Microemulsion cyclosporin formulation, in contrast to the old formulation, widens the T lymphocyte subsets differences between stable and acute rejection of kidney transplants

José G. G. Oliveira1,, João P. Ramos2, Paula Xavier3, Susana Sampaio1, Maria C. Magalhães2, Armando A. Mendes3 and Manuel Pestana1

1 Renal and 2 Immunology Departments, Hospital S. João and 3 Tissue Typing Center, Porto, Portugal



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The new cyclosporin (CsA) formulation, Neoral®, has different pharmacokinetics compared with Sandimmune® (SIM). Larger area under the curve (AUC) values with equivalent trough blood values are reached when Neoral is administered at equivalent doses to SIM. Previously, we showed a great diagnostic reliability when using cytofluorometric analysis from fine-needle aspiration biopsy (FNAB) samples. We investigated possible changes brought about by Neoral on lymphocyte subsets and the repercussions on the activation score cut-off for acute rejection, defined under SIM treatment.

Methods. Of 63 patients that received SIM, 40 remained rejection-free and 23 suffered one episode of rejection. Of 52 patients that received Neoral, 38 remained rejection-free. Peripheral blood lymphocytes (PBL) and lymphocytes from FNAB taken on days 7 and 14 post-transplantation and on the first day of acute rejection were analysed by flow cytometry.

Results. Trough blood CsA levels were not different between SIM and Neoral treatments. Among rejection-free patients, a significant down-regulation of CD3DR and of CD8DR expression on both graft-infiltrating lymphocytes (GIL) and PBL, and significant up-regulation of naïve T cells on GIL were observed with Neoral. These changes were followed by a significant down-regulation of the activation score with Neoral. Conversely, within the acute rejection group, the activation score was significantly higher with Neoral than with SIM. The activation score performed equally well in Neoral transplants compared with what we had reported with SIM.

Conclusions. Our study indicates that Neoral elicits stronger immunosuppressive effects in stable patients, which eventually should translate into better clinical efficiency. However, when acute rejection supervenes, the treatment breakthrough seems stronger with Neoral. Cytofluorometric studies from FNAB samples showed that diagnostic reliability was maintained at a high level under Neoral therapy.

Keywords: cyclosporin formulation; fine-needle aspiration biopsy; graft-infiltrating lymphocytes; Neoral; peripheral blood lymphocytes; Sandimmune



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The new cyclosporin (CsA) formulation, Neoral, is endowed with different and more predictable pharmacokinetics [1] compared with Sandimmune (SIM). Several studies have reported higher maximum blood concentrations, a shorter time to reach maximum concentration and higher area under the blood concentration vs time curve (AUC/dose) [1,2]. Interestingly, trough blood concentrations were not significantly different when Neoral and SIM were given at identical doses [1]. In the past, AUC/dose was identified as the most important parameter of clinical efficiency amongst SIM-treated patients [3,4]. The first reports of renal transplant patients treated with Neoral have thus far suggested a tendency for a lower rate and lesser severity of rejection episodes [1].

Cytofluorometric studies of peripheral blood T lymphocytes (PBL) and graft-infiltrating lymphocytes (GIL) showed several differences in various T subsets between stable and rejecting kidney transplant patients. IL-2 receptor expression increases at the peripheral blood level [6], while CD3DR is up-regulated also, at least in late acute rejections [7]. The changes observed inside the renal grafts present a more consistent pattern; notably, an enhanced IL-2 receptor and CD3DR expression have been reported during rejection crisis when analysed by both flow cytometry [8] and with an indirect immunoperoxidase method [9]. Ibrahim et al. reported an increased CD45RO+ to CD45RA+ ratio, in a diffuse pattern within renal graft biopsies, with the diagnosis of acute rejection [10]. CsA, through its inhibition on calcineurin activity, down-regulates IL-2 and IFN-{gamma} synthesis [11], and as a consequence causes decreases in T lymphocyte receptor and HLA-DR expression, respectively [11]. It has been suggested that CsA spares suppressor cells and predominantly affects helper T subsets [12]. Furthermore, by inhibiting IL-2 synthesis, CsA may down-regulate the transition from ‘naïve’ to ‘memory’ T cells [13].

Our group has been studying fine-needle aspiration biopsy (FNAB) samples by flow cytometry. We reported several significant differences between FNAB samples from acute rejection renal transplants compared with samples in stable recipients. Furthermore, by following an empirically defined activation score in FNAB T lymphocytes, we obtained very high positive and negative predictive values for acute rejection [14]. This activation score is defined as the sum of the percentage of DRx8 plus CD8DRx32 plus CD3DRx16 plus CD8CD57x4 present in FNAB samples, plus the ratios of FNAB/PBL CD3CD25x40 plus FNAB/PBL CD3DRx100 minus FNAB CDCD45RAx4 [14]; when this score is >=630 the negative and positive predictive values for acute rejection are 93.6 and 76.9%, respectively.

We hypothesized that lymphocyte subsets from PBL and GIL populations would change when Neoral was substituted for SIM, and we specifically looked at differences in the activation score caused by Neoral, which might mandate either a different cut-off or even a different activation score composition in order to maintain a good diagnostic performance.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Of 63 kidney transplant patients treated with SIM, 40 were male and 23 female, with an age range of 16–62 years. They were the last transplant recipients treated with SIM in our unit. Their original renal diseases were unknown/chronic glomerulonephritis (19), hereditary nephritis/tubulointerstitial nephritis (14), polycystic renal disease (10), diabetic nephropathy (9), IgA nephritis (8), focal segmental glomerulosclerosis (1) and rapidly progressive glomerulonephritis (2). All but one were first cadaver kidney transplants, the exception being the recipient of a second transplant, and their panel reactive antibodies (PRA) were always <10%. Twenty-three patients suffered 29 acute rejection episodes, all starting within the first 6 weeks post-surgery. Of the first 52 Neoral-treated transplants entering this study, 31 were male and 21 female, all within the age range of 19–60 years. Their original renal diseases consisted of unknown/chronic glomerulonephritis (20), hereditary nephritis/tubulointerstitial nephritis (11), IgA nephritis (7), polycystic renal disease (5), diabetic nephropathy (5), focal segmental glomerulosclerosis (2) and rapidly progressive glomerulonephritis (2). All were first cadaver kidney transplants, except three patients who were recipients of a second transplant. PRA were <10% in all but two cases. Fourteen patients suffered 17 acute rejection episodes.

The donors and recipients were typed by micro-lymphocytotoxicity tests using well standardized alloantisera. The immunosuppressive regimen was the same for both groups, except for SIM vs Neoral, and included triple therapy (with azathioprine) for first transplants and quadruple therapy with horse ATG for second renal grafts.

GIL were obtained by FNAB, according to the methods described by Häyry [15], and were performed between 90 and 150 min following immunosuppressive drugs administration. The samples were obtained on days 7 and 14 post-transplantation in stable recipients and on the first day of acute rejection diagnosis. Acute rejection was always defined on the basis of a core renal biopsy, following the Banff criteria [16], and complemented by a positive response to anti-rejection therapy. The corresponding patients’ peripheral blood was drawn 2 h earlier, just before administration of immunosuppressive drugs.

One millilitre from the FNAB sample was analysed using a FACScan from Becton-Dickinson, and monoclonals from Becton-Dickinson and Coulter. Our interest was focused on the phenotype of T cells that were studied previously [14]. To characterize these T cells, monoclonal antibodies recognizing differentiation antigens (CD2, pan T cell marker; CD3, T cell receptor-associated molecule on T cells; CD4, helper T cell subset; CD8, suppressor/cytotoxic T cell subset; CD57, present on natural killer and T cell subsets), early activation markers (CD25, interleukin-2 receptor-{alpha}; CD69, member of the natural killer-gene complex; CD71, transferrin receptor on proliferating cells) and a late activation marker (DR, an HLA class II antigen) were used. Also, we studied CD4 subsets expressing CD45RA (‘naïve’ or ‘helper-suppressor’), CD29 (ß-integrin adhesion molecule–ß chain of very late antigen-4, ‘memory’ or ‘helper-inducer’) and CD54 (inter-cellular adhesion molecule-1). All samples remained at room temperature until they were prepared within 2 h of collection. Briefly, a 10 min incubation at room temperature was performed with an average amount of 100 µl of the following Becton-Dickinson (BD) and Coulter (C) monoclonal antibodies: Leucogate standard Simulset product line (BD-S), fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/PERCP (BD) and FITC/PE (C) isotypic controls, CD4-FITC/CD45RA-PE standard Cytostat product line (C-CS), CD4-FITC/CD29-PE (C-CS), CD3-FITC/CD8-PE (C-CS) plus HLADR-PERCP (BD), CD3-FITC/CD25-PE (BD), CD57-FITC/CD8-PE (BD-S), CD3-FITC/CD69-PE (BD), CD3-FITC/CD71-PE (BD) and CD2-FITC/CD54-PE (BD). All were directly conjugated and standard for use in flow cytometry. Erythrocytes were lysed and cells were preserved by Coulter Q-Prep reagents in a Coulter Multi-Q-Prep workstation. Samples were then refrigerated at 5°C until acquisition in a Becton-Dickinson FACScan, using Lysys II software and analysis using BD PcLysys version 1.1. All samples were washed twice with phosphate-buffered saline just before acquisition. The same methods were used with whole-blood samples. The following lymphocyte subsets were studied: CD2, CD2CD54, CD54, DR, CD3, CD3DR, CD8DR, CD3CD25, CD25, CD3CD69, CD69, CD3CD71, CD71, CD8, CD3CD8, CD8CD57, CD57, CD4, CD4CD45RA, CD45RA, CD4CD29 and CD29.

For calculating sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), we defined:

true positive (TP): activation score of >630 points and acute rejection;
true negative (TN): activation score of <630 points and absence of acute rejection;
false positive (FP): activation score of >630 points and absence of acute rejection;
false negative: activation score of <630 points and acute rejection.

The sensitivity was calculated as TP/TP+FN, specificity as TN/TN+FP, PPV as TP/TP+FP, and NPV as TN/TN+FN.

CsA trough blood levels were measured by TDx monoclonal from Abbott. The results were analysed using unpaired Student t-tests for CsA levels, Mann–Whitney U-test for lymphocyte subsets, and Pearson correlation between CsA and lymphocyte phenotypes. The activation score was analysed by Kruskal–Wallis analysis of variance (ANOVA). Furthermore, we introduced a correction for multiple comparisons using the Edward formula: Pc=1-(1-Po)n, where Pc is the corrected P value, Po is the observed P value, and n is the number of comparisons (n=25).

This study was approved by the local Committee of Ethics, and informed consent was obtained in all cases.



   Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Each FNAB sample was submitted to cytocentrifugation, and following a modified Romanowski stain, was observed using light microscopy. If they did not meet the criteria for adequacy by Häyry classification, i.e. <10 parenchymal cells per 100 granulocytes [15], they were discarded. Also, if each tube of monoclonal antibody combination did not contain a comfortable number of T lymphocytes, they were not used. Thus, the number of FNAB lymphocytes available for analysis in each monoclonal combination ranged from 160 to 4580 (median±SD: 741±555) cells. Following the exclusion of stable patients who eventually developed cytomegalovirus infection during the first month post-transplantation, we were left with 54 samples from stable SIM-treated patients, 28 samples from stable Neoral patients, and with all the samples from rejections, i.e. 29 from SIM and 17 from Neoral patients.

The mean doses of SIM and Neoral on day 7 (7.8 vs 7.5 mg/kg/day, respectively) and on day 30 (7.2 vs 6.9 mg/kg/day, respectively) did not differ significantly. CsA blood levels, with day 7 and day 14 measures mixed, were 221±94.6 {eta}g/ml for SIM and 248± 80.9 {eta}g/ml for Neoral, which were not significantly different (P=0.76). We did not observe any clinically significant CsA acute nephrotoxicity episode in either group. In the rejection groups there were no significant differences between SIM and Neoral, although CsA levels were significantly lower for SIM (P=0.030) and for Neoral (P=0.045) at the start of acute rejection compared with the whole rejection-free group. Serum creatinine was not significantly different between rejection-free patients treated with SIM vs Neoral, and was not different between acute rejection patients treated with SIM vs Neoral.

We began our study by comparing rejection-free patients treated with SIM vs Neoral. We present both P and corrected P (Pc) values for multiple comparisons. Both in PBL and FNAB samples, several significant differences were found. In Table 1Go, we present T lymphocyte subsets in PBL. In Figure 1Go, we present T subsets present in FNAB samples. The following GIL phenotypes were down-regulated with Neoral: CD3CD8 (P=0.011), CD8DR (P<0.0001), CD3DR (P<0.0001) and CD69 (P=0.010). However, following correction for multiple comparisons, only CD3DR and CD8DR maintained a level of significance (both Pc values <0.0001). The ratio FNAB/PBL for CD3CD25 was higher during Neoral treatment (P=0.042, Pc=0.66), and CD4CD45RA was up-regulated with Neoral (P=0.002, Pc=0.049). As a consequence, the activation score was 418 points in SIM-treated patients, which was significantly higher than the 365 points in Neoral-treated recipients (P=0.031).


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Table 1. T lymphocyte subsets in peripheral blood (%), comparing SIM vs Neoral in rejection-free patients

 


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Fig. 1. GIL subsets in rejection-free transplants treated with either SIM or Neoral (Neo). F/P, FNAB/peripheral blood ratio. The differences between SIM and Neoral are significant for each subset.

 
We analysed T subsets among acute rejection transplants treated with SIM vs Neoral. We observed four changes associated with PBL and only three with GIL. DR expression on PBL was up-regulated during Neoral treatment (P=0.040, Pc=0.64), whereas CD4 (P=0.050, Pc=0.72), CD4CD29 (P=0.030, Pc=0.53) and CD3CD25 (P=0.006, Pc=0.14) were down-regulated. In relation to GIL, CD3CD25 was down-regulated with Neoral (P=0.009, Pc=0.21) while both CD54 (P=0.049, Pc=0.72) and CD29 (P=0.033, Pc=0.56) were up-regulated with Neoral. Interestingly, the activation score was significantly higher with Neoral (1441 points) compared with SIM (967 points) (P=0.025).

We then examined whether the activation score maintained the reliability described in SIM-treated transplants [14]. In Figure 2Go, we present T phenotypes expressed in PBL that showed significant differences comparing rejection-free vs acute rejection. As far as GIL is concerned, more than half of the subsets analysed displayed significant differences, and eight T subsets showed significant differences even after correction for multiple comparisons (Table 2Go). The activation scores in rejection-free and acute rejection episodes were 365±160 and 1441±619 points, respectively (P<0.0001). In spite of the differences found when comparing SIM with Neoral, we selected as a cut-off for the activation score a single value, which was 630 points. Again, good results were obtained. The sensitivity and specificity for acute rejection was 88.8 and 88.2%, respectively; the positive predictive value reached 80% and the negative predictive value was 93.7%. These values are very similar to what we have reported while studying SIM-treated transplants [14], and were actually slightly improved in PPV and NPV with Neoral compared with SIM.



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Fig. 2. PBL T subsets in transplant patients treated with Neoral. s, stable; r, acute rejection.

 

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Table 2. T subsets present in FNAB samples in transplants treated with Neoral, both rejection-free and acute rejection patients; FNAB/PBL: ratio of FNAB over PBL; (a): P corrected for multiple comparisons, (n=25), by the formula from Edwards

 
We did not find significant correlations between trough cyclosporin blood levels and any of the analysed T cell subsets, either in stable or in acute rejection kidney transplants.



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This study confirms our working hypothesis that, compared with SIM, Neoral changed T subsets present in either PBL or GIL, independent of whether recipients were stable or developing an acute rejection episode after kidney transplantation.

In stable patients, 19 significant differences were found, and seven remained when correction for multiple comparisons was applied. Most importantly, some of these changes affected the most relevant T subsets for acute rejection diagnosis following our methodology [14]. These subsets were CD3DR, CD8DR and CD4CD45RA. Furthermore, the activation score decreased significantly with Neoral within this stable recipients group. DR is a late activation marker and has consistently been shown to be associated with acute rejection [7,8,10]. The significant up-regulation of CD4CD45RA and CD45RA in GIL, together with a significant down-regulated CD29 and CD4CD45RA/CD4CD29 ratio in PBL, may constitute an additional indication of an improved immunosuppressive efficacy by Neoral. Actually, CD29 (memory) can be activated on a broader range of antigen-presenting-cells than CD45RA (naïve) T lymphocytes, and CD4CD29 T cells are endowed with an enhanced expression of several adhesion/ accessory molecules [16]. As a rule, naïve and memory T cells are efficiently stimulated by dendritic cells and activated B lymphocytes, but memory cells can respond further to resting B lymphocytes, to unprimed macrophages, and to T cell receptor stimulation only. All of these properties may be important in a direct antigen presentation, especially during the early stages of post-transplantation [17]. The small but significant rise in the ratio of FNAB/PBL for CD3CD25 (not significant when corrected for multiple comparisons) was explained by a stronger CD3CD25 down-regulation at the PBL level, since GIL CD3CD25 were lower with Neoral (12.3%) compared with SIM (12.8%). We believe that this may reflect the ability of cyclosporin to accumulate inside the kidney, where it reaches values approximately four times its blood concentration [11], thus dampening the consequences of a superior Neoral bioavailability.

We were pleased to reproduce the very high reliability of our activation score to diagnose acute rejection. When restricting the analysis to Neoral, we found 18 (eight when corrected for multiple comparisons) significant differences comparing GIL from stable transplants with those from acute rejection. However, when we looked at PBL populations, we found only five significant differences comparing stable with acute rejection (Figure 2Go). This is a strong confirmation of the differences between these two T cell populations, and indicates that the small FNAB contamination by blood in the aspirate does not decrease its utility.

We were puzzled by the significant increment in the activation score with Neoral in comparison with SIM when we analysed the two acute rejection groups. This necessitated a greater need to reinstate antibody anti-rejection treatments with Neoral, which was used in 75% compared with 55% of acute rejections treated with antibodies in the SIM group. There are no definitive data on acute rejection grades with Neoral, although they seem not to be significantly different [19,20]. It was not within the scope of this study to evaluate acute rejection prevalence and severity under Neoral treatment, especially as the number of patients would not permit it. However, we speculate that this apparent contradiction with presumed better Neoral efficiency may simply indicate that once patients under Neoral therapy develop acute rejection, they may mount a more powerful anti-allograft response than when the event arises under SIM.

New immunosuppressive drugs are currently being introduced into clinical transplantation. Mycophenolic acid will probably be substituted for azathioprine in almost all kidney transplant patients. Therefore, we speculate that some changes may develop in both GIL and PBL subsets. Interestingly, our preliminary data on rejection-free patients treated with Neoral, mycophenolic acid and prednisolone do show a few significant differences; however, the activation score did not change [21]. The small number of acute rejection cases analysed thus far prevent us from making further comments.

In summary, we describe several significant T cell subsets changes, either in PBL or in GIL, when kidney transplants under Neoral are compared with SIM-treated patients. As a whole, these changes point to stronger immunosuppression with Neoral. We reconfirm the validity of our activation score for GIL for the diagnosis of acute rejection using cytofluorometric studies in Neoral-treated kidney transplant patients.



   Acknowledgments
 
This work was supported by a grant from project PRAXIS/P/SAU/14020/1998.



   Notes
 
Correspondence and offprint requests to: J. G. G. Oliveira, Trav. das Antas, 84, 5°C, 4350-046 Porto, Portugal. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 10. 7.00
Revision received 30.11.00.



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