Tair-Long Pan

Assessment of donor fatty livers for liver transplantation

Aim. The effect of fatty liver on graft survival, especially with reference to macrovesicular and microvesicular steatosis, is still uncertain. This preliminarily study was designed to create a noninvasive method for the quantification of the hepatic fat content in vivo and to establish provisional criteria for the assessment of fatty donor livers before liver transplantation among transplant surgeons, radiologists, and pathologists. Methods and Materials. Different degrees of rat fatty liver model were established by feeding rats a diet deficient in choline and methionine for different periods of time. Computed tomography (CT) with test tubes containing variable percentages of fat equivalent substance were used to assess the severity of fatty change of the rat liver. This was then correlated with the histological classification, level of hepatic enzymes, and graft survival. Results. Linear correlation between the fat volume fraction added to the test tubes and CT density were found. The process of producing a fatty liver via diet alteration peaked at week 3. At this time hepatic enzymes, radiological fat content, and posttransplantation survival were worse (P =0.013), compared with other time points. Radiological assessment of fatty liver correlated well with survival and serum glutamic oxaloacetic transaminase and glutamic pyruvate transaminase levels. Conclusion. Severe microvesicular steatosis does not influence recipient survival, however, macrovesicular steatosis affects graft survival. Caliber CT is a practical and simple method that allows an accurate noninvasive quantitative assessment of hepatic fatty infiltration. It has potential to be a useful parameter for the assessment of donor livers for clinical liver transplantation.

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 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.

Major hepatic resection may suppress the growth of tumours remaining in the residual liver

Little is known as to how hepatectomy is associated with the growth of hepatic tumours, which may reside in the remaining liver after curative resection for hepatocellular carcinoma. Using an intra-hepatic tumour implantation model in rats, the effects of hepatectomy on tumour growth in the remaining liver were investigated. On post-operative day 7, the tumour weight in the remaining liver following 30% hepatectomy was 0.321 ± 0.058 g (mean ± SD) which was significantly greater than that (0.245 ± 0.040 g) in sham operations (P < 0.05). However, the tumour weight (0.156 ± 0.067 g) in the remaining liver following 60% hepatectomy was significantly lower than that in sham animals (P < 0.005). The number of TdT-mediated dUTP nick-end labelling (TUNEL) positive tumour cells was significantly increased in 60% hepatectomy as compared with the sham and 30% hepatectomy group. The mRNA expression of TGF-β1, TNF-α and Fas in the tumour portion of 60% hepatectomy, was higher than that in 30% hepatectomy group. Plasma levels of TGF-β1 were inversely correlated with intra-hepatic tumour weights. These results suggest that major hepatic resection may lead to an increased induction of apoptosis for the remaining hepatic tumour.

Identification of the indoleamine 2,3-dioxygenase nucleotide sequence in a rat liver transplant model

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.

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.

Repeated hypotensive episodes due to hepatic outflow obstruction during liver transplantation in adult patients

We report two cases of unusual repeated hypotension, decreased cardiac output, decreased mixed venous oxygen saturation, decreased central venous pressure, pulmonary artery pressure, and pulmonary wedge pressure after the completion of all vascular anastamoses of liver transplantation. These unstable hemodynamics appear to reflect a clinically relevant picture of hypovolemia. However, the real cause was partial hepatic outflow obstruction. The obstruction was suspected because hypotension was alleviated by elevating the full-sized liver graft ventrally and to the left. Doppler ultrasound examination confirmed that the flow velocity of the hepatic vein outflow was insufficient when the liver fell to its resting position in the right hepatic fossa. An additional side-to-side cavo-caval anastomosis resolved the problem in one patient, whereas the other required not only the additional anastomosis, but also application of a tissue expander filled with 770 mL normal saline beneath the liver to eliminate the obstruction. We emphasize that obstruction of the hepatic outflow causes only temporal hypovolemia because of a decrease of venous return and that treatment of this complication should be surgical intervention to relieve the obstruction. Blind resuscitation with fluids will not solve the problem and, in fact, may result in fluid overload with subsequent complications.

Telomerase activity in rat liver allografts.

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.

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.

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.