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), which play an essential role in cell interactions and the initiation of immune responses. Male Brown Norway (BN) and Lewis (LEW) rats were used as donors and recipients, respectively. Orthotopic liver transplantation (OLTX) was done using the cuff technique of Kamada and Calne. Animals were killed on days 3, 5, and 7 after OLTX, and a piece of tissue from each of the liver grafts was obtained. Immunohistochemical staining was used to investigate the expression of CD44, ICAM-1, and LFA-1. CD44 was strongly expressed in portal areas of the rejected liver, and LFA-1 and ICAM-1 were expressed mainly on sinusoids and hepatocytes. These findings indicate that CD44 is closely involved in lymphocyte infiltration, which is dominant in portal areas, and that lymphocyte infiltration during the rejection process may involve a homing mechanism.     

Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice

Allogeneic transplantation of islets of Langerhans was facilitated by the cotransplantation of syngeneic myoblasts genetically engineered to express the Fas ligand (FasL). Composite grafting of allogeneic islets with syngeneic myoblasts expressing FasL protected the islet graft from immune rejection and maintained normoglycemia for more than 80 days in mice with streptozotocin-induced diabetes. Graft survival was not prolonged with composite grafts of unmodified myoblasts or Fas-expressing myoblasts. Islet allografts transplanted separately from FasL-expressing myoblasts into the contralateral kidney were rejected, as were similarly transplanted third-party thyroid allografts. Thus, the FasL signal provided site- and immune-specific protection of islet allografts.     

Mechanisms of intrathymic tolerance induction to isolated rat hepatocyte allografts

Intrathymic injection of alloantigen in young adult rats is capable of mediating long-lived transplantation tolerance. In this study, we use a well-defined model of isolated hepatocyte transplantation to define the mechanisms of intrathymic induced tolerance. The recipient rats are Nagase analbuminemic rats (NAR) that are deficient in albumin, to allow for following transplant acceptance using metabolic and genetic markers. Tolerance to allogeneic hepatocyte transplants could be mediated by intrathymic injection of live allogeneic splenocytes, lethally irradiated splenocytes, or isolated hepatocytes. Intrathymic injection of live allogeneic splenocytes, but not of hepatocytes or irradiated splenocytes, resulted in donor microchimerism in peripheral lymphoid organs, with preferential expansion of CD4-positive T cells in the recipient spleens. Tolerance could be adoptively transferred from tolerant animals to naive recipients, but only from those animals that had been inoculated with intrathymic donor splenocytes. We conclude that donor microchimerism is found after intrathymic inoculation of live splenocytes, but is not required for tolerance induction and that microchimerism is not an absolute requirement for the generation of regulatory cells.     

Graft-versus-host-disease-associated lymphoid hypoplasia and B cell dysfunction is dependent upon donor T cell-mediated Fas-ligand function, but not perforin function

Allogeneic bone marrow transplant recipients often exhibit a graft-versus-host-disease (GVHD)-associated immune deficiency that can be prolonged and lead to life-threatening infections. We have examined the role of donor T cell-mediated cytotoxic function in the development of GVHD-associated immune deficiency. A major histocompatibility complex-matched model of allogeneic bone marrow transplantation was employed in which lethally irradiated C3H.SW mice received a nonlethal dose of T cells from either perforin-deficient (B6-perforin 0/0), Fas-ligand (FasL)-defective (B6-gld), or normal (B6) allogeneic donor mice. T cell-depleted marrow from B6-Ly-5.1 congenic donor mice was transplanted along with the donor T cell populations to determine the effects of donor T cell-mediated cytotoxicity on engraftment. Our results demonstrate that recipients of perforin-deficient or normal allogeneic T cells exhibit profound lymphoid hypoplasia and severely reduced splenic proliferative responses to lipopolysaccharide in vitro. In contrast, GVHD-associated lymphoid hypoplasia is dramatically reduced and in vitro B cell function is intact in recipients of FasL-defective allogeneic T cells. Engraftment of myeloid and erythroid lineage cells occurs irrespective of donor T cell cytotoxic function. Although recipients of perforin-deficient or normal allogeneic T cells exhibited hematopoietic engraftment exclusively of donor origin, recipients of FasL-defective donor T cells exhibited significant mixed chimerism (Ly-5.1/Ly-5.2). Because only marrow of donor origin was transplanted, this finding suggests that Fas-mediated antirecipient cytotoxicity is required for clearance of residual hematopoietic stem cells of host origin that persist following lethal irradiation.     

Heterotopic liver transplantation in rats: effect of intrahepatic islet isografts and split portal blood flow on liver integrity after auxiliary liver isotransplantation

BACKGROUND: Auxiliary heterotopic liver grafts atrophy in the absence of portal venous inflow; evidence suggests that an islet-derived hepatotrophic factor may exist in the portal drainage. Here we examine the effects of intrahepatic islet isografts in maintaining hepatocyte integrity in Wistar Furth rats with one of several types of arterialized auxiliary liver isografts. METHODS: In type 1 procedures the auxiliary liver was interposed into the recipient infrarenal vena cava and perfused through the graft portal vein with caval blood. In type 2 procedures the donor infrahepatic vena cava was anastomosed end-to-side to the recipient vena cava and the recipient portal vein was diverted to the graft portal vein. Both types of auxiliary grafts were arterialized; bile duct drainage was through the duodenum. Syngeneic islets were isolated and embolized into the portal veins of one half of the donor type 1 or native type 2 livers (1500 to 1700 islets). Finally, we performed six type 3 procedures in which a type 2 procedure was performed except that the portal blood flow was split so that the portal vein receiving the splenic, gastric, pancreatic, and duodenal drainage supplied the native liver and that the common mesenteric vein supplied the auxiliary graft with equivalent portal blood flow. Atrophy in heterotopic and native livers were compared for the three models after 3 months. RESULTS: Intrahepatic islets in type 1 auxiliary liver isografts without portal venous inflow did not prevent graft atrophy. Conversely, native livers deprived of portal venous inflow in our type 2 procedures, regardless of the presence of intrahepatic islet isografts, atrophied relative to auxiliary liver grafts in which portal venous inflow was provided by diverting the recipient's portal vein to the graft. In type 3 recipients atrophy was greater in the native livers than in the grafts. CONCLUSIONS: The results of our study suggest that islet-derived factors are not sufficient to prevent hepatocellular atrophy in auxiliary rat liver transplantation models and that a potent hepatotrophic factor may exist in the venous drainage of the bowel distal to the duodenum.     

Arthroscopic repair of full-thickness tears of the rotator cuff

OBJECTIVE: The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. SUMMARY BACKGROUND DATA: Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. METHODS: To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. RESULTS: In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). CONCLUSIONS: Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.     

Induction of donor-specific ACAID can prolong orthotopic corneal allograft survival in "high-risk" eyes

PURPOSE: To examine the effect of donor-specific anterior chamber-associated immune deviation (ACAID) induction on the survival of orthotopic corneal allografts in neovascularized graft beds. METHODS: To induce donor-specific ACAID in recipients, peritoneal exudate cells (PEC) from C57BL/6 mice were incubated overnight with transforming growth factor (TGF)-beta. Cultured PEC were injected intravenously (i.v.) into BALB/c mice, and, 1 week later, these animals received orthotopic corneal allografts from C57BL/6 donors into neovascularized graft beds. Control mice received i.v. injection of syngeneic (BALB/c) PEC, cultured overnight with TGF-beta, and then received orthotopic corneal allografts from C57BL/6 donors. RESULTS: All corneal allografts (15 out of 15) were rejected within 2 weeks after grafting in the neovascularized graft beds of control animals. However, only 6 out of 16 (37.5%) of corneal allografts were rejected in recipients in which donor-specific ACAID had been induced by injection of allogeneic PEC cultured with TGF-beta. CONCLUSION: Previous studies revealed that rejection of orthotopic corneal allografts in neovascularized graft beds in mice correlated with acquisition of donor-specific delayed hypersensitivity (DH). The results of this study suggest that induction of donor-specific ACAID, which selectively impairs DH responses to donor antigens, effectively prolongs corneal allograft survival in "high-risk" eyes.     

Cationic lipid-mediated transfer of the hIL-10 gene prolongs survival of allogeneic hepatocytes in Nagase analbuminemic rats

Gene transfer techniques can be used as a drug delivery system to achieve local immunosuppression. We performed a series of experiments to identify the cationic lipid that most efficiently transfects isolated, cultured, rat hepatocytes; to optimize conditions for efficient transfection; to determine the duration of gene expression in vitro; and finally, to determine the survival of allogeneic hepatocytes transplanted into Nagase rats. Our results suggest that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. In addition, the following conditions appear to optimize transfection efficiency: a DNA:DOTAP ratio of 1:6; a 24 exposure time of the hepatocytes to the DNA-DOTAP complex; a DNA dose of 4 microg/35 mm culture plate seeded with 2.5x10(5) rat hepatocytes. When transfected as described above, cultured hepatocytes expressed the hIL-10 gene for approximately 14 days. Accordingly, Nagase rats transplanted with 4x10(7) DOTAP-hIL-10 transfected, allogeneic hepatocytes had an abrupt rise in serum albumin levels that peaked within 7 days of the transplant, decreased abruptly after 15 days, and approached baseline by day 40. In contrast, control animals had a smaller albumin peak that returned to baseline within 10 days (P<0.01). In all animals, serum hIL-10 levels were undetectable when tested. We conclude that DOTAP is the best cationic lipid for transfection of cultured rat hepatocytes. Furthermore, hIL-10 transfected hepatocytes have a prolonged survival in an allogeneic host which is probably limited by loss of gene expression. Further studies using other vectors capable of prolonged gene expression will help determine if indefinite hIL-10 gene expression leads to indefinite graft survival.     

Hepatotoxins and liver transplantation decrease pulmonary metastases in rats with hepatoma

Results following liver transplantation for hepatocellular carcinoma have been dismal, attributed largely to recurrent disease locally or at distance sites. Undetected micrometastases or tumor that embolizes at the time of liver transplant from manipulation of the liver may account for these recurrences. A model and treatment protocol were developed to address this clinical problem. The protocol is modeled on the concept of bone marrow transplantation for leukemia. Hepatotoxins that are lethal to both normal hepatocytes and hepatoma cells are administered followed by liver transplantation to "rescue" the failing liver. The feasibility of this protocol was examined in a rat model. Male Buffalo rats were injected with 1 million Morris hepatoma MH-7777 cells intravenously at Day 0 as a model for micrometastatic disease. Three treatment groups were established. Group 1 received no treatment. Group 2 received 5% dextrose in water (D5W) followed by a syngeneic orthotopic liver transplant (OLTX). Group 3 received the hepatotoxin pyrazofuin (10 mg/kg) followed by OLTX. Animals were followed to Day 35, at which time they were sacrificed and examined for evidence of pulmonary metastases and quantitation of nodules with India ink insufflation. There was a significant decrease in the number with pulmonary nodules as well as the number of animals with pulmonary metastatic disease in the pyrazofurin-treated group compared with groups 1 and 2 (4.8 +/- 4.0 nodules/animal vs 45.2 +/- 11.2 nodules/animal--no treatment and 60.8 +/- 21.4 nodules animal--D5W/OLTX group) These data indicate that this model is reliable for examining metastatic hepatoma and that pyrazofurin is effective in preventing hematogenous micrometastases of hepatoma cells. Other hepatotoxins and the effect of allogeneic transplantation and immunosuppression could be examined in this model.