C Y. Lai

The Fas and Fas ligand pathways in liver allograft 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 14u2003, 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.

The role of a nuclear protein, histone H1, on signalling pathways for the maturation of dendritic cells.

We have demonstrated previously that liver allograft tolerance is associated with the immunosuppressive activity of anti‐histone H1 autoreactive antibodies induced in the serum of liver transplantation. Furthermore, we and others have shown that nuclear proteins such as histone H1 and high mobility group box 1 play an important role in maturation of dendritic cells (DCs), although the precise mechanisms are still unknown. In the present study, we focus upon the significance of histone H1 on DCs in terms of the intracellular signalling pathway of DCs. Our immunostaining and immunoblot studies demonstrated that histone H1 was detected in cytoplasm and culture supernatants upon the activation of DCs. Histone H1 blockage by anti‐histone H1 antibody down‐regulated the intracellular activation of mitogen‐activated protein kinases (MAPKs) (p38) and IκBα of DCs, and inhibited DC activity in the proliferation of CD4+ T cells. On the other hand, the addition of histone H1 without endotoxin stimulation up‐regulated major histocompatibility complex class II, the CD80 and CD86 surface markers of DCs and the activation of MAPKs (p38 and extracellular‐regulated kinase 1/2) and IκBα. These results suggest that the translocation of histone H1 from nuclei to cytoplasm and the release of their own histone H1 are necessary for the maturation of DCs and the activation for T lymphocytes.

Clusterin may be involved in rat liver allograft tolerance.

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.

Proteome Analysis in Liver Transplantation

THE USE of the DNA database does not allow for an understanding of to how a cell or whole tissue functions at the molecular level before or after transplantation. Therefore, attention has increasingly turned toward the identification and characterization of the functional protein products encoded by the genome of an organism or tissue, and thus the process of proteomics has been coined to describe this approach. In the present study, proteome analysis is applied to liver transplant research.

Expression of Clusterin in a Rat Tolerogenic OLT Model

IN RAT orthotopic liver transplantation (OLT), PVG recipients of DA livers (tolerogenic model) survive without immunosuppression and become tolerant of subsequent grafts of other DA organs such as skin, heart, and kidney. Many aspects of the mechanism regarding this naturally achieved tolerance have been discussed in our previous reports. However, the mechanism of liver allograft tolerance from the aspect of the complement system and its regulatory proteins has not yet been investigated. The complement system comprises a group of proteins that interact with other immune system molecules to provide many of the effector functions of humoral immunity and inflammation. Clusterin, which is a plasma glycoprotein, regulates the complement system by inhibiting the membrane attack complex (MAC) formation. Here, we studied the kinetics of clusterin expression after transplantation in the tolerogenic model (DA-PVG) compared with syngenic model (DA-DA) and acute rejector model (DA-LEW) by immunoblotting and Northern blotting.

CHARACTERIZATION OF IMMUNOSUPPRESSIVE FACTORS EXPRESSED IN SERUM BY RAT TOLEROGENIC LIVER TRANSPLANTATION

BACKGROUNDnIn 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.nnnMETHODSnImmunosuppressive 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.nnnRESULTSnRecipient 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.nnnCONCLUSIONSnIn 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.

Activation of telomerase by liver transplantation in rats.

ALTERATION of cellular senescence following transplantation may be associated with posttransplant carcinogenesis. A study of the aging of donor grafts also may be important for the donor selection in living-related or cadeveric orthotopic liver transplantation (OLT). Telomere and telomerase are involved in the central timing mechanisms for cellular aging. Here, we studied telomerase activity in livers from various rat OLT models.