In our experience, immunosuppression regimen with tacrolimus in monotherapy after LT is feasible and safe. Patient survival, acute rejection rates and immune graft loss were similar to our standard regimen of tacrolimus and steroids. Sixty per cent of patients in the study group never received steroids in the post-LT period whereas 40% were treated with steroids for rejection. Steroid withdrawal was started at 3 months post-LT in the TACRO + ST group, and at 10 months of follow-up 90% of patients in both groups were free of steroids. The incidence of rejection in our study was lower than in previous studies with monotherapy with either neoral or tacrolimus [4,5], probably because of the high proportion of older patients, all of whom had postnecrotic cirrhosis mainly of alcohol or HCV origin. It is well known that senior and alcoholic patients have lower immunoreactivity and a lower incidence of acute rejection. This protocol may not be applicable to younger patients with autoimmune or cholestatic diseases who have higher inmunoreactivity and, consequently, suffer more frequent and severe rejection episodes. In terms of safety, we could not demonstrate a significant benefit of avoiding steroids, probably because of the low number of patients included in the study. However, a trend was observed towards less de novo arterial hypertension and diabetes in the monotherapy group, whereas renal insufficiency was higher in the TACRO group.
Concerning pharmacokinetics studies, in the TACRO + ST group, higher doses of tacrolimus were required to reach the same blood levels achieved with tacrolimus alone. This finding confirms the increase in tacrolimus metabolism due to cytochrome P450 3A4 iso-enzyme (CYP3A4) induction by steroids, and shown in the report of van Duijnhoven et al. .
HCV subgroup of patients
Recurrent hepatitis C viral infection is universal post-LT. Almost half the patients present acute graft hepatitis early after LT and 90% develop chronic active hepatitis with evolution to cirrhosis in 30% of cases at 5 years post-LT . Evolution to liver failure and death is very rapid once these patients present decompensated cirrhosis. Results obtained with retransplantation for recurrent HCV infections are poor. Recent data from UNOS and the Spanish Liver Transplant Registry have shown lower patient and graft survival in HCV patients [16,17,24]. Moreover, reports suggested that graft outcome of LT for HCV may have deteriorated in recent years. Hence, the subgroup of hepatitis C patients was evaluated independently to assess the potential benefit of a steroid-free immunosuppression regimen.
In our experience, there was a tendency for the subgroup of HCV patients to have more acute rejections diagnosed than HCV-negative patients. Difficulty in liver biopsy interpretation between acute rejection and HCV hepatitis, or the coexistence of both, has sometimes led to patients with recurrent hepatitis C being treated for rejection with steroid boluses. TACRO and TACRO + ST groups had similar demographic characteristics and acute rejection rates. Analysis of HCV recurrence in the graft showed a lower, although not significant, incidence of graft hepatitis in the TACRO monotherapy group as well as a higher proportion of patients with good outcome. However, these differences did not reach statistical significance, probably because of the scant number of patients studied.
Although further grouping down patients into real-TACRO and real-TACRO + ST leaves only few patients in each group, we were interested in knowing the evolution of graft hepatitis in the patients who never received steroids and compared this group with the remaining patients who received steroids either for protocol or to treat acute rejection episodes. Significantly lower levels of hepatitis C viraemia were found during the early weeks and months in patients without steroids, thereby confirming the findings of Garcia Retortillo et al.  and Boker et al. . This may have been the reason for the lower incidence of acute graft hepatitis and its milder course in all 45% of steroid-free patients who presented it. We observed that a cut-off point in viral load pre-LT of >log10 5 was predictive of a poor outcome. At 12 months, viral loads in our study were similar in both groups, probably because at that time point most of the patients in both groups were free of steroids and received the same amount of immunosuppression. In contrast to our findings, Papatheodoridis et al.  reported higher levels of HCV RNA at 3 months in patients with single initial therapy compared with those receiving double therapy. However, a correlation was found between viral load at 12 months, duration of steroid treatment and extent of fibrosis. All these reports indicate a correlation between viral load and the amount of immunosuppression.
After a mean follow up of 44 months, evolution of HCV graft infection was significantly better in the steroid-free group. Only one patient presented a poor outcome (compensated cirrhosis), whereas the rest had good outcome (normal liver function or mild chronic active hepatitis). These findings were confirmed by a significantly lower fibrosis score in the liver biopsy study. Nevertheless, the possible effects of steroids on HCV recurrence and fibrosis remain controversial. A recent study by Fasole et al.  defended the beneficial effect of steroids on avoiding fibrosis in graft HCV recurrence. The hypothesis of Berenguer  is that the immune system becomes reconstituted following steroid withdrawal and dose reduction in calcineurin inhibitors and immune-mediated liver damage may then occur, together with progression to severe forms of chronic hepatitis.
There are many factors which influence the outcome of graft hepatitis C re-infection. Liver graft quality is extremely important: suboptimal donor liver either due to steatosis, old donor age, fibrosis, etc. results in important ischaemic–reperfusion injury, activation of inflammatory processes, greater susceptibility to acute rejection and probably more severe HCV graft re-infection. Recipient age over 60 years appeared to be significant in our study as well as in other studies [16,29–33]. In a previous study  concomitant CMV hepatitis was found to aggravate the evolution of hepatitis C; no case of CMV hepatitis was found in the present study. The link between anti-rejection therapy and worsening of hepatitis C seems very clear. Many authors and ourselves have demonstrated an association between more aggressive recurrence of hepatitis and evolution to fibrosis and cirrhosis and episodes of treated rejection [31,35,36].
Finally, patient survival was much better in real-TACRO, although the low number of patients in each group did not allow to reach statistical significance. Altogether, we can affirm that outcome of HCV graft infection was better in patients who could be maintained without steroids after LT. All HCV-related deaths occurred in the group receiving steroids from the beginning, or during follow-up. This study is one of the few prospective and randomized trial of immunosuppression regimen based on tacrolimus in monotherapy since the first post-transplant day, and although it was not originally designated to look at hepatitis C this is the first prospective randomized study of monotherapy regimen and recurrent hepatitis C infections. Another strong point of the trial is the long-term follow-up presented in patients without steroids. Other authors have reported differences in recurrent hepatitis but not long-term evolution of the graft. The few number of patients enrolled and the lack of periodically protocol biopsies in HCV patients are the weak points of the study. However, we should be able to learn about these findings and in fact, this study has prompt us to change our standard immunosuppressive regimen in HCV positive patients, which is based at the moment on tacrolimus plus MMF.
In summary, (i) a steroid-free immunosuppression protocol based on tacrolimus monotherapy is safe in a cohort of senior patients transplanted mainly for alcoholic or viral cirrhosis. Patient and graft survival and acute rejection and steroid-resistant rejection rates were similar to those of our standard tacrolimus short-term steroid protocol. (ii) No significant benefit was obtained in terms of side-effects of this steroid-free protocol although a trend was observed towards less hypertension and diabetes but more nephrotoxicity. (iii) The principal benefit could be in patients transplanted for HCV cirrhosis, as a trend towards lower incidence and milder course of acute recurrent HCV hepatitis and better long-term outcome was found in the group without steroids. (iv) This benefit was more evident in patients receiving no steroids at all after LT. Therefore, the key of future protocols for HCV patients would be a more effective steroid-free immunosuppression to achieve acute rejection rates of <10%; consequently, more than 90% of HCV LT patients will be steroid-free.