**5. Idiopathic post-transplant hepatitis**

Some groups have also called chronic hepatitis of unknown cause, such as idiopathic posttransplant hepatitis, *de novo* autoimmune hepatitis. This condition is commonly associated with positive autoantibodies, such as antinuclear antibody and elevation of IgG levels, and biochemically and histopathologically resembles autoimmune hepatitis in patients who did not receive transplants [78-79]. Increasing evidence suggests that late acute rejection, *de novo* autoimmune hepatitis, and idiopathic post-transplant hepatitis are part of an overlapping spectrum of immune-mediated late allograft damage occurring in long-term post-transplant patients [62]. Together with idiopathic post-transplant hepatitis, immune-mediated late allograft damage can cause late graft dysfunction leading to cirrhosis. Telomere length observed in chronic hepatitis or cirrhosis is significantly lower than that in normal livers of the same age. Sustained cellular turnover in chronic liver disease accelerates cellular senescence or a severe damage because of telomere shortening. We initially hypothesized that idiopathic post-transplant hepatitis may show more progressive telomere shortening due to higher cell turnover [3-4]. Cellular senescence in the explanted livers of young children was reported to be associated with hepatocyte damage rather than to a corresponding age-dependent phe‐ nomenon [80]. However, we observed no significant telomere reduction in hepatocytes taken from patients with idiopathic post-transplant hepatitis at late biopsy. Telomere shortening does not necessarily reflect the long-term graft status in idiopathic post-transplant hepatitis, which differs clinically and histologically. Telomere length in hepatocytes already shortened during the early post-transplant period. Increasing number of senescent cells associated with telomere shortening confirmed in a mouse model of ischemia–reperfusion injury [81]. Therefore, hepatocyte damage related to ischemia–reperfusion injury is likely to be a major factor in the accelerated telomere decline observed in the early post-transplant period. On the other hand, from the standpoint of telomere shortening in the early post-transplantation phase, telomere decline is considered a risk factor for late dysfunction of the graft. This finding is clinically significant in follow-up examinations of high-risk allografts.

#### **6. Tolerance**

Tolerance is a condition in which an allograft functions normally and lacks histological evidence of rejection in the absence of immunosuppression [82]. Tolerated grafts are suitable study materials for evaluating the biological organ age of grafts unaffected by inflammation and immunosuppression. We have previously reported a significant reduction in hepatocyte telomere signal intensity compared to the predicted age-dependent decline in the tolerated liver allograft, using quantitative fluorescence in situ hybridization [80,21]. Recently it has been demonstrated that measurement of relative average telomere lengths can be accomplished by real-time polymerase chain reaction (PCR) using a carefully designed pair of oligonucleotide primers [83]. In a larger number of cases, we performed quantitative real-time PCR, and confirmed accelerated telomere shortening relative to the chronological graft age in tolerated grafts. It is possible that a significant proportion of liver transplantation recipients are tolerant [84-86]. Accelerated telomere intensity decline occurred in hepatocytes in tolerated graft within a year of transplantation. The results of the previous study have suggested that even tolerated grafts might undergo a lowering of renewal capacity and a decrease in function as the recipients become older [2]. According to our previous study, the allograft could be older than the predicted age of the allograft even in tolerated grafts, and the telomere length shortened based on the graft age.

#### **7. Oxidative stress after living-donor liver transplantation**

Ischemia and reperfusion during transplantation produce a transient increase of reactive oxygen species in the organ, which are potent inducers of DNA breaks. In a rat model, both allogeneic and syngeneic transplants were characterized by shortened telomeres during ischemia at transplantation [87]. Oxidative stress accelerates telomere shortening [88-89]. Low ambient oxygen conditions can extend the life span of cells in culture [90]. In cell culture protected from oxidative stress through low ambient oxygen tension, the addition of antioxi‐ dants, or overexpression of antioxidant enzymes delays telomere shortening [91-93]. Further data have demonstrated the important interaction between telomere-induced senescence and oxidative stress. Senescence leads to the development of oxidative stress that reinforces the senescent state of the cell and causes further oxidative stress. The telomere decline is probably due to premature aging of the graft that might occur during ischemia–reperfusion injury or graft regeneration immediately after transplantation [81]. Thus, telomere shortening in grafts could reflect not only the proliferative history of a cell, but also the accumulation of oxidative damage during the early post-transplant period [94]. Telomere reduction is presumably accelerated by the transplantation process, in both young and old tissues, modification of perior post-transplantation environmental stress may probably reverse aging-dependent factors.
