The introduction of cyclosporine (CsA) more than one decade ago clearly improved the results of renal transplantation. Nowadays most immunosuppressive regimens used early after transplantation are based on CsA, usually combined with other pharmacological drugs and, in many instances, also with biological antilymphocyte agents. However, in spite of the amelioration in graft and patient survival, the use of CsA is associated with nephrotoxicity, acute rejection was not completely prevented, and in addition its appearance seems to be associated with the occurrence of chronic rejection, which is one of the leading causes of long-term renal allograft failure. So, several new immunossupressive agents have entered in clinical trials and some of them have already been approved. The different immunosuppressive agents interfere with the T-cell activation cascade and the allograft reaction at different levels, as summarised in figure

1. The first signal for T-cell activation starts in the T-cell receptor complex (TCR) as a result of antigen recognition, presented by the antigen presenting cell (APC) as a peptide bound to an MHC molecule. The second signal comes from the interaction of costimulatory molecules after the close contact between T-cell and APC. These two signals promote T-cell activation and trigger intracellular reactions that lead to the synthesis and secretion of IL-2, which binds to the IL-2 receptor that induces ADN synthesis and cell proliferation. The biological anti T-cell antibodies directed against different surface receptors interfere with the interaction ligand-receptor.

The pharmacological agents mainly interfere with the intracellular reactions.

The recently described immunosuppressive regimens have been mainly based on the use of the new xenobiotics (tacrolimus, mycophenolate mofetil (MMF), and rapamycin) and on the other hand the new monoclonal antibodies against anti-IL-2R, anti-adhesion molecules (anti-LFA1, anti-ICAM-1) has also been studied, and even the classical polyclonal preparations have been re-evaluated as well. Most clinical trials have been designed to prevent acute rejection, some of them aimed to treat resistant or refractory rejection and some attempts have been made to manage late allograft dysfunction.The majority of the new immunosuppresive therapies in the prevention of acute rejection have been based on the use of anti-calcineurin agents, CsA or tacrolimus because both inhibit the synthesis of IL-2, a mechanism that has been considered the key stone in transplant immunosuppression.

The prohylactic potency of tacrolimus was compared to CsA in prospective and randomised trials. In an American study (1), tacrolimus significantly reduced the incidence of biopsy-proven acute rejection from 46% to 31% (p=0.001) within the first year after transplantation, and the proportion of rejections with vascular involvement (< 10% with tacrolimus), which resulted in a less frequent use of antilymphocyte antibodies (11% vs 25%, p<0.001). In the European study (2), a similar reduction in the appearance of biopsy-proven acute rejection was observed (45.7% vs 25.9%, p<0.001).

The introduction of new xenobiotics has allowed studying the utility of these new agents in conjunction with calcineurin inhibitors in the prevention of acute rejection.

The immunosuppressive potency of MMF has been clearly demonstrated in three large prospective, randomised, controlled trials in renal transplantation involving nearly 1500 patients (3-5). The European trial of MMF for the prevention of allograft rejection (3), a double blind placebo-controlled study, demonstrated that MMF in conjunction with CsA and corticosteroids significantly reduces the incidence of the composite end-point of biopsy-proven acute rejection/treatment failure (defined as graft loss, death or premature withdrawal from the study) at 6 months post-transplant from 56% for placebo-treated patients to 38.8% for MMF 3 g/day-treated patients and 30.3% for MMF 2 g/day-treated patients.

This considerable reduction in rejection or treatment failure was consistent with the results obtained in the American (4) and Tricontinental (5) studies in which MMF was compared with the control drug azathioprine as a component of a triple therapy regimen including CsA and corticosteroids. In addition, a pooled efficacy analysis for these three clinical trials at 1 year after transplantation (6) confirmed the ability of MMF to reduce the incidence of biopsy-proven acute rejection in comparison with placebo or azathioprine.

The use of MMF resulted in a significant reduction of biopsy-proven acute rejection from 40.8% in the azathioprine/placebo-treated patients to 19.8% and 16.5% in MMF 2 g/day and MMF 3 g/day-treated patients, respectively. MMF also reduced the histological severity of rejection and consequently the need for antilymphocyte antibodies. Interestingly, at 1 year post-transplant, MMF significantly reduced graft losses due to rejection from 6.3% in the placebo/azathioprine group to 2.6% and 3.5% in the MMF 2/day and MMF 3 g/day, respectively.

This positive impact in the reduction of immunological failures persisted at 3 years after transplantation. In the Tricontinental study (7), using azathioprine in the control group, MMF reduced graft losses due to rejection from 9.9% in the azathioprine group to 5.8% and 3% in the MMF 2 and 3 g groups, respectively.

Similarly, in the European study graft losses due to rejection decreased from 10.8% in the placebo group to 4.8% and 6.3% in the MMF 2 and 3 g groups, respectively (8). In this study, an intent-to-treat analysis of patient and graft survival over 3-year follow-up showed that the cumulative incidence of graft loss (including graft loss as a result of death) for the MMF 2 g, MMF 3 g, and placebo groups were 15.2%, 18.8% and 22% respectively. Patient deaths in the respective groups were 7.3%, 8.2%, and 11.1%, being acute rejection the principal cause of graft loss in all groups. The differences in the 3-year graft loss rates (including death) for comparisons of MMF 2 g and MMF 3 g vs placebo were respectively 6.9% and 2.9%. Censoring for death, the differences in 3-year graft loss rates were 7.6% and, 3.2%, respectively.

For the MMF 2 g group, this represents a relative risk of 0.55 (p=0.04), or a 45% reduction in graft loss compared with the placebo group. An analysis of the Tricontinental study also showed a trend in favour of MMF 2 g and MMF 3 g over 3 years in graft and patient survival compared with treatment with azathioprine (7). The 3-year data from the European and Tricontinental study also confirmed the deleterious impact of early acute rejection on long-term patient and graft survival. In these two studies, patients who experienced a biopsy-proven acute rejection in the first 6 months post-transplant were 5 and 4 times, respectively, more likely to lose their graft to those who were free of such rejection.

At 3 years after transplantation the cumulative incidence of adverse effects of MMF (7, 8) was similar to and consistent with the results previously reported (3-5). MMF is associated with slight increases, in a dose-dependent manner, in gastrointestinal and haematology adverse events as well as infections and malignancies. However, the cumulative incidence of cytomegalovirus invasive disease in azathioprine-treated patients from the

Tricontinental study (6.8%) is higher than that observed in the MMF 2 g-treated patients (3.6%), and similar to that of MMF 3 g-treated recipients (8.1%) in the European study. These data suggest that the increased incidence of opportunistic infections in MMF-treated patients cannot be only attributed to the administration of MMF but to the cumulative doses of conventional immunosuppressives. We have recently shown that by reducing the doses of CsA and steroids in patients treated with 3 g of MMF, the incidence of cytomegalovirus disease remains similar to that observed in patients treated with conventional doses of CsA and steroids, without an increased risk for rejection (9). A similar observation is also true for the overall incidence of malignancies. It is higher in all groups from the Tricontinental study in comparison with that reported in the European study (7, 8).

After 3 years the mortality rates and causes of death in the three therapeutic groups were similar in both studies. Vascular diseases were the most common cause of death followed by infection and cancer.

According to these positive results, many transplant centers have replaced azathioprine for MMF in triple regimens or added MMF to dual therapies in the prophylaxis of acute rejection in renal transplantation. However, long-term observations are required to demonstrate the long-term benefits of this agent.

The association of tacrolimus and MMF at 1 g/day and 2 g/day has also been recently evaluated in the prevention of acute rejection. In a retrospective study (10), in which the majority of patients received induction therapy, the association of tacrolimus, MMF and steroids greatly decreased the incidence of acute rejection to a minimal 8.2% in comparison to 21% in patients treated with tacrolimus and steroids. Besides, the use of MMF resulted in a significant reduction in the doses of tacrolimus. The ability of a combination of tacrolimus and mycophenolate to reduce the incidence of acute rejection has also been studied in two recent prospective trials.

As in the case of CsA-based regimens, the addition of 1 or 2 g/day of MMF to tacrolimus and steroids reduced by half the incidence of acute rejection from 48% to 25% and 23%, respectively, in the European study. The use of MMF also decreased the proportion of steroid-resistant acute rejections (from 16% to 6.5% and 4%), and the rate of recurrent rejection episodes (12%, 3%, and 3%), with excellent graft survival rates 6 months after transplantation (11). Similar data can be draw from the American study (12). In this trial, the immunosuppression in the control arm consisted of tacrolimus, steroids and azathioprine and induction was employed in all patients. As in the European study, 2 g/day of MMF greatly reduced the occurrence of biopsy-proven acute rejection (from 33% in the control group to 30% in the MMF 1 g, and to 10% in the MMF 2 g) and delayed the onset of the first rejection episode.

The combination of tacrolimus and MMF is associated with an augmentation in the MPA levels (the active metabolite of MMF) and a concomitant decrease of MPAG due the interference of tacrolimus with the MPA glucoronidation because of the inhibitory effect of the drug on the human UDPGT, which is 60 times more potent than that of CsA(13). This interesting effect may allow reducing the concomitant doses of MMF given in conjunction with tacrolimus. The immunosuppressive efficacy of tacrolimus may help to reduce or stop steroids in a high proportion of adult (14) or paediatric transplant recipients (15).

Rapamycin is another macrolide with immunosuppressive effects that inhibit cell proliferation driven by growth factors. In a phase I/II dose-escalation trial, rapamycin reduced the overall incidence of acute rejection to 7.5% from 32% in the CsA/prednisone-treated patients (16). Moreover, in rapamycin-treated patients steroid withdrawal was successfully accomplished in 78% of cases 12 months after transplantation. In two large phase III multicenter studies rapamycin at 2 mg/day or 5 mg/day was associated with CsA and steroids and compared to a dual therapy consisting of CsA, steroids and placebo (Global study) or CsA, steroids and azathioprine (US study).

In the Global study (n= 576), the use of rapamaycin at 2 and 5 mg/day reduced the incidence of acute rejection from 29.6% to 19.4% and 11%, respectively. In the US study (n=719), a similar decrement was observed from 24% in the control group to 14.8% and 10.6%, in the 2 mg/day and 5 mg/day, respectively. In these two studies the graft survival rates at 1 year were 90% or higher in the rapamycin groups. The immunosuppressive potency of rapamycin led to design a trial on rapamycin-based therapy in combination with azathioprine and steroids in comparison with a standard triple therapy regimen based on CsA in the prophylaxis of acute rejection (17). The incidence of biopsy-confirmed acute rejection was similar in both groups (41% vs 38%), but renal function was better in rapamycin-treated patients, because this macrolide is not nephrotoxic. In the next few years rapamycin may constitute an alternative to calcineurin inhibitors in the design of non-nephrotoxic regimens. The main adverse effects of rapamycin are hipercholesterolemia, hipertriglyderidemia and thrombocytopenia that appear to be concentration-dependent. In the first trials with rapamycin it was observed an augmentation of Pneumocystis carinii infection, and consequently a systematic prophylaxis, mainly with trimetoprim-sulphametoxazol, is recommended. To adjust the doses of rapamycin it is recommended to monitor blood levels which target varies according to the administered concomitant immunosuppression .

In summary, the incidence of acute rejection can be greatly reduced to less than 20% the fist 6 month after transplantation by combining a calcineurin inhibitor with MMF or rapamycin and with high graft and patient survival rates and an adequate risk/benefit balance.

In parallel with the progression of the above mentioned trials mainly combining the new xenobiotics, the new monoclonal antibodies have been tested in the prevention of acute rejection, usually associated with CsA and steroids with or without azathioprine.

The interaction of IL-2 with its cellular receptor triggers the DNA synthesis and cellular proliferation (18). The IL-2 receptor consists of three subunits: IL-2R alfa (55 kDa), IL-2R beta (70/75 kDa), and IL-2R gamma (64 kDa). The noncovalent association of all receptor subunits forms a high affinity receptor for IL-2. The a chain is only expressed on activated lymphocytes, and hence targeting this chain may be a selective manner to block the immune response.

Murine monoclonal antibodies, that block the interaction of IL-2 with its receptor, were initially studied in the prophylaxis of renal allograft rejection. 33B3.1 and anti-Tac are monoclonal antibodies directed against the a chain, and they were used in clinical transplantation (19, 20). In these studies, the reported incidence of rejection was 31% and 35% respectively, with few side effects. However, these murine antibodies elicit the development of human antibodies against murine proteins that neutralizes their therapeutic effect. To overcome these limitations a chimeric (basiliximab) and humanised (daclizumab) monoclonal antibodies directed against the a chain of IL-2R were developed and studied in preventive regimens. The administration of these antibodies result in prolonged saturation IL-2 a receptors on circulating lymphocytes. Basiliximab associated with CsA microemulsion and steroids reduced to 35% the proportion of patients who experienced biopsy-confirmed acute rejection episodes from 49% in patients treated with CsA and steroids (p=0.009), within the first year after transplantation. (21). Despite the diminution of acute rejection, the use of this selective antibody did not increase the occurrence of opportunistic infections or the overall incidence of adverse events.

Similarly, the effect of daclizumab in addition to dual immunosuppression significantly reduced biopsy-proven acute rejection from 47% to 28% (p=0.001), the need for additional antilymphocyte therapy (16% vs 8%, p=0.02) after renal transplantation, improved patient survival, and did not add to the toxicity of the immunosuppressive regimen (22). This antibody has also been associated with a triple therapy regimen (23). In this study 22% of daclizumab-treated patients developed biopsy-proven acute rejection compared with 47% of the patients treated with triple therapy (p=0.03). The patients given daclizumab did not have adverse reactions to the drug, and at six months, there were no significant differences between the two groups with respect infections or malignancies. In association with MMF, CsA and steroids, daclizumab further reduced the incidence of acute rejection to 12%. The excellent safety profile and immunosuppressive efficacy of these new anti-IL2R monoclonal antibodies suggest that they may be very helpful in sparing or avoiding the use of calcineurin inhibitors and steroids in renal transplantation (24).

The immune response to recognised alloantigens requires the migration of immune cells to the sites of antigenic stimulation. In this process, adhesion molecules play a crucial role. Adhesion molecules mediate the attachment of circulating lymphocytes to endothelial cells and extracellular matrix. Hence, monoclonal antibodies against these molecules may result immunosuppresive because they could interfere with cell recruitment and migration to the target tissues. The therapeutic efficacy of these monoclonal antibodies was proven in experimental models. However, an anti-ICAM-1 murine monoclonal antibody (enlimomab) have failed to prevent either rejection or delayed graft function in a phase III clinical trial (25). On the other, an anti-LFA-1 murine monoclonal antibody did not reduce the occurrence of acute rejection in a phase III trial (26) a trend for less delayed graft function with the use of this antibody was observed. The potential utility of this agent for such indication is currently under study in a large multicenter trial.

The classical polyclonal anti-lymphocyte antibodies have recently been evaluated in prohylactic protocols. Rabbit anti-thymocyte globulin (Thymoglobulin) was compared with horse antisera (Atgam) in the prevention of acute rejection (27), being both agents associated with a calcineurin inhibitor, steroids, and azathioprine or MMF. By one year after transplantation, 4% of Thymoglobulin-treated patients experienced acute rejection compared with 25% of Atgam-treated patients (p=0.014). Patients treated with Thymoglobulin had less cytomegalovirus disease, no recurrent rejection episodes and higher graft survival.

Thymoglobulin and MMF have each demonstrated to be potent immunosuppressants. On the other hand, kidneys from suboptimal donors may be more susceptible to CsA nephrotoxicity. In order to avoid the use of calcineurin inhibitors, we studied the combination of MMF, Thymoglobulin and low-dose steroids in the prevention of acute rejection in low-risk transplant recipients of a suboptimal graft (28). Four out of 17 patient entered in this study experienced biopsy-proven acute rejection, and 70% of the recipients remained free of cyclosporine 3 months after transplantation. These preliminary results suggest that it is possible to avoid the systematic use of calcineurine inhibitors in renal transplantation, and 3 new studies using polyclonal or the new anti-IL2R monoclonal antibodies are in progress in this direction. Another potential utility of the combination of MMF and polyclonal preparation might be the avoidance of steroids (29). Treatment of acute rejection

The first line treatment of acute rejection has been the use of high doses of steroids. Corticorresistant or refractory rejections were rescued with polyclonal or monoclonal antilymphocyte antibodies. Two polyclonal preparations have recently been studied in the treatment of corticorresistant grade I or grade II and III acute rejections (30). Thymoglobulin successfully reversed acute rejection in 88% of cases in comparison with a 76% of patients treated with Atgam (p=0.027), and the proportion of patients with recurrent rejection episodes were much lower in Thymoglobulin-treated patients (17% vs 36%, p=0.011).

In contrast with the first calcineurin inhibitor (CsA) or the first antipurinic agent (azathioprine) used in renal transplantation, the new anti-calcineurin agent, tacrolimus, and the new antipurinic, MMF, both are useful in the treatment of ongoing rejection. In a single center study, tacrolimus was successfully employed as a rescue agent for resistant rejections that occurred under CsA (31). Tacrolimus rescued 85% of acute cellular rejections, 65% of cellular and vascular rejections and 40% of cellular rejections with primary non-function of allograft. In a multicenter trial (32), tacrolimus was evaluated in the treatment of rejection refractory to steroids and previous antilymphocyte therapy in most instances (81%). The switch from CsA to tacrolimus resulted in an improvement of renal function in 78% of the cases, stabilisation in 11% and progressive deterioration in 11%. The risk of experiencing progressive deterioration was related to the pretacrolimus serum creatinine, suggesting that the sooner the treatment started the better is the therapeutic response. Fourteen percent of cases experienced recurrent rejection episodes. The global incidence of infection was 15%, and the 1-year graft and patient survival were 75% and 93%, respectively. These data indicate that tacrolimus is an important tool to manage allograft rejection in established patients.

MMF has been shown to be effective for the treatment of acute refractory (33) or first (34) cellular allograft rejection. Because patients with acute cellular rejection episodes refractory to standard therapy with steroids and antilymphocyte antibodies are at high risk of losing their grafts, MMF was used in these cases in comparison with high doses of intravenous steroids in a prospective randomised trial. (35).

In this study, the proportion of MMF-treated patients who experienced a biopsy-proven or presumptive acute rejection or were classified as a treatment failure was significantly lower than in steroids-treated recipients (39% vs 64%) six months after the enrolment in the study. This resulted in a clinically significant reduction in the use of antilymphocyte agents from 24.7% in steroids-treated patients to 10.4% in MMF-treated patients subsequent to enrolment, and also the use of MMF was associated with a significant reduction, by more than 40%, in graft loss or death one year after the entry in the study. These positive results in the treatment of refractory rejection are in agreement with those recently reported on the utility of MMF in the treatment of first acute cellular rejection (34). In this double-blind, double-dummy controlled study, renal allograft recipients experiencing the first biopsy-proven cellular rejection within 6 months of transplant were treated with MMF (3 g/day) and intravenous steroids, or azathioprine (1-2 mg/kg/day) and intravenous steroids. In comparison with intravenous steroids, MMF decreased the subsequent use of antilymphocyte therapy (41.7% vs 16.8%), and the proportion of patients who lost their graft or died (14.8% vs 8.9%) at 6 months. In MMF-treated patients there was a trend for better renal function that may result from a more rapid and complete resolution of the rejection in these patients. This study has an extended follow-up to determine whether the use of MMF in the treatment of a first acute rejection will have an impact to reduce late graft loss and chronic rejection. However, in both studies MMF was associated with a higher incidence of adverse events. Cytomegalovirus tissue invasive disease was higher in MMF-treated patients in comparison with steroid-treated patients in the Refractory Rejection study (9.1% vs 1.4%), although it was similar in both groups in the Acute Renal Rejection study. In routine clinical practice, with less constraint than in study conditions, dose adjustments might help to reduce MMF side effects.

On the other hand, the combination of two agents effectively used in rescue therapy, such as MMF and tacrolimus, may constitute a promising association in the treatment of corticoresistant, refractory or humoral rejection (35, 36).

Maintenance immunosuppression

The ideal maintenance immunosuppressive regimen is not established. The majority of centers use CsA, steroids, and at times azathioprine as maintenance immunosuppression. The main objectives of maintenance immunsupression are to avoid late acute rejection episodes and chronic rejection/chronic transplant nephropathy, which is the leading cause of late graft failure. However, and although it is still too early to evaluate the utility of the new agents, the new therapeutic combinations seem to have a modest impact on allograft half-life. On the other hand, the long-term use of immunosuppressants is associated with nephrotoxicity, in calcineurin inhibitors regimens, steroid morbidity, and increased risk for neoplasia. The concomitant use of CsA and azathioprine (37) or MMF (38, 39) allows to drastically decreasing CsA doses to minimise its nephrotoxicty. It has been recently reported that the conversion from CsA to azathioprine at one year after transplantation results in improvement in long-term renal function and blood pressure control without a negative impact on graft or patient survival (40). These data suggest that similar strategies can be undertaken with the more potent novel immunosuppressants, such as MMF or rapamycin. These agents may also help to eliminate steroids (41, 16).

Conclusions and perspectives

In the last years it appears that transplantation has entered in a modern therapeutic era with the introduction of several immunosuppressants that act on different steps of the allograft reaction. So, it has been possible to design drug combinations that have resulted additive or synergistic. Such potent associations have dramatically reduced the incidence of acute rejection to less than 20% early after transplantation. Having seen these recent data, the first question that arises is whether it is worth it to try to further reduce acute rejection to lower rates at any cost, and whether these potential reductions will be clinically relevant. Probably, the best option will be to keep low the incidence of acute rejection and at the same time try to reduce the co-morbidity related to drug use, namely to steroid related side effects and to minimise or avoid nephrotoxicity. In this regard, the increasing proportion of elderly donors makes necessary to design protocols according to the quality of the graft, and not only according to the characteristics of the recipient. The prevention and treatment of chronic transplant nephropathy is still an issue, specially if we remind that immune and non-immune factors have been implied in its pathogenesis, which makes evident that the role of immunosuppressants will be limited. The use of anti-fibrogenic ancillary drugs may help.

Finally, one of the most challenging issues in organ transplantation is the induction of donor-specific immune tolerance to promote permanent engraftment to allografts. Transplantation clinical trials on the induction of tolerance pose a number of critical scientific and ethical questions and issues (42). There is an increasing body of evidence that standard immunosuppressive therapy based on the use of calcineurine inhibitors blocks the intracellular signals necessary to induce at least some types of tolerance. This raises the question of what xenobiotic use in tolerogenic studies in humans. Costimulation blockade seems highly promising in the induction of transplant tolerance. In experimental models of induction of tolerance by means of costimulatory blockade, CsA seems to dampen, rather than enhance, tolerance (43). Rapamycin or MMF may be good alternatives to calcineurine inhibitors in clinical trials based on costimulatory blockade.

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