1 Division of Nephrology and Dialysis, Department of Internal Medicine III, University of Vienna, Austria, 2 Department of Laboratory Medicine, University of Vienna, Austria and 3 Division of Ultrastructural Pathology and Cell Biology, Department of Pathology, University of Vienna, Austria
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Abstract |
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Methods. Twenty re-transplanted and broadly immunized cadaver kidney recipients (median PRA reactivity 87%, range 55100%) were treated with IA (protein A) immediately before transplantation and during the early post-transplantation period (median number of IA sessions 11, range 124). Patients received additional prophylactic anti-lymphocyte antibody therapy. Nineteen patients had a negative pre-transplant cross-match. In one patient, a positive cross-match was rendered negative by the pre-transplant IA session.
Results. One-year graft survival was 80% and patient survival 95%. Median (range) serum creatinine in functioning grafts was 1.6 (0.82.7) mg/dl at discharge and 1.5 (1.05.8) mg/dl at 1 year. Two grafts were lost due to acute vascular rejection, whereby one rejection occurred after withdrawal of immunosuppression due to septicaemia. One patient had acute cellular rejection, which was reversed by a second course of anti-lymphocyte antibody therapy. Thrombotic microangiopathy and surgical complications were the causes for one graft loss each. Retrospective immunohistochemistry revealed peritubular C4d staining, a presumed marker for humoral alloreactivity, in 12 out of 15 biopsies.
Conclusions. These results suggest that prophylactic peri-operative IA and anti-lymphocyte antibody therapy might be an effective therapeutic strategy for the prevention of early graft failure in sensitized re-transplant recipients.
Keywords: HLA-antibodies; humoral response; hypersensitized patients; immunoadsorption; kidney re-transplant; kidney transplantation; renal graft rejection
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Introduction |
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An increasing number of studies emphasize the important pathogenetic role of alloantibodies, which can have a detrimental influence on graft survival [1218]. Particularly broadly sensitized recipients are susceptible to humoral graft injury [12,13,15,16,18]. On this basis we hypothesized that a more efficient prophylaxis against acute rejection in sensitized patients might be achieved by targeting not only cellular but also humoral immune responses.
Extracorporeal immunoadsorption (IA) is considered to be the most efficient way to remove antibodies. A single treatment session is able to decrease IgG levels by 80% [19]. IA has thus been used to effectively reverse acute humoral rejection in refractory patients [2024], which is why we hypothesized that prophylactic IA therapy might be equally efficient in broadly sensitized patients prone to antibody-mediated graft loss.
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Subjects and methods |
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Immunological techniques
Cytotoxic cross-match testing was performed with donor peripheral blood mononuclear cells according to the rules of the Eurotransplant Organization using the standard microcytotoxicity technique described by Terasaki and McClelland [25]. Cytotoxic PRA reactivity was assessed using a panel of cells from 20 to 50 phenotyped donors. For exclusion of IgM, tested sera were pre-treated with dithiotreitol.
Immunosuppression
The procedure of IA is reported elsewhere in detail [26]. Briefly, the patients underwent plasma separation using the autopheresis-CTM therapeutic plasma system (Baxter, IL). During each session, 2.5 plasma volumes were run alternatively over two cartridges containing staphylococcal protein A (Excorim, Lund, Sweden). For removal of bound immunoglobulin, the column was flushed with sodium citrate after each cycle and for storage with phosphate-buffered saline and 0.9% NaCl solution. IA was initiated and performed once immediately before renal transplantation and then every 23 days until stabilization of graft function (serum creatinine less than 2 mg/dl) or graft loss. The number of sessions ranged between 1 and 24 (median 11), since the time to graft function differed. Total serum IgG levels were effectively reduced by IA in all patients and were below the detectable level after the last IA session (less than 1.95 g/l) in most of them (Table 2). As assessed in 16 out of 20 patients, PRA levels, determined simultaneously with the IgG levels, were significantly reduced upon IA treatment (87%, range 55100% before vs 53%, range 13100% after IA; P<0.001). The median PRA titre of 14 out of 20 patients decreased from 1:32 (1:21:32) to 1:3 (1:21:8) (Table 2
). The basal immunosuppressive therapy consisted of cyclosporine (adjusted to trough levels between 200 and 250 ng/ml for the early post-transplant period (8 weeks) and then reduced to 100150 ng/ml), steroids (according to a local tapering regimen), and mycophenolate mofetil at 2 g/day (dose-adjusted according to white blood cells). Nineteen patients received prophylactic therapy with anti-lymphocyte antibody (rabbit, or horse, anti-lymphocyte serum (ALS); initial dose 510 mg/kg/day; Pasteur Merieux S.V., France) for a treatment course of 1014 days. The dose was adjusted according to the white blood cell count. To minimize reduction of ALS levels by IA as a result of antibody binding to staphylococcal protein A, anti-lymphocyte antibody was infused shortly after each IA treatment session. One patient (no. 16) with a history of allergic reactions towards mono- (OKT3) and polyclonal anti-lymphocyte antibodies received prophylactic anti-IL-2R antibody (20 mg at transplantation and after 5 days; basiliximab (Simulect®), Novartis, NJ, USA). Ganciclovir (tapered according to renal function) was administered to all patients as prophylaxis against CMV infection. Anti-cellular rejection therapy consisted of high-dose steroids (dexamethasone, 100 mg/day for 3 days) with or without an additional course of poly- or monoclonal anti-lymphocyte antibody. Three patients were converted from cyclosporine to tacrolimus, in one case because of severe rejection, in the other two cases because of suspected cyclosporine-induced haemolytic uraemic syndrome (HUS).
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Renal allograft biopsy
Percutaneous renal biopsy was performed in 15 patients (three with initial non-function and 12 with delayed graft function). For histopathological analysis, paraffin-embedded sections were stained with haematoxylin and eosin, Periodic Acid-Schiff, Methenamin-Silver and with the trichrome stain Acidic Fuchsin-Orange-G. The histopathological classification was done according to the Banff Working Classification of Kidney Transplant Pathology [30]. C4d staining on paraffin-embedded sections was made according to a protocol described previously [18].
Statistical analysis
All results are expressed as mean±SD or median and range when appropriate. A two-tailed Student's t-test for paired data was used to assess differences between two time points within the group. A P value <0.05 was considered to indicate statistical significance.
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Results |
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Another three recipients lost their graft during the early post-transplantation period. One allograft (no. 13) was lost because of surgical complication (renal vein thrombosis) two days after transplantation. In another patient (no. 4) with graft loss, a first biopsy (day 16) performed because of initial non-function of the transplanted kidney revealed Banff borderline lesion. The presence of intracapillary granulocytes in this specimen pointed to humoral graft injury. In a subsequent biopsy (day 21), borderline changes had progressed to acute vascular rejection (Banff IIA), whereon monoclonal antibody therapy with OKT3 (5 mg/day for 10 days) was initiated. Two subsequent biopsies revealed a complete remission of cellular rejection and disappearance of intracapillary granulocytes. Nevertheless, graft function did not recover and the patient remained on dialysis. The fourth graft loss occurred in a patient (no. 5) with an initially well functioning transplant. At day 5, severe graft dysfunction occurred together with thrombocytopenia and laboratory signs of haemolysis. This and the histologic finding of thrombotic microangiopathy (TMA) led to the diagnosis of cyclosporine-induced HUS. IA and cyclosporine were converted to plasma exchange and tacrolimus, however, graft function did not recover and the patient remained on haemodialysis.
C4d staining
C4d staining was performed in all 15 histologically examined patients (in 10 of them immunohistochemistry was done retrospectively). In 12 recipients (80%) at least one biopsy stained positive for C4d in PTC. In nine C4d-positive patients, biopsy revealed no unequivocal signs of cellular rejection (ATN, n=6; TMA, n=2; Banff borderline, n=1). The remaining three C4d-positive patients showed features of vascular rejection. Three recipients were C4d negative. One of them had renal vein thrombosis. In the other two patients biopsy revealed normal histology or ATN, respectively. All grafts lost, except the one with renal vein thrombosis, were C4d positive.
Infectious complications
With the exception of the patient dying from severe septicaemia, no severe infection episodes occurred. Two patients developed mild CMV infection. Both patients were successfully treated with i.v. ganciclovir. Urinary tract infection in five and bacteraemia in two patients responded well to antibiotic therapy.
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Discussion |
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Our reported 1-year graft survival of 80% compares favourably to the 6070% graft survival of sensitized recipients shown by previous studies aimed mainly at inhibiting cellular rejection [48,10,11]. Schroeder et al. [10] described an early graft loss due to hyperacute or acute rejection in 22% of the sensitized recipients treated with prophylactic quadruple therapy, and a 1-year graft survival of only 70%. Apparently, different results were presented by Thibaudin et al. [9], who reported a high graft survival in patients with more than 5% PRA treated with prophylactic quadruple therapy, but the data cannot be extrapolated to broadly sensitized recipients. Despite the classification of their patients in different groups of sensitization, the authors did not specify the graft survival for each group. Thus, it remains unclear how many of the approximately 50% acute rejections in the broadly sensitized recipients led to graft failure [9]. Furthermore the majority of the patients were not re-transplanted and thus probably less prone to graft failure than our population. In those reports describing the outcome in sensitized recipients receiving a second or subsequent graft, 1-year survival was reported to be even worse than in sensitized first transplants. In the study of Gaston et al. [4], 40% of the patients receiving a re-transplant, in the presence of a negative pre-transplant CDC-cross-match, lost the graft within 1 year. As quadruple therapy did not improve the outcome, it was concluded that induction therapy with ALS is without benefit in patients undergoing repetitive transplantation. The data are in accordance with our own experiences. Between the years 1993 and 1995 highly sensitized and re-transplanted patients at our transplant centre were regularly treated with prophylactic quadruple immunosuppressive therapy. The 1-year graft survival during this period was similarly only 60%.
It could be argued that transplant outcomes have been improving as immunosuppressive strategies have been changing in the latter years. In fact, according to USRDS, 1-year graft survival for patients with a PRA greater than 30% was 84% in 1997 and that of repeat transplants 82%. However, transplant survival of patients with both risk factors, i.e. high PRA concentrations and re-transplantation, remains clearly below non sensitized recipients. The Eurotransplant Foundation reports a 1-year graft survival of 70% (95% CI 6773) in 1045 sensitized (PRA greater than 50%) recipients receiving a second or subsequent graft during the years 19951999 (Drs J. Smits and G. Persijn, Eurotransplant Foundation, personal communication). This outcome underlines the importance of alternative immunosuppressive strategies in sensitized re-transplant recipients.
The remarkable low incidence of immunologic graft losses seems particularly unusual given that 80% of the patients had a positive C4d staining. The high sensitivity of C4d in the diagnosis of acute humoral rejection and the close correlation to donor-specific antibodies has only recently been confirmed [28,29]. Thus, graft survival in C4d-positive patients is markedly decreased and has been reported to be as low as 60% [30]. Even if an aggressive post-transplant anti-rejection therapy is applied, including mycophenolate mofetil, tacrolimus and plasmapheresis, graft survival has been increased to at most 70% [28]. Our results stand in clear contrast to the reported low transplant survival in patients with anti-donor antibodies. Although the number of C4d-positive biopsies was almost three times as high as usual [28,29], 83% of the C4d-positive grafts survived beyond 1 year. In only two patients immune-mediated graft loss occurred. In both of them graft loss was attributed to vascular rejection, occurring after the end of induction therapy. Our finding of positive C4d staining in these recipients, in one of them associated with an accumulation of granulocytes in peritubulary capillaries, points at a contribution of humoral immune mechanisms to graft failure. In a third recipient, the finding of thrombotic microangiopathy together with distinct laboratory features led to the diagnosis of cyclosporine-induced HUS. Retrospective analysis, however, revealed endothelial C4d deposits. Capillary microthrombi with or without C4d deposits have earlier been described to be associated with antibody-positive rejection [1,30,31]. Accordingly, thrombotic microangiopathy in this patient could also be interpreted as a sign of refractory humoral injury.
The general paucity of additional histological features for acute rejection (i.e. interstitial inflammatory infiltrates, vasculitis, tubulitis, polymorphonuclear cells in the peritubulary capillaries) could be explained by prophylactic anti-cellular and anti-humoral therapy, which might have mitigated the histological picture. ATN was a common biopsy finding in our patients and was often associated with capillary C4d deposits. Previously, we were able to demonstrate that C4d deposits in acute humoral rejection persist despite successful anti-rejection therapy with IA [24]. Hence, C4d-positive ATN without other features of humoral rejection might represent a discrete lesion of antibody-mediated graft injury. In these patients, the complete manifestation of humoral rejection might have been prevented by early anti-humoral therapy.
Although IA is highly effective in removing immunoglobulins, other methods might prove to be similarly successful. Plasma exchange has been shown to effectively reverse humoral rejection [32] and might be a promising prophylactic regimen when combined with i.v. IgG [33]. In addition, administration of a specific anti-CD20 monoclonal antibody (rituximab) directed against B cells might hinder donor-specific antibody synthesis. So far, however, none of these treatments has been administered to a larger transplant population as prophylactic post-transplant therapy and their efficacy in this setting still needs to be ascertained.
In conclusion, our data suggest a beneficial effect of prophylactic peri-operative IA and anti-lymphocyte antibody therapy on graft survival in broadly sensitized renal re-transplant recipients. Inhibition of both the humoral and cellular arm of the immune system might comprise an increased graft survival in this specific group of high-risk patients, however, a prospective and randomized trial is warranted to definitely answer this question.
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Notes |
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References |
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