Paula Xavier

Compared to mycophenolate mofetil, rapamycin induces significant changes on growth factors and growth factor receptors in the early days post-kidney transplantation.

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Analysis of fine-needle aspiration biopsies by flow cytometry in kidney transplant patients

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.

XM-ONE Detection of Endothelium Cell Antibodies Identifies a Subgroup of HLA-Antibody Negative Patients Undergoing Acute Rejection

BACKGROUND Kidney transplant recipients exhibiting antibodies (Ab) against either HLA or non-HLA antigens undergo frequent episodes of rejection and exhibit decreased long-term graft survival. The novel flow cytometry crossmatch kit XM-ONE, detects Abs to HLA antigens plus those directed to Tie-2-positive precursor endothelial cells (anti-endothelial cell antibodies, AECA). We studied the clinical importance of these lesser known antibodies. METHODS We retrospectively analyzed 208 sera from 160 recipients of deceased donor grafts for AECA using non-donor peripheral blood endothelial progenitor cells as targets and Luminex methodology for HLA antibodies. RESULTS AECA were detected in 64 patients (40%). A significantly higher proportion of patients showing a positive endothelial crossmatch experienced rejection (31 AECA-positive among 43 rejection cases, 72%) compared with those without rejection (33/117, 28.2%). Immunoglobulin M(IgM) predominated (66%) over IgG (14%) and IgG plus IgM (20%). HLA antibodies positively and significantly associated with rejection as expected. Of special interest were the 19 patients who presented with acute rejection episodes along with restricted AECA positivity. The relative-risk for an acute rejection episode with either AECA or HLA--13.87 and 2.43, respectively--was significant. When HLA was already positive, the relative risk for AECA was 1.24, a non-significant increase. CONCLUSIONS Our data identified AECA-positive patients that showed an increased risk to develop an acute rejection episode early after transplantation. Moreover, restricted AECA-positive patients with acute rejection are an important subgroup which otherwise may be wrongly labeled as non-humoral rejection. Among HLA-negative cases, AECA conferred a significantly greater risk for rejection.

The synthesis by fine-needle aspiration biopsy cultures of IL-7, IL-16 and IL-18 is significantly associated with acute rejection in kidney transplants.

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.

sTNFRI and sTNFRII synthesis by fine-needle aspiration biopsy sample cultures is significantly associated with acute rejection in kidney transplantation.

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-&ggr;, 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-&agr; (TNF-&agr;) and its two soluble receptors, sTNFRI and sTNFRII, IL-1&bgr; 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-1&bgr;, sIL-1RII, TNF-&agr;, 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-1&bgr; and sIL-1RII were synthesized by all groups but without differences. No trace of LIF and TNF-&agr; 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.

Cultures of aspiration biopsy specimens in the immunological monitoring of renal transplants

OBJECTIVE 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. PATIENTS AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

Cultures of Kidney Transplant Fine-Needle Aspiration Samples from Rejection-Free Patients Produce a Specific Antidonor Response Suppressive Factor

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.

Flow cytometry assessment of graft-infiltrating lymphocytes can accurately identify acute rejection in kidney transplants

Previously, we have reported that flow cytometry analysis of fine‐needle aspirates can accurately predict rejection in kidney transplants treated with cyclosporine–azathioprine–prednisolone. In this study, we examined this techniques accuracy using current immunosuppression.

Direct downregulation of Toll-like receptors by anti-IL-2 alpha chain-receptor antibody in cadaver kidney transplant recipients.

Antibodies against the chain of IL-2 receptor (IL-2R Ab) seems to act by preventing signaling after IL-2 receptor ligation by IL-2 (1), an adaptive immune reaction-associated cytokine. However, as the effect of IL-2R Ab seems to be stronger when delayed graft function supervenes (2), we explored whether IL-2R Ab modulates innate inflammatory reaction by looking at tolllike receptors (TLR)-2, -4, and -9. The first 20 cadaver kidney graft recipients with original renal diseases, not deviated from the pattern observed in a white European population and treated with calcineurin inhibitors, mycophenolate mofetil, and prednisolone, were divided into group I (n 13), no further immunosuppressive therapy, and group II (n 7), which received IL2R Ab. Every case remained rejection free. No differences were observed by comparing demographic data of donorrecipient pairs of both groups and neither serum creatinine nor calcineurin inhibitors levels were different on days 7 and 30 postsurgery between groups. Fine-needle aspiration biopsy (FNAB) were performed on days 7 and 30, and total mRNA samples were extracted using Quiagen Rneasy. Experiments were performed in duplicate for each gene, and mRNA quantification results were expressed in an arbitrary unit set as the average value of the no antibody treatment group. Specific polymerase chain reaction primers were TLR-2-fw 5 -TGG CCA CAC CGG AAT AAG-3 and rev 5 -CAA GCC AGG ATG AGG ACT-3 ; TLR-4-fw 5 CTA AAC CAG CCA GAC CTT GAA-3 and rev 5 -ACC TGT CCC TGA ACC CTA TGA-3 ; and TLR-9-fw 5 -GCC CGG ACT GCC ACA CTT-3 and rev 5 -GAT GTA AGC GCC AAC CCT CTG-3 . Four milliliters of the FNAB sample was used for the culture process, which followed the procedures we published previously (3). Immunocytochemistry of every FNAB sample was studied using avidinbiotin methodology. TLR-2, -4, and -9 monoclonal antibodies at 40 g/mL were purchased from Santa Cruz Biotechnology. This study was approved by the local ethics committee, and informed consent was obtained in all cases On day 7 post-KTX, both TLR-2 and -9 mRNAs were significantly reduced in group II compared with group I, P 0.026, and 0.027, respectively. A close to significant downregulation for mRNA of TLR-4 was observed among II, P 0.091. TLR-2 and -4 mRNA expression displayed a significant positive correlation within group II (R 0.75, P 0.01). TLR-9 mRNA values were negatively correlated with the number of DR mismatches in groups I and II (R 0.52, P 0.038). On day 30 post-KTX, no mRNA amplification was observed for TLR-4 and -9 from both groups in 70% of samples. In the remaining cases, only minor amounts were observed and no significant differences were found between groups. As for TLR-2, a significant downregulation within group II persisted, P 0.03. In immunocytochemistry of FNAB samples, each positive cell and the number of renal cells, lymphocytes, and monocytes was counted to calculate the ratios. Only seven samples were positive for TLR-2 and -4, always less than five cells. TLR-9 was significantly higher in group I, whether for absolute number of cells (P 0.000), or ratio of positive over renal cells (P 0.0001) and ratio of positive ones over lymphocytes plus monocytes (P 0.0001). Day 30 samples were negative for TLR-9. FNAB samples cultures displayed a significantly higher cell proliferation when comparing group I versus groups II on day 7 post-KTX, P 0.039, and a significantly lower proliferation result was found when comparing day 30 with day 7 samples from group I, P 0.016. We tested whether IL-2R Ab directly modulates TLR-4 and -9 expression. Eleven healthy blood donors provided the mononuclear cells separated by Lymphoprep, with cultures manipulated through lipopolysaccharide (LPS; for TLR-4) and CpG oligodeoxynucteotide (ODN) from Coley (for TLR-9). The immunocytochemistry studies for TLR-4 and -9 were performed as described for FNAB samples. LPS significantly enhanced TLR-4 expression (P 0.004) and IL-2R antibody significantly down-regulated TLR-4 expression (P 0.004), whereas a preincubation with IL-2R antibody significantly decreased the ability of LPS to upregulate TLR (P 0.012). CPG ODN close to significant rise of TLR-9 expression (P 0.09), and again IL-2R antibody was able to significantly suppress the CPG ODN promoted TLR expression when both added simultaneously (P 0.015) or when CPG ODN provision was delayed by 48 hr (P 0.049). As far as we are aware, this is the first report of a significant downregulation in FNAB samples of TLR-2 and -9 in KTX treated with IL-2R Ab (with a close to significant downregulation of TLR-4). IL-2R antibody is able by itself, without the concourse of other immunosuppressive drugs, to modulate TLR-4 and -9 upregulation by LPS and CPG ODN, respectively. Our data may be of clinical significance as TLR are a powerful link of innate to adaptive immunity.