Maria Gerbase-DeLima

Association of high post-transplant soluble CD30 serum levels with chronic allograft nephropathy.

The purpose of this study was to evaluate the association of post-transplant soluble CD30 (sCD30) levels, isolated or in combination with of anti-HLA class II antibodies and of serum creatinine levels, with kidney graft loss due to chronic allograft nephropathy (CAN), and type of lesions in graft biopsies for cause. The study comprised 511 first kidney graft recipients, transplanted at a single center, with a graft functioning for at least 2.8 years. A single blood sample was collected from each patient. sCD30 levels were determined by ELISA, and HLA antibodies by Luminex assay. The minimum follow-up after testing was 9.3 years. High sCD30 levels, set at sCD30 ≥ 34.15 ng/mL, the presence of HLA class II antibodies, and serum creatinine ≥ 1.9 mg/dL were independently associated with CAN-graft loss (P values <0.0001, 0.05, <0.0001, respectively), and the combined hazard ratio for CAN-graft loss was 20.2. Analyses of 166 biopsies for cause showed that high sCD30 levels and creatinine were independently associated with interstitial lesions. Post-transplant sCD30 serum levels, especially in conjunction with information regarding HLA class II antibodies and serum creatinine levels, provide valuable information regarding graft outcome and could be useful for the management of kidney transplant recipients.

Investigation of apoptosis-related gene expression levels in preimplantation biopsies as predictors of delayed kidney graft function.

Background The purpose of this study was to investigate the expression of the gene coding for the antiapoptotic molecule Bcl-2, the proapoptotic molecule Bax, and the apoptosis executor enzyme caspase-3 in preimplantation renal biopsies (PIB) as markers for delayed graft function. Methods In this prospective single-center study, gene expression levels were evaluated using real-time TaqMan polymerase chain reaction in PIB of kidneys from 72 deceased donors (DDs) and 18 living donors (LDs). Results CASP3 and BAX expression levels were higher, whereas those of BCL2 were lower, in DD than in LD PIB. In biopsies from DD, BCL2 levels were lower in cases with DGF, whereas no differences were observed concerning CASP3 and BAX. The BAX/BCL2 gene expression ratio greater than 2.29 associated with DGF with an odds ratio of 2.00. A multiple regression analysis including data of TLR4 expression in the first day posttransplant PB from a previous study of our group conducted in the same patients revealed a very strong association of the combination of BAX/BCL2 greater than 2.3 in PIB and TLR4 of 0.95 uRE or lesser in PB with the occurrence of DGF, with OR of 120 and positive and negative predictive values of 91% and 92%, respectively. Conclusions The power to predict DGF of the combination of high BAX/BCL2 expression in PIB and low TLR4 expression in the first day posttransplant peripheral blood observed in the present study is extremely high, in comparison to any other marker or combinations of markers so far published in the literature.

Post-transplant soluble CD30 levels are associated with early subclinical rejection in kidney transplantation.

Several studies have shown association of high pre- or post-transplant levels of soluble CD30 (sCD30) with acute rejection and poor late kidney transplant outcome. Our goal was to investigate whether sCD30 levels at month-3 post-transplant are associated with subclinical rejection, presence of CD30(+) cells within the graft, and expression of immune response genes in peripheral blood mononuclear cells. The study comprised 118 adult first kidney graft recipients, transplanted at a single center, receiving tacrolimus in low concentration. All were submitted to a protocol biopsy at month-3. Subclinical rejection was identified in 10 biopsies and sCD30 levels ≥ 61.88 ng/mL (P = 0.004), younger recipient age (P = 0.030) and non-Caucasian ethnicity (P = 0.011) were independently associated with this outcome. Rare CD30(+) cells were present in only two biopsies. There was a correlation between sCD30 levels and CD30 gene expression in peripheral blood mononuclear cells (r = 0.385, P = 0.043). These results show that high sCD30 levels are independent predictors of graft dysfunction and may contribute to patient selection protocols by indicating those who could benefit from a more thorough evaluation.

HLA-B*15:04:04, a novel HLA allele identified during proficiency testing in Brazil.

HLA‐B*15:04:04 differs from HLA‐B*15:04:01 by one nucleotide at codon 238 (gat > gac).

Influence of immunosuppressive drugs on the CD30 molecule in kidney transplanted patients

BACKGROUND Soluble CD30 (sCD30) is a suggested marker for kidney transplantation outcomes. We investigated whether sCD30 serum levels are influenced by immunosuppression and whether they correlate with findings in protocol biopsies and with CD30 gene expression in peripheral blood mononuclear cells (PBMC). METHODS We studied 118 kidney transplant recipients that initially received tacrolimus (TAC) and, at month-3, were converted or not to sirolimus (SRL). RESULTS sCD30 serum levels gradually declined after transplantation, being the decline more pronounced in the SRL group. CD30 gene expression in PBMC was higher in the SRL group than in the TAC group. Patients with IF/TA ≥ I in the month-24 protocol biopsy had higher sCD30 levels than patients without IF/TA, in the SRL group (P = .03) and in the TAC group (P = .07). CD30+ cells were observed in three out of 10 biopsies with inflammatory infiltrate from the SRL group. In mixed lymphocyte cultures, SRL and TAC diminished the number of CD30+ T cells and the sCD30 levels in the supernatant, but the effect of SRL was stronger. CONCLUSIONS Overall, sCD30 levels are lower in SRL-treated patients, but the association between increased sCD30 levels and IF/TA at month-24 post-transplantation is stronger in SRL than in TAC-treated patients.

Heightened expression of HLA-DQB1 and HLA-DQB2 in pre-implantation biopsies predicts poor late kidney graft function

BACKGROUND Accurate pre-transplant prediction of late graft function remains an unmet need in kidney transplantation. The aim of this study was to evaluate HLA genes expression levels in pre-implantation biopsies (PIB) of deceased donor kidneys as markers for long-term graft outcome. METHODS HLA genes expression analysis was initially performed using microarray data of 53 PIB, previously generated by our laboratory. The validation analysis was performed by real-time PCR in 116 PIB from an independent cohort. RESULTS The microarray data showed association between high expression levels of HLA class II genes, especially HLA-DQB1 and -DQB2, in kidneys from young (18 to 49-year-old) donors and poor (eGFR < 45 mL/min/1.73 m2) 1- and 5-year graft function. A subsequent study in an independent cohort, in which only HLA-DQB2 expression was evaluated, validated the association between increased HLA-DQB2 expression in PIB of kidneys from young donors and poor 1-year graft function: expression levels ≥0.0025 relative units conferred an odds ratio of 22.5, with positive and negative predictive values of 71.4% and 90.0%, respectively. CONCLUSION Heightened expression of HLA-DQB1 and -DQB2 in PIB are promising tools for pre-transplant risk assessment of poor late graft function in transplants with kidneys from 18 to 49-year-old donors.

Predicting delayed kidney graft function with gene expression in preimplantation biopsies and first-day posttransplant blood

The purpose of this study was to investigate possible markers for predicting delayed graft function (DGF). To this end we analyzed, in pre-implantation biopsies (PIB) and in first-day post-Tx peripheral blood mononuclear cells (PBMC), the expression of five genes (ACSL4, CUBN, DEFB1, FABP3, GK) through real-time TaqMan PCR assays. These genes were selected from a large scale gene expression study in PIB. DEFB1, FABP3 and GK expression levels in PIB were lower in cases with DGF and, in a multivariate analysis which included these genes and clinical variables, only FABP3 expression remained independently associated with DGF. FABP3 expression lower than -1.32 units of relative expression conferred an odds ratio for DGF of 41.1. Compared to the PBMC of recipients without DGF, recipients with prolonged DGF (pDGF) had lower ACSL4 and higher DEFB1 expression levels. In a multivariate analysis, including PBMC gene expression levels of ACSL4, DEFB1 and TLR4 (data from a previous study with the same patients) and clinical variables, only TLR4 remained independently associated with pDGF. In summary, this study revealed FABP3 expression in PIB as a marker for DGF and disclosed new genes possibly involved in the pathogenesis of DGF.

Failure to Up-Regulate BCL2 Gene Expression in Deceased Donors Kidneys Associated with Occurrence of Delayed Graft Function: 541

This study was designed to investigate apoptotic molecular pathways in the pathogenesis of delayed graft function (DGF) in human kidney transplantation (Tx). For this, we evaluated expression levels of the genes BCL2, BAX, and CASP3 in pre-implantation biopsies (PIB) from kidneys of 72 deceased donors (DD) and from 18 living donors (LD). DGF was defined as dialysis requirement within the first week after TX. Total RNA was extracted from PIB with RNeasy Mini Kit (Qiagen). Target and endogenous control (TATA-binding protein) genes expression levels were quantified by TaqMan® assays (Applied Biosystems) and results are presented as relative expression units (RU) calculated with the 2-(delta delta Ct) method. The reference group consisted of PIB from kidneys of 18 living donors (LD). Statistics: Mann-Whitney and Fisher ́s exact tests; significance was set at p< 0.05. Tx with DD kidneys that presented (n=27) or not (n=45) DGF did not differ in respect to donors‘ age, % of expanded criteria donors, recipients‘ age, and cold ischemia time (CIT). BCL2 expression in PIB was lower in kidneys from DD than from LD (median values: 0.6 vs 1.0 RU, p=0.007), did not differ between DD with and without expanded criteria nor between grafts with CIT < 30h and between 30 and 38h, and was lower in DD Tx with than without DGF (median values: 0.5 vs 0.8 RU, p=0.04). BAX expression in PIB was higher in kidneys from DD than from LD (median values: 2.0 vs 1.0 RU, p< 0.0001), did not differ between DD with and without expanded criteria, was higher in grafts with CIT between 30 and 38h (median values: 2.2 vs 1.7 RU, p=0.02), and did not differ between cases with than without DGF; BAX/BCL2 ratio was higher in kidneys from DD than from LD and in cases with DGF (median values: 3.0 vs 1.3, p< 0.0001); CASP3 expression was higher in kidneys from DD than from LD (median values: 1.2 vs 1.0 RU, p=0.005), did not differ between DD with and without expanded criteria, between grafts with CIT < 30h and from 30 to 38h, nor between cases with and without DGF. In conclusion, up-regulation of the anti-apoptotic gene BCL2 in PIB from DD conferred protection against DGF, whilst the expression of the executor caspase CASP3 and the pro-apoptotic BAX genes, although higher in DD than in LD, did not correlate with DGF. This finding is in line with experimental data (Gobe, 2000) showing that the protection of acute renal failure conferred by BCL2 probably results both from an anti-apoptotic effect and from a positive stimulus on the synthesis of several growth factors by cells of the distal tubule 561

Impact of Different Levels of Preformed Anti-HLA Donor Specific Antibodies Detected by Single Antigen Luminex Assay on Kidney Transplants Performed with Negative Complement Dependent Cytotoxicity T and B Crossmatches: 737

This study aimed to evaluate the clinical relevance of different levels of preformed anti-HLA donor specific antibodies (DSA), revealed by Luminex single antigen beads assay (SAB) in kidney transplants performed with negative complement dependent cytotoxicity (CDC) crossmatches against donor T (CDC-anti-human globulin) and B lymphocytes. The study comprised 298 recipients of first (N=256) or second (N=42) kidney grafts, transplanted in a single center, between 2001 and 2006. At the time of transplantation no information regarding Luminex-detected antibodies was available. In this study, stored pretransplant sera were screened for anti-HLA antibodies (Labscreen® Mixed, One Lambda) and the positive ones were further analyzed with the SAB assay (Labscreen® Single Antigen, One Lambda). In the SAB assay, all reactions with normalized mean fluorescence intensity (nMFI) >300 were classified as positive and DSA were defined as antibodies against HLA-A, B or DR mismatches. Based on these results, the recipients were divided into three groups: PRA+,DSA+, PRA+,DSA-, and PRA-. Females, non-white patients, retransplants, and ATG induction therapy were significantly (p< 0.001) more frequent among PRA+ recipients. The proportion of transplants with deceased donors was the same in the three groups (42, 43 and 36%, in PRA+,DSA+, PRA+,DSA-, and PRArecipients, respectively). The mean time of post-transplant follow-up was 57.8, 59.0 and 54.4 months for PRA+,DSA+, PRA+,DSA-, and PRArecipients, respectively. Graft survival curves were constructed with Kaplan-Meier method and compared with the log-rank test; Cox regression analysis was used to calculate risk of graft loss. Graft survival did not differ between the three above mentioned groups of recipients. We then sub-divided the PRA+,DSA+ recipients into the following DSA nMFI ranges: 300-1,500, >1,500-3,000, >3,000-6,000 and >6,000. No difference in graft survival was observed among PRA+,DSA+ with nMFIs 300-1,500, PRA+,DSA-, and PRArecipients. On the other hand, no graft survival difference was observed among recipients with DSA with nMFIs >1,500-3,000, >3,000-6,000 or >6,000. Therefore, further analyses were conducted considering only two groups of recipients: one comprising all recipients without DSA or with DSA < 1500 nMFI (N=228), and the other comprising recipients with DSA >1,500 MFI (N=70). Graft survival was significantly lower (p< 0.001) in recipients with DSA >1,500 MFI (one-year graft survival: 78.6% vs 92.1%; 5-year graft survival: 64.3% vs 78.9%). DSA >1500 MFI conferred a relative risk of 4.4 (95% CI 1.7-11.1) for graft loss at one year and of 3.0 (95% CI 1.4-6.4) for late graft loss in recipients with functional kidneys at one year. Recipients transplanted with DSA >1500 nMFI also presented lower estimated glomerular filtration rates (Cockroft-Gault formula), at the first month (43.0±19.9 vs. 56.0±20.3 ml/min, p< 0.001) and first year (56.6±17.2 vs. 65.0±21.2 ml/min, p< 0.01). In conclusion, the results of this study showed that DSAs directed to HLA-A, B and DR mismatches with nMFIs from 300 to 1,500 did not have any influence on the transplant outcome, whereas DSAs with nMFIs >1,500, although not associated with immediate graft losses, conferred a risk for graft dysfunction already at the end of the first month, and a risk for long-term graft loss. 900

High Soluble CD30 Serum Levels Are Associated with Inflammatory Infiltrate in Renal Graft Protocol Biopsies and Predict Dysfunction of the Graft: 509

In a previous study we showed, in recipients (R) with a functional kidney graft for at least three years, that high sCD30 serum levels (≥70 U/mL) are markers for long-term graft loss. In the present study we evaluated the relationship between sCD30 levels in R with stable renal function three months after transplantation (Tx) and the presence of inflammatory infiltrate in protocol biopsies (PBx) and renal function 3 and 6 months thereafter. The study was conducted in 127 first graft R with pre-Tx peak PRA < 30%, negative T and B CDC crossmatches and no donor-specific antibodies detected with Luminex assays. sCD30 levels were determined by ELISA (Bender MedSystems), in a sample drawn at the day or up to 28 days before the PBx. Graft lesions were graded according to Banff ́07, C4d was evaluated by immunofluorescence. Serum creatinine levels (Cr) and proteinuria/Cr ratios (PCR) nearest to the PBx and 3 and 9 months thereafter were recorded. Estimated glomerular filtration rate (eGFR) was calculated with the Cockroft-Gault formula. Statistics: Mann-Whitney and Fisher ́s exact tests. Results: Inflammatory infiltrate was present in 30 (23.6%) biopsies (9 had acute cellular rejection; 7, borderline rejection; 14, tubulo-interstitial nephritis/ inflammatory infiltrate without quantification; none had antibody mediated rejection). R with and without inflammatory infiltrate did not differ regarding serum Cr or PCR. sCD30 levels were higher in patients with inflammatory infiltrate (69.7 vs 37.5 U/mL, p< 0.05). sCD30 levels ≥70 U/mL were associated with the presence of inflammatory infiltrate (p< 0.02, RR=2.2), but not with Cr >1.5 mg/dL, proteinuria/Cr ≥ 0.1, or CMV infection. The eGFR did not differ between cases with sCD30 ≥ or < 70 U/mL at the time of the PBx, but was lower 3 and 9 months thereafter in R with sCD30 ≥ 70 U/mL, while no significant differences in eGFR were observed in relation to the presence of inflammatory infiltrate or acute rejection. In conclusion, the results showed that high sCD30 serum levels are associated with inflammatory infiltrate in PBx and may predict graft dysfunction 6 to 9 months after the biopsy, independently of the histological findings. 510