R. Bardi

Cytokine Gene Polymorphisms in Kidney Transplantation

BACKGROUND Acute and chronic rejections remain an important cause of graft loss after renal transplantation. It has been suggested that cytokine genotyping may have a predictive role to identify patients at greater risk of rejection regardless of human leukocyte antigen (HLA) compatibility and/or the presence of anti-HLA antibodies before the renal allograft. OBJECTIVES We sought to investigate polymorphisms of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, interleukin (IL)-10, IL-6, and interferon (IFN)-γ as indices of differential cytokine production in kidney transplantation and to examine their predictive roles for acute or chronic rejection. PATIENTS AND METHODS TNF-α (G/A -308), TGF -β1 (haplotype codon 10/codon 25), IL-10 (haplotype-1082, -819, -592), IL-6 (C/G -174), and IFN-γ (T/A +874) single nucleotide polymorphisms (SNPs) were detected using polymerase chain reaction (PCR)-specific sequence primers (SSP) in 231 kidney transplant recipients (KTR) including 106 treated with mycophenolate mofetil (MMF+). RESULTS We observed no significant associations of any of investigated polymorphism taken alone with acute rejection episodes (ARE) or chronic allograft dysfunction (CAD). Nevertheless, TGF-β1 Low (L) production was correlated with greater graft survival at 20 years (81.8%) compared with intermediate (L) or high (H) levels (56.1%), affect that the difference was not significant (P = .2). Cytokine haplotype analysis in KTR (MMF-) without HLA-mismatches or presynthesized anti-HLA antibodies (n = 32) showed ARE to be significantly more prevalent among the TNF-α*H/TGF- β1*H/IL-10*H production haplotype (75%) compared with the other haplotypes (16%; P = .03). CONCLUSION The presence of TGF-β1-H secretion profile may protect the kidney graft. TNF-α*H/TGF-β1*H/IL-10*H haplotype was associated with a higher risk of ARE and with poorer graft survival.

Risk factors and consequences of delayed graft function.

The impact of delayed graft function (DGF) on the outcome of renal transplantation remains controversial. We analyzed the risk factors for DGF and its impact on graft and patient survival. A total of 354 renal transplants performed between June 1986 and April 2000 were analyzed. Variables analyzed included donor and recipient age, method and duration of renal replacement therapy, HLA mismatch, cold and warm ischemia times, biopsy-confirmed acute rejection, length of stay in the hospital, serum creatinine at the end of first hospitalization as well as graft and patient survival at one, three, five and ten years. The study patients were divided into two groups: patients with DGF (G1) and those without DGF (G2). DGF occurred in 50 patients (14.1%), and it was seen more frequently in patients transplanted from deceased donors (60% vs. 40%, P <0.0001). The cause of DGF was acute tubular necrosis, seen in 98% of the cases. Univariate analysis showed a statistically significant difference between the two groups G1 and G2 in the following parameters: average duration on dialysis (52.3 vs. 36.4 months, P = 0.006), HLA mismatch (44.9% vs. 32.11% P = 0.015), donor age (35.9 vs. 40.2 years, P = 0.026), cold ischemia time (23 vs. 18.2 h, P = 0.0016), warm ischemia time (41.9 vs. 38.6 mn, P = 0.046), length of stay in the hospital during first hospitalization (54.7 vs. 33.2 days, P <0.0001), serum creatinine at the end of first hospitalization (140 vs. 112 μmol/L, P <0.0001) and at three months following transplantation (159 vs. 119 μmol/L, P = 0.0002). Multivariate analysis revealed the following independent risk factors for DGF: deceased donor (RR = 13.2, P <0.0001) and cold ischemia time (RR = 1.17, P = 0.008). The graft survival at one, three, five and ten years was 100%, 93%, 88.3% and 78.3% in G1 versus 100%, 95.9% 92.8% and 82.3% in G2; there was no statistically significant difference. The patient survival at one, three, five and ten years was 100%, 91.3%, 83.6% and 74.4% in G1 versus 100%, 95.9%, 94% and 82.6% in G2 with a statistically significant difference (P = 0.04). Prolonged cold ischemia time and transplantation of kidneys from deceased donors were the main risk factors for DGF in our study. Also, DGF significantly affected patient survival but had no influence on graft survival.

The first year renal function as a predictor of long-term graft survival after kidney transplantation.

This study examined the impact of graft function at the end of the first year after kidney transplantation on long-term graft survival. We analyzed the roles of serum creatinine (Scr) and other variables as predictors of graft survival among 235 adult kidney transplant patients. The subjects were divided into 3 groups according to their Scr at the end of the first year: group 1, Scr < 100 micromol/L; group 2, 100 micromol/L < or = Scr < or = 150 micromol/L; and group 3, Scr >150 micromol/L. The annual rate of graft loss of 0.7% (95% confidence interval [CI], 0.63-0.77) in group 1, was lower than those in group 2 (2.1%; 95% CI, 2.02-2.18; P < .0001) and group 3 (6%; 5.74-6.26; P < .0001). Regression analysis showed the role of recipient age at the time of operation, and Scr level at the end of the first year to be independent predictors of graft loss. Graft survival was not influenced by any other studied parameter, including donor age, year of procedure, warm ischemia time, history of acute tubular necrosis, and occurrence of an acute rejection episode. We conclude that the 1-year Scr value predicts long-term renal graft survival, representing a simple, practical tool to identify recipients with an high risk for late graft failure.

THE INVOLVEMENT OF HLA -DRB1, DQA1, DQB1 AND COMPLEMENT C4A LOCI IN DIAGNOSING SYSTEMIC LUPUS ERYTHEMATOSUS AMONG TUNISIANS

Background Genetic susceptibility to systemic lupus erythematosus (SLE) varies among populations. Few data exist on associations of HLA class II and class III alleles of the major histocompatibility complex (MHC) and susceptibility to SLE in Tunisians. Patients and Methods We compared HLA-DRB1*, DQA1, DQB1* and C4 allotypes in 62 Tunisian SLE patients and 100 matched controls. We also assessed the association of specific alleles with distinct autoantibody profiles in SLE patients. Results HLA-DRB1*0301, -DRB1*1501 and C4AQO alleles were increased in the SLE patients, while the frequencies of HLA-DRB1*04 and DQB1*03 were decreased. HLA-DQA1*0102 and DQA1*0501 were significantly increased in the SLE patients. HLA-DQB1*0201 and DQB1*0602 were more frequent in the SLE patients. C4A*QO and C4B*QO were increased in frequency in the SLE patients compared to the controls, but only C4A null was significantly increased. Eleven of 17 SLE patients with the C4 null allele were HLA-DRB1 *0301 positive. Three of 16 SLE patients with HLA-DRB1*1501 were associated with HLA-DQB1*0501 rather than DQB1 *0602, as has been reported in European SLE patients. Conclusions The MHC class II alleles (DRB1, DQA1, DQB1) and C4 null associations noted in other ethnic groups are also found in Tunisians, suggesting shared susceptibility factors across ethnic lines in predisposition to SLE. In contrast to other ethnic groups, MHC class II alleles are not associated with the presence of specific autoantibodies in Tunisian SLE patients.

(AT) Repeat in the 3′ Untranslated Region of the CTLA-4 Gene and Susceptibility to Acute Allograft Rejection in Tunisian Renal Transplantation

Allograft rejection is an immune response relying on the proliferation and the differentiation of T cells. CTLA-4 is a co-stimulatory molecule, expressed on activated T lymphocytes, which has been shown to play a crucial role in the down-regulation of T-cell activation. Herein, we have examined the impart of a genetic marker in the CTLA-4 gene on renal transplant outcomes. A cohort of 144 renal recipients and 100 healthy subjects were genotyped by the fragments analysis method using an automated sequencer. Patients were classified into two groups: Group I included 31 HLA-identical haplotype allograft recipients and Group II, 113 showing one or more HLA haplotype mismatches. Forty patients (27.78%) developed at least one acute rejection episode (ARE): 9 in Group I and 31 in Group II. Before transplantation, 20 patients were lymphocytotoxic antibodies (LCT) positive: 4 Group I, 2 of whom developed an ARE, and sixty in Group II, including 8 with an ARE. The occurrence of an ARE was associated with the presence of LCT before transplantation among the entire cohort of patients (P = .032) and among Group II (P = .037). The allelic frequencies of (AT)n polymorphism did not reveal significant differences between patients and controls. The most prevalent alleles were the 88 bp (51% in controls and 44.44% in patients) and the 106 bp (8% and 10.76%, respectively). We noticed an increase of the 120 bp allele frequency among patients who had undergone an ARE compared with those who did not display this complication (8.75% vs 3.85%). Likewise, among LCT-negative Group I, recipients the incidence of the 120 bp allele was higher in ARE than non-ARE patients. Although the differences were not statistically significant, we propose that the 120 bp allele of the CTLA-4 gene (AT)n microsatellite a predisposes to acute rejection episodes in renal transplantation.

Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production.

Leishmania-specific cytotoxic T cell response is part of the acquired immune response developed against the parasite and contributes to resistance to reinfection. Herein, we have used an immune-informatic approach for the identification, among Leishmania major potentially excreted/secreted proteins previously described, those generating peptides that could be targeted by the cytotoxic immune response. Seventy-eight nonameric peptides that are predicted to be loaded by HLA-A*0201 molecule were generated and their binding capacity to HLA-A2 was evaluated. These peptides were grouped into 20 pools and their immunogenicity was evaluated by in vitro stimulation of peripheral blood mononuclear cells from HLA-A2+-immune individuals with a history of zoonotic cutaneous leishmaniasis. Six peptides were identified according to their ability to elicit production of granzyme B. Furthermore, among these peptides 3 showed highest affinity to HLA-A*0201, one derived from an elongation factor 1-alpha and two from an unknown protein. These proteins could constitute potential vaccine candidates against leishmaniasis.

Genetic Polymorphisms of Inflammatory Molecules in Tunisian Kidney Transplantation

As chemokines and adhesion molecules play major roles in the process by which leukocytes are recruited from the bloodstream into sites of inflammation, genetic variations in the production or activity of molecules may influence susceptibility to acute rejection episodes. This study sought to determine the impact of recipient monocyte chemoattractant protein-1 (MCP-1), chemokine receptor (CCR2, CCR5), and adhesion molecule (ICAM-1, PECAM-1 and L/E selectin) polymorphisms on acute rejection after renal transplantation. We selected 169 healthy blood donors and 173 renal transplant recipients for analysis according to the presence or absence of graft rejection in the first 30 days after transplantation. Using molecular methods DNA was genotyped for 11 polymorphisms of these inflammatory molecules genes. Results were stratified by the incidence of rejection episodes and by human leukocyte antigen (HLA) mismatching. No association was detected between adhesion molecule polymorphisms and the incidence of acute rejection episodes. However, a significant risk of acute renal loss was observed among HLA-identical recipients who possessed the CCR2-64I allele (odds ratio 0.24, 95% confidence interval, 0.05 to 1.06; P=.035). In conclusion, the observed association of CCR2-64I with acute rejection episodes should be added to the spectrum of immunogenetic factors known to be involved in renal allograft rejection.

Kidney Transplantation: Charles Nicolle Hospital Experience

The aim of our retrospective study was to analyze the short- and long-term follow-up of 298 renal transplantations performed between June 1986 and May 2005. All were first transplantations except 4 cases, with 54 from cadaveric and 244 from living donors. The recipients included 196 males and 102 females of overall mean age of 31.21 +/- 8.9 years (range, 16-61 years). A combination of prednisolone and azathioprine was presented for 212 patients or mycophenolate mofetil for 86 patients. Polyclonal or monoclonal antibodies were used as induction therapy in 183 cases. Cyclosporine was administered to 188 cases and tacrolimus only to 16. HLA matching was 0 mismatches (MM) in 65 cases; 1 or 2 MM in 113; 3 MM in 99; and > or =4 MM in 21. Acute tubular necrosis occurred in 45 cases. One hundred eighteen patients experienced at least 1 acute rejection episode: 102 cases (41.8%) among living and 16 (29.6%) among cadaveric kidneys donor (P = .0007). The actuarial patient and graft survival rates at 1, 5, 10, 15, and 20 years were 95.9%, 87.4%, 77.5%, 65.6%, and 60.8%, and 94.9%, 84.5%, 75.4%, 65.4%, and 53%, respectively. Sixty-three patients died and 72 patients returned to dialysis. Our results were comparable to experienced centers. However, the member of kidney transplantations does not match the increased number of patients on renal replacement therapy. It is advisable to promote obtaining organs from brain-dead donors.

Toll-Like Receptor 4 and CD14 Gene Polymorphisms in Tunisian Kidney Transplantation

BACKGROUND Acute and chronic rejections remain an important cause of graft loss after renal transplantation. Currently, activation of innate immune responses through Toll-like receptors (TLRs) is suspected to be implied in the loss of the transplant tolerance. OBJECTIVES We investigated functional single nucleotide polymorphisms (SNPs) of TLR4 and its coreceptor CD14 in kidney transplantation and looked for any potential role in acute rejection (AR) and chronic allograft nephropathy (CAN) and impact on graft survival. PATIENTS AND METHODS TLR4 (Asp299Gly) and CD14 (C/T -159) SNPs were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 209 kidney transplant recipients (KTRs) including 132 treated with mycophenolate mofetil (MMF+). AR occurred in 59 patients and 24 were identified as having CAN by biopsy and scored according to the Banff criteria. RESULTS There were no significant associations between TLR4 and CD14 genotypes and alleles and the occurrence of both AR episodes and CAN. Moreover, TLR4 and CD14 SNPs did not seem to influence kidney graft survival. Analysis according to human leukocyte antigen (HLA) compatibility status, positivity of anti-HLA antibodies, and immunosuppression by MMF confirmed the absence of correlation of the investigated SNPs with the graft outcome. In addition, incidence of post-transplantation infections, including cytomegalovirus (CMV) infections, was not influenced by both TLR4 and CD14 SNPs. CONCLUSION These results suggest that TLR4 (Asp299Gly) and CD14 (C/T -159) functional SNPs do not play a major role in AR, CAN, and kidney graft survival. Therefore, intragraft monitoring of TLR4/CD14 genes expression by messenger RNA (mRNA) would provide clarity on the exact role of these receptors in graft injuries.

The PTPN22 C1858T (R620W) Functional Polymorphism in Kidney Transplantation

To investigate the association between kidney transplant rejection and PTPN22 (protein tyrosine phosphatase non-receptor 22) polymorphism, genomic DNA of 175 renal transplant recipients and 100 healthy blood donors were genotyped by restriction fragment length polymorphism-polymerase chain reaction. The patients were classified in two groups: G1 included 33 HLA-identical recipients and G2 included 142 with one or more HLA mismatches. Forty-nine patients developed an acute rejection episode (ARE): 8 in G1 and 41 in G2. The allelic frequencies of PTPN22 R620W revealed a significant difference between patients and controls. In fact, the W-allele was significantly more frequent in graft recipients than in blood donors (0.05 vs 0.01, P < .05). Furthermore, the frequency of this allele was increased in G1 patients with an ARE (0.188) compared with those without an ARE (0.040), but the difference was not statistically significant. Thus, we concluded that the PTPN22 W-variant allele could be involved in the susceptibility to acute allograft rejection in Tunisian kidney transplant patients.