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Objective: The aims of the study were to determine the effects of denervation on the function of the liver transplantation as a blood reservoir and to define its vulnerability to ischemic-reperfusion (I/R) injury after hemorrhagic shock., Materials and Methods: Hemorrhagic shock with a mean arterial blood pressure (MAP) of 40 to 50 mm Hg was induced by withdrawing blood at a rate of approximately 1 mL/min among 10 posttransplant denervated rats and 10 sham rats for 1 hour. The rats were then resuscitated by retransfusing the drawn blood with sacrifice under deep anesthesia at 1 hour after resuscitation. The total amount of blood required to achieve hemorrhagic shock was compared between groups as well as the vulnerability and reactions of the posttransplant denervated liver to I/R injury after hemorrhagic shock as assessed by gene expressions of c-jun, c-fos, tumor necrosis factor (TNF)-alpha, interleukin (IL)6, IL-10, and heat-shock protein 70 (HSP70)., Results: The volume of blood that had to be drawn to reach a MAP of 40 to 50 mm Hg was not significantly different between the groups. One hour of hemorrhagic shock followed by resuscitation resulted in significant increases in the genes expression of c-fos, TNF-alpha, IL-6, IL-10, and HSP70 in comparison to the control values, but no difference was observed between experimental and sham groups., Conclusion: We suggest that the function of the liver as a blood reservoir and the gene expressions of c-fos and pro- and anti-inflammatory cytokines, as well as the protective protein HSP70 in response to I/R injury, were not altered by liver transplantation.

Objective: In a rat tolerogenic orthotopic liver transplantation (OLT) model, the recipient serum (post-OLT serum) shows strong immunosuppressive activity. In our previous reports, we suggested that autoreactive antibody (Ab) against histone H1 is a major immunosuppressive factor in this serum. The present study sought to determine whether up-regulation of anti-histone H1 Ab by histone H1 vaccination led to tolerance., Materials and Methods: Using mixed lymphocyte reactions (MLR) and heterotopic heart transplantations (HHT), the alloreactive T-cell responses and allograft survivals of histone H1-immunized rats were compared with those of control rats. Cytokine and cellular profiles were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry., Results: The alloreactive T-cell response of histone H1-immunized rats was significantly lower than that of control rats, although there was no difference in nonspecific T-cell activation between the 2 groups. The allograft survival of histone H1-immunized rats was significantly prolonged after HHT. The major histocompatibility complex (MHC) class II and CD25 molecules of histone H1-immunized rats were significantly down-regulated compared with those of control rats. Moreover, the serum cytokine profile was modified by the immunization with histone H1., Conclusions: These results suggest that histone H1 vaccination of transplant recipients leads to the production of immunosuppressive factors and the modification of cytokine/cellular profiles.

Objective: We recently reported that autoreactive antibodies (Abs) against nuclear histone H1 was transiently induced at an early phase after orthotopic liver transplantation (OLT) in a tolerogenic rat OLT model and possessed immunosuppressive activity. It was also reported that nuclear antigen, high-mobility group box 1 (HMGB1) protein was one of the initiators of the immune reaction. The present study sought to evaluate the role of antinuclear Abs in experimental and clinical liver transplantation., Materials and Methods: We prepared 3 animal models: natural tolerance model (DA liver into PVG); acute rejection model (DA liver into LEW); and drug-induced tolerance model (acute rejection model + cyclosporine [CsA]). In addition, we examined clinical samples, including 1 drug-free patient, to measure the antihistone H1/HMGB1 titers at various times after OLT., Results: In a natural tolerance model, antihistone H1 and HMGB1 Ab was induced during the rejection and the tolerance induction phases, respectively. Those Ab responses were also confirmed in a drug-induced tolerance model, whereas no such responses were shown in an acute rejection model. In our clinical drug-free patient, antihistone H1/HMGB1 titer was significantly higher after cessation of CsA than that in healthy volunteers., Conclusions: Antinuclear Ab is actively expressed in accordance with overcoming rejection episodes with subsequent tolerance induction in both a natural tolerance model and a drug-induced tolerance model. We also observed a similar tendency in our clinical drug-free patient. These results suggested that antinuclear Abs may be useful markers to determine the timing to withdraw immunosuppressants.

Background: In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum., Methods: Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing., Results: Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction., Conclusions: In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1a) donor livers were transplanted into DA, PVG (RT1c), and LEW (RT1l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as alpha-glutathione sulfotransferase (alpha-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7-26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance.

A tryptophan catabolizer, indoleamine 2,3-dioxygenase (IDO) is highly expressed in the placenta and plays an essential role in maternal tolerance. Recent data have shown that the administration of an IDO inhibitor blocked not only maternal tolerance but also liver allograft tolerance. However, little is known about the induction of IDO in liver allografts, although a gene specific for tryptophan 2,3-dioxygenase (TDO) is believed to be expressed in the liver. In the present study, we investigated whether IDO is induced in liver allografts. Synthetic oligonucleotide primers based on the mouse IDO cDNA sequence were used to amplify RNA derived from livers of donor, syngeneic or allogeneic OLT rats. RNA encoding IDO was induced in the rat allogeneic liver after orthotopic liver transplantation (OLT), but not in syngeneic OLT. The rat nucleotide sequence of the RT-PCR products obtained from OLT livers revealed identities of 89% homology to the mouse IDO and of 68% to the human IDO. This study demonstrated the presence of RNA encoding IDO in allogeneic OLT livers, which may be involved in the immune response after liver transplantation.

Little is known about the possible role of complement inhibitors on tolerance induced by liver allografts. Clusterin, which is a plasma glycoprotein, inhibits cytolytic membrane attack complex (MAC) of complement by binding to soluble C5b-7 complex. The role of clusterin in relation to the naturally achieved tolerance in a rat orthotopic liver transplantation (OLT) has not been investigated before. Here we determined the kinetics of clusterin expression at different post-transplantation time points in a tolerogenic model (DA-PVG) where rejection was naturally overcome without any immunosuppressive drugs in comparison with the syngenic OLT model (DA-DA). Peripheral blood and liver tissues were taken from OLT at various post-operative time points. A strong expression of soluble clusterin was observed on post-transplantation day 7, which occurred at the peak of the rejection in this tolerogenic OLT model. The expression of clusterin remained strong even after tolerance was achieved. The intensity of clusterin expression was much stronger when compared with the syngenic OLT (DA-DA) model after OLT. A strong expression of clusterin mRNA was also observed in the tolerogenic model on post-OLT day (POD) 7 and the expression persisted when compared with the syngenic model on post-OLT day 60. Our data have shown that the strongest levels of clusterin during the reaction phase in tolerogenic OLT may be involved in tolerance induction.

Background: Telomerase activity in grafts may be involved in the alteration of cellular senescence after transplantation or its relevant immunological events., Methods: At the age of 20 weeks, donor livers harvested from DA (RT1a) were orthotopically transplanted into PVG (RT1c) or LEW (RT1(1)) rats. Rats having undergone orthotopic liver transplantation (OLT; DA-PVG) naturally overcome rejection, whereas all OLT (DA-LEW) rats die from acute rejection within 14 days. Telomerase activity in liver allografts was measured at various intervals post OLT., Results: At day 7 when the most severe rejection episode was observed in OLT (DA-LEW) and OLT (DA-PVG), the telomerase activity was significantly higher than in syngeneic OLT (DA-DA) rats, in which no rejection occurred. Telomerase activity in tolerogenic OLT (DA-PVG) livers remained elevated for at least 2 months., Conclusion: These results suggest that telomerase activity in allogeneic OLT livers may reflect regenerating hepatocytes or activation of lymphocytes and/or hematopoietic stem cells associated with rejection or tolerance.

The Fas and Fas ligand (Fas/FasL) pathways may play a central role in cytotoxicity or immunoregulation in liver transplantation. Here, in an attempt to examine the role of Fas/FasL on drug-free tolerance, we measured mRNA levels of Fas/FasL in livers by reverse transcriptase-polymerase chain reaction (RT-PCR), and also protein levels of Fas/FasL in livers by immunohistochemistry and in serum by dot blot assay. PVG recipients bearing DA livers showed serious rejection between post-operative (POD) days 7 and 14, but this rejection was naturally overcome without any immunosuppression. Fas gene and protein products were expressed on almost every cell in livers taken from naive rats, and at any time point in both syngeneic and allogeneic orthotopic liver transplantation (OLT) rats. In contrast, FasL mRNA in DA livers was detectable at POD 2, peaked at POD 14, and declined at POD 63 in allogeneic OLT (DA-PVG). Although the FasL gene was detectable in isografts at POD 14, its expression was much lower than in allografts. The time course and localization of FasL expression indicated that the expression of FasL gradually switched from infiltrating cells to hepatocytes when the rejection was naturally overcome and tolerance was induced in this OLT model. Soluble Fas could constitutively be detected at any time point in the serum of the tolerogenic OLT (DA-PVG) rats and was not diminished during the rejection phase. Soluble FasL peaked at POD 14 in allogeneic OLT, while sFasL was significantly lower in the serum of normal and syngeneic OLT rats. These findings suggest that the Fas and FasL pathways, including soluble forms, may contribute to the control of the immune response in this drug-free tolerance OLT model.

Abstract: Objectives: Our previous study noticed remarkably elevated titers of anti–high-mobility group box 1 (HMGB1) antibodies in sera during the tolerance induction phase of a rat tolerogenic orthotopic liver transplantation (OLT) as well as in sera of clinically drug-free patients. We hypothesized that the release of nonhistone nuclear protein HMGB1 during rejection may play a pathogenic role in deteriorating post-OLT graft functions, such as inducing liver fibrosis. This study sought to investigate whether HMGB1 can directly activate hepatic stellate cells (HSCs) and drive them toward fibrogenesis. Methods: The cultured HSCs were treated with recombinant HMGB1. RT-PCR and Western blotting analysis were used to measure α-smooth muscle actin (α-SMA) expression. Conditioned media were collected for gelatin zymography to monitor the activities of collagen-degrading matrix metalloproteinases (MMPs). Results: HMGB1 at concentrations >1 ng/mL significantly stimulated HSC growth as revealed by proliferation and BrdU assays. α-SMA gene and protein expression were significantly up-regulated by HMGB1, whereas the MMP-2, but not MMP-9, activity was suppressed by HMGB1 treatment. Conclusion: Our data suggested that HMGB1 protein, once released during the rejection phase of OLT, activated HSCs and exhibited profibrogenic effects on liver grafts either by increasing the HSC population and extracellular matrix content in liver grafts, or by transforming HSCs into myofibroblasts. Neutralization with anti-HMGB1 antibody was suggested to be a therapeutic modality applicable to prevent fibrogenesis in post-OLT liver grafts. [Copyright &y& Elsevier]