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BACKGROUND Previously we reported that cultures of fine-needle aspiration biopsy (FNAB) samples synthesize different cytokine pattern depending on the alloimmune response towards the kidney graft. However, we failed to find a clear picture for growth factors implicated in early T-cell activation (interferon-gamma, interleukin [IL]-4, IL-12), although we observed that interleukin-1 receptor antagonist (IL-1ra) was associated with absence of acute rejection. We have now studied tumor necrosis factor-alpha (TNF-alpha) and its two soluble receptors, sTNFRI and sTNFRII, IL-1beta and soluble IL-1 receptor II (sIL-1RII), and leukemia inhibitory factor (LIF), all potential modulators of T-cell activation. METHODS Sixty-six cadaver kidney transplants (KTX) were divided into four groups: group 1, day 7 after KTX, stable (n=30); group 2, day 7 after KTX, 8+/-4.5 days before acute rejection (n=12); group 3, first day of acute rejection (n=17); and group 4, day 14 after KTX, stable (n=32). Patients from groups 1 and 4 remained rejection-free for the first 6 months. All rejection episodes were confirmed by core renal biopsy. FNAB samples were cultured according to our published methodology, and culture supernatants were collected at 48 hr and analyzed by ELISA for IL-1beta, sIL-1RII, TNF-alpha, sTNFRI, sTNFRII, and LIF. Serum levels for sIL-1RII, sTNFRI, and sTNFRII were also measured. RESULTS FNAB cultures from groups 1 and 4 synthesized significantly lower amounts of sTNFRI and sTNFRII than those from either groups 2 or 3. Both sTNFRI and sTNFRII reached high positive and negative predictive values for acute rejection. IL-1beta and sIL-1RII were synthesized by all groups but without differences. No trace of LIF and TNF-alpha was found. sTNFRII was significantly higher in serum from group 3. CONCLUSIONS Both TNF receptors were positively associated with acute rejection and were good predictors of impending acute rejection. The ratio of sIL-1RII over IL-1 (together with IL-1ra that we previously measured in FNAB cultures) suggests that IL-1 actions may be inhibited with current immunosuppression early after transplantation.

Background: T-cell activation, the key event in the development of acute allograft rejection, depends on co-stimulatory signals delivered by antigen-presenting cells (APC). APC-derived cytokines may provide co-stimulation and modulate alloimmune reaction. We have studied cytokine synthesis by fine-needle aspiration biopsy (FNAB) culture and we found significant differences for interleukin (IL)-2, IL-6, IL-10, M-CSF and IL-1ra on comparing acute rejection versus stable kidney transplant patients. We report our findings on FNAB cultures synthesis of IL-7, IL-15, IL-16, IL-17, IL-18 and RANTES (regulated upon activation, normal T-cell expressed and secreted), all potential modulators of anti-graft reaction. Patients and Methods: Kidney transplants (KTX) treated with CsA-AZA-Pred from the beginning, were divided into four groups. Group I: day 7 post-KTX, stable; II: day 7 post-KTX, 6.5 ± 5.5 days before acute rejection; III: first day of acute rejection; IV: day 14 post-KTX, stable. Patients from I and IV remained rejection-free for the first 6 months, at least. All rejection episodes were confirmed by classical core renal biopsy. FNAB samples were cultured according to our published methodology and culture supernatants were collected at 48 h and analysed by ELISA for IL-7, IL-15, IL-16, IL-17, IL-18 and RANTES. Results: Group III synthesized significantly higher amounts of IL-7, IL-16 and IL-18 than stable patients (groups I and IV). RANTES production did not show significant differences among the four groups. We did not find any trace of IL-15. Conclusions: IL-18 may play the activation role that has been attributed to IL-12 which previously, we did not find to correlate significantly with acute rejection in KTX. IL-16 seems to play an activation role rather than an inhibition of anti-graft reaction. We confirm that RANTES is not significantly associated with acute rejection in KTX.

Background/Aims: Growth of T cell lines from kidney graft biopsy specimens that suppress the antidonor response, either directly or through a soluble factor, both specific and nonspecific to donor, has been reported. Fine-needle aspiration biopsy sample cultures synthesize a large array of cytokines/chemokines with significant differences between stable versus acute rejection transplants. We hypothesize that the products of such cultures may be endowed with antidonor response modulation abilities in kidney transplantation. Methods: From 46 patients, 21 rejection free (group A) and 25 developing 28 acute rejection crises (group B), samples were obtained on days 7 and 30 after transplantation in group A and on day 7 and on acute rejection day in group B. The supernatants obtained after 48 h of culture were studied for IL-1 receptor antagonist, IL-4, IL-4 soluble receptor alpha, IL-12, IL-13, IFN-γ, TGF-β1, TGF-β2, GM-CSF, and PGE2 and for their effects on mixed lymphocyte reactions, donor-recipient and recipient-third-party combinations. At the end, analysis by flow cytometry of donor-recipient cultures complemented the analysis done before cultures. Results: Day 7 supernatants from group A specifically and significantly inhibited the antidonor response; supernatants from group B nonspecifically stimulated the antidonor response. The IL-1 receptor antagonist production was significantly higher by day 7 in group A, and the PGE2 production was significantly higher in group B. Flow cytometry analysis did not disclose significant differences between inhibited versus stimulated antidonor responses. Conclusions: Cultures of aspiration biopsy samples done early after transplantation in rejection-free patients produce soluble suppression-specific antidonor response factor(s), not present in cultures from biopsy specimens taken before and during rejection. This was associated with IL-1 receptor antagonist synthesis.

The new immunosuppressive drug Rapamycin (Rapa) is endowed with a mechanism of action that is distinct from that of calcineurin inhibitors. It has been claimed that Rapa does not significantly modulate either the cytokine expression or the transcription of several growth factors in mitogen-activated T cells. Previously, we reported that fine-needle aspiration biopsy (FNAB) sample cultures synthesize a large array of cytokines, and some of them may be powerful predictors of acute rejection in renal transplants. We hypothesized that Rapa may induce significant changes on cytokine production by FNAB sample cultures and on serum cytokine receptors when compared to other immunosuppressive drugs.Kidney transplants treated with CsA-Rapa-Pred (Rapa group) were compared with transplants treated with CsA-mycophenolate mofetil-Pred (MMF group). They were studied on day 7 posttransplantation, and they remained rejection free for at least the first 6 months. FNAB samples were cultured and the supernatants were collected at 48 hr of incubation and analyzed by ELISA for interleukin 1 receptor antagonist (IL-1ra), IL-2, IL-6, IL-10, IL-18, monocyte chemotactic protein 1 (MCP-1), soluble tumor necrosis factor I, and transforming growth factor (TGF)-beta(1). The soluble receptors for IL-1, IL-2, IL-6, and tumor necrosis factor alpha, together with IL-2 and IL-18 were also measured in serum.Significant differences were observed when comparing Rapa with the MMF group. IL-18 and TGF-beta(1) synthesis were up-regulated, whereas IL-6 and MCP-1 were down-regulated in FNAB sample cultures. The Rapa group showed a significant down-regulation of each cytokine receptor and of IL-2 in serum.Rapa was associated with a decreased synthesis of primarily monocyte-derived cytokines and enhanced production of TGF-beta(1), which in an appropriate cytokine milieu may promote allograft tolerance. The down-regulation of cytokine receptors and IL-2 may be associated with a depressed immune response towards the kidney allograft.

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 naive 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.

of cytokine production: Th0 cells (IL-2, IL-4, IFN-c, Methods. Fifty-six adult cadaver kidney transplants lymphotoxin), Th1 (IL-2, IFN-c, lymphotoxin) and were subdivided into 21 stable patients (group A), 22 Th2 (IL-4, IL-5, IL-6, IL-10). Th0 is the common suVering acute rejection (group B), 10 with chronic precursor to both Th1 and Th2 [2,3]. The cytokine rejection (group C ) and three with CMV disease milieu at the time of antigenic stimulation can greatly (group D). Fine-needle aspiration biopsies were cul- influence the development of Th1 and Th2 subsets as tured and their supernatants analysed for IL-2, IL-4, there is no Th0 commitment to either subset [3]. Th1 IL-10 and IFN-c. maturation requires IL-12/IFN-c while Th2 needs IL-4 Results. Group A produced small amounts of both [4]. Several groups have presented data linking a Th1 IL-2 and IL-10 while group B synthetized significantly cytokine pattern to allograft rejection and Th2 cytokine higher IL-2 and significantly lower IL-10 amounts than to transplant tolerance [5‐12]. However, others have group A. Group B produced significantly more IL-2 reported conflicting results [13‐15], which suggests than A on day 7 post-transplantation, several days that the Th1/Th2 paradigm may not be suYcient to before rejection supervened. Group C produced IL-10 fully explain the mechanisms underlying allograft rejecand very low amount of IL-2. Group D produced both tion and tolerance. IL-2 and IL-10. We did not find any IL-4, and IFN-c The measurements of cytokines from renal transwas present in a few samples. For IL-2, sensitivity, plant patients either in peripheral blood or in urine specificity, negative and positive predictive values for have produced variable results, with some pointing to acute rejection were 100, 87.2, 94.7 and 83.3%, their potential use in the diagnosis of renal graft respectively. rejection [16,17], and others failing to reproduce these Conclusions. Cytokine analysis in fine-needle aspira- findings [18,19]. tion biopsy cultures supernatants is a very useful Fine-needle aspiration biopsy (FNAB) is a reliable immunological screening method for kidney trans- and safe diagnostic tool for the immunologic monplants. IL-2 synthesis on day 7 post-transplantation itoring of kidney transplants [20‐22]. Previously we reliably predicted the risk of impending acute rejection have shown that FNAB cell cultures have significant during the first weeks. The cytokine pattern suggests diVerent proliferative abilities whether they come from that acute rejection is associated with Th1, stable stable kidney transplant patients or from acute patients with Th0/Th2, and chronic rejection with Th2 rejecting patients [23]. We hypothesized that patterns. these diVerent proliferative responses could reflect discriminant cytokines patterns of production, following our previous report on chemokines synthesis by FNAB cell cultures [24], which could help both

Background. Peripheral blood lymphocyte (PBL) analysis by flow cytometry has been inconsistently reported as an adjunctive method for diagnosing acute kidney transplant rejection. However, there is good evidence that lymphocytes infiltrating renal grafts differ from those found at the peripheral level. We hypothesized that the study of aspiration biopsy samples in conjunction with PBL by flow cytometry would enable us to diagnose acute rejection crisis reliably. Methods. Lymphocytes from PBL and aspiration biopsies of kidney transplant patients were analyzed. Fifty-one stable patients, rejection-free for the first 6 months, were studied on day 7 and day 30 after transplantation and were compared with 32 patients with 40 acute rejection episodes. Results. Significant differences were observed for several lymphocyte subpopulations on aspiration biopsy samples comparing stable patients with rejection patients. In contrast, PBL analysis was not helpful in differentiating the two groups of patients. By combining the expression of several activation markers inside the graft with CD3DR and CD3CD25 aspiration biopsy to peripheral blood ratios, we obtained very good values for sensitivity and specificity-83.9% and 90.5%, respectively. The positive predictive value for rejection among dysfunctional grafts reached 85.8%. Conclusions. Flow cytometry study of aspiration biopsy samples of kidney transplant patients is a reliable and powerful method to diagnose acute rejection episodes, although it is needed to consider several lymphocyte phenotypes; cytofluorometric analysis of PBL is important because it provides graft-infiltrating cell to peripheral blood lymphocyte ratios. This safe and rapid test may significantly improve the management of kidney transplant patients.

Background Monocytes-macrophages are found within kidney allografts during the first days after surgery, where they perform "housekeeping" tasks, participate in postreperfusion injury, and act as antigen-presenting cells, as well as become involved in the effector phase of acute rejection. They also seem to play a prominent role in chronic rejection. We quantified their presence in fine-needle aspiration biopsies and studied the growth factors that, we hypothesized, would mark the different implications of the presence of monocytes-macrophages. Methods Fine-needle aspiration biopsies were obtained from 56 adult renal transplants and analyzed for CD14+ using the alkaline phosphatase-anti-alkaline phosphatase procedure. Thirty-three patients were studied on the production of interleukin (IL)-1 receptor antagonist (IL-1ra), IL-6, IL-8, macrophage colony-stimulating factor, monocyte chemotactic protein 1, and macrophage inflammatory protein 1alpha by aspiration biopsies cultures using enzyme-linked immunosorbent assay techniques. Results CD14+ cells were present at significantly higher numbers in steroid-resistant acute rejections but also during the first days after surgery, especially if acute tubular necrosis was present. We found a significantly higher production of IL-1ra by rejection-free patients compared with acutely rejecting patients, and this difference was already established on day 7 after surgery (10+/-10.5 days before rejection). Conclusions Monocytes-macrophages are present at higher numbers in aspiration biopsies of kidney transplant patients suffering either acute tubular necrosis or steroid-resistant rejections, but they are present during the first days after transplant in stable patients, too. The production of IL-1ra is significantly up-regulated in stable patients, which suggests that monocytes-macrophages may constitute an early key factor in the down-regulation of the anti-allograft immune response.

Graft-infiltrating cells (GIC) have been studied in heart, lung, and liver transplants and have been shown to have greater proliferative ability when taken from rejecting allografts. Our aim was to study GIC harvested by fine-needle aspiration biopsy (FNAB) in renal transplant recipients.93 adult patients entered the study. The FNABs were done on the 7th, 14th, and 30th day after transplantation in stable cases and whenever a rejection crisis supervened.The proliferation responses of GIC were significantly higher in rejection than in stable cases during the 1st month after transplantation. The sensitivity for rejection was 96.4%, the specificity 91.3%, the negative predictive value 98.7%, and the positive predictive value was 93.3% among dysfunctioning grafts.The study of the proliferative capacity of graft-infiltrating cells in renal transplants is a safe and very useful immunologic monitoring tool, and it could improve the FNAB diagnostic accuracy.