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.

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)-ß1, 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 -ß1 (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-ß1 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- ß1*H/IL-10*H production haplotype (75%) compared with the other haplotypes (16%; P = .03). CONCLUSION: The presence of TGF-ß1-H secretion profile may protect the kidney graft. TNF-α*H/TGF-ß1*H/IL-10*H haplotype was associated with a higher risk of ARE and with poorer graft survival.

Y402H polymorphism in complement factor H and age-related macular degeneration in the Tunisian population.

To evaluate a possible association between the complement factor H (CFH) Y402H polymorphism and susceptibility to age-related macular degeneration (AMD) in the Tunisian population, as well as the impact of the genotype distribution among different phenotypes and the response to treatment with intravitreal bevacizumab, exon 9 of CFH was analyzed for the Y402H polymorphism by direct sequencing in 135 healthy controls and 127 sporadic unrelated AMD patients classified into the following groups: 12 atrophic AMD (group G1), 115 exudative AMD (G2) and 10 AMD patients who had fibrovascular scarring (G3) that did not allow a precise grading of the phenotype. Seventy patients in G2 were treated with 1.25 mg intravitreal bevacizumab at 6-week intervals until choroidal neovascularization (CNV) was no longer active. The frequency of the CFH 402H allele was significantly higher in AMD patients than in controls (p = 2.62 × 10(-16)). However, subgroup analysis does not reveal any association between the variant allele H and phenotypes of AMD or CNV. Also, there was no significant difference in response to bevacizumab treatment according to Y402H CFH genotype (p = 0.59). A strong association of the 402H allele with susceptibility to AMD in the Tunisian population was confirmed; however, this variant does not appear to be involved in the clinical progression of this disease or in the postintravitreal bevacizumab response.

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.

Allograft renal rejection and chemokine polymorphism.

Chemokines play a major role in the process by which leukocytes are recruited from the bloodstream into the sites of inflammation. Genes for the chemokine receptors CCR5, CCR2 and MCP-1 are characterized by functional polymorphisms implicated in transplant rejection. To investigate this association, we analyzed polymorphisms of CCR5-∆32, CCR5-59029-A/G, CCR2-V64I and MCP-1 G/A (-2518) in 173 renal transplant recipients and 169 healthy blood donors. The patients were classified in two groups: Group-1 (G-1) included 33 HLA-identical recipients and Group-2 (G-2) included 140 (one or more) mismatched graft recipients. Forty-two patients had developed acute rejection episodes (ARs): seven in G-1 and 35 in G-2. Thirteen G-2 patients developed chronic allograft dysfunction (CAD). The genotypic and allelic frequencies of all polymorphisms studied did not reveal significant differences between patients and controls and among G-1 and G-2 recipients. However, a significant risk of acute renal transplant rejection was found in G-1 patients who possessed the CCR2-64I allele (odds ratio 0.24, 95% confidence inter-val [CI], 0.05-1.06; P = 0.035). There was no significant association of this polymorphism and CAD. In conclusion, the observed association of CCR2-64I with AR should be added to the spectrum of immunogenetic factors known to be involved in allograft renal loss.

Association of chemokine and chemokine receptor polymorphisms with activity degree of IBD in Tunisian patients.

Crohn's disease (CD) and ulcerative colitis (UC) have complex genetic background that is characterised by more than one susceptibility locus. To detect a possible association between the functional polymorphisms of the chemokine receptors CCR5, CCR2 and MCP-1 genes and susceptibility to CD and UC in Tunisian population, polymorphisms of CCR5-delta32, CCR5-59029-A/G, CCR2-V641 and MCP-1-2518-G/A were analysed in 194 Inflammatory bowel disease (IBD) patients and 169 healthy blood donors using PCR-RFLP and PCR-SSP methods. The patients were classified in 126 patients with CD and 68 patients with UC. The genotypic and allelic frequencies of all polymorphisms studied, did not reveal significant differences between patients and conrols and among CD and UC patients. However, analysis of CD patients revealed that those without homozygosous G/G genotype are more frequently in remission compared to those with this genotype (OR: 0.4, 95% CI: [0.174-0.928]; p = 0.03). Also, the frequency of the CCR2-641 muted allele was statistically higher in CD patients in remission disease than those in active form (OR: 0.267 95% CI: [0.09-0.78]; p = 0.01). Adjustment for known covariates factors (age, gender and immunosuppressive regimen) confirmed these univariate findings and revealed that the CCR5-59029-A/G and CCR2-V64I genotype were associated to remission form of CD (OR: 263; 95% CI: [1.01-6.80]; p = 0.047 and OR: 4.64; 95% CI: [1.01-21.31]; p = 0.049 respectively). In conclusion, the present study supports the involvement of chemokine receptor (CCR2 and CCR5) polymorphisms in activity degree of the IBD disease in Tunisian 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.

Association of specific amino acid sequence (QRRAA) of HLA-DRB1*0405 with rheumatoid arthritis in a Tunisian population.

This study aimed to investigate HLA-DRB1 alleles in rheumatoid arthritis (RA) patients from Tunisia and to examine the effect of these alleles on disease severity. HLA-DRBI alleles and sub-typing of DRBI*04 and *01 were determined in 90 patients and 100 healthy controls, by PCR-SSP. HLA-DRB1*04 was significantly higher in patients (51.1%) than in controls (27%) [OR=2.83, p=0.00066]. DRBJ*0405 was found to be the unique DR4 allele associated with RA (28.88% vs 6%) [OR=6.36, p=0.000059]. A significant decrease in the frequency of HLA-DRB1*0701 was observed in RA patients (16.66%) compared to controls (36%) [p=0.0026]. However, the frequency of patients carrying the shared epitope (SE) QRRAA, was slightly increased compared with controls (37.8% vs 23%) [OR=2.03, p=0.039]. We found that the presence of rheumatoid factor, HLA-DR4 and HLA-DRBI*0405 were not significantly associated with bone erosions or the presence of extra-joint involvement. In our population, the SE (QRRAA) expressed in DRBI*04 alleles is related to the susceptibility to RA but it is not involved in RA severity in Tunisia, while DRBI*0701 might protect against this disease.

Genetic polymorphisms of immunoregulatory proteins in acute renal allograft rejection.

CTLA-4 and CD28 are T lymphocyte receptors involved in the regulation of T-cell activation. Allograft rejection is an alloimune response which is strongly dependent on T-cell proliferation. Thus, we examined the relationship between CTLA-4 and CD28 gene polymorphisms and renal transplant outcomes. We genotyped 141 renal recipients and 229 healthy controls using PCR-SSP methods for the (-318) C/T polymorphism in the promoter region of the CTLA-4 gene and IVS3 (+17) T/C on intron 3 of the CD28 gene, and by PCR-RFLP method for exon 1 (+49) A/G and CT60 G/A within the 3'-untranslated region (UTR) of the CTLA-4 gene. Patients were classified into two groups: Group I included 23 HLA-identical haplotype allograft recipients and group II, 118 recipients with one or more mismatches in HLA haplotypes. Thirty-six patients developed at least one acute rejection episode (ARE). No significant differences were observed between the genotypes or the allele distribution between ARE and non-ARE patients. However, in group I, (+49) A and CT60 (G) allele frequencies were lower in patients with ARE than those without ARE (0.100 and 0.400 vs 0.361 and 0.722 respectively). However, the difference was not significant. Our study suggested that these alleles may confer protection against renal allograft loss.

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.

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.

Functional polymorphisms of PTPN22 and FcgR genes in Tunisian patients with rheumatoid arthritis.

To investigate a possible association between functional polymorphisms of the protein tyrosine phosphatase nonreceptor type 22 (PTPN22-R620W) and receptors for the Fc fragment of IgG (FcgRIIa-H131R, FcgRIIIa-F158V FcgRIIIb-NA1/NA2), and rheumatoid arthritis (RA), 133 Tunisian patients with RA and 100 controls were genotyped. We found strong evidence of an association of PTPN22 620W allele and RA. However, analysis does not detect an association between auto-antibodies seropositivity, presence of nodules or erosions and this allele. No significant skewing of any of the three FcgR polymorphisms was seen in this RA group. Nevertheless, we identified FcgRIIIa-V/V158 as the most important FcgR genotype for severe disease subset with joint erosions and observed that patients with FcgRIIIb-NA2/NA2 genotype had an earlier incidence of clinical symptoms. In conclusion, we have confirmed that PTPN22 620W allele is associated with Tunisian RA but does not constitute a factor influencing clinical manifestations. Conversely, this study supports that the FcgRIIa/IIIa and IIIb polymorphisms could influence the course and the severity of this disease. A large number of samples are required to provide independent confirmation of these findings.

Adhesion molecule polymorphisms in acute renal allograft rejection.

Acute rejection is the main cause of early renal allograft failure. Adhesion molecules provide signals for activation and recruitment of effector cells leading to graft infiltration by host T cells, which are key to allograft rejection. This study was undertaken to analyze the adhesion molecule gene polymorphisms in renal transplant recipients and to investigate their potential association with the development of acute allograft rejection. A total of 120 renal transplant recipients and 100 controls were retrospectively genotyped. Seven nucleotide polymorphisms in intracellular adhesion molecule (ICAM)-1, platelet endothelial cell adhesion molecule (PECAM)-1, L-selectin, and E-selectin were analyzed using allele-specific polymerase chain reaction (PCR)-SSP assay and PCR-restriction fragment length polymorphism (RFLP). Recipients were selected on the basis of the development of acute allograft rejection in the first 6 months after renal transplantation. Forty-one patients developed acute allograft rejection and 79 showed uneventful courses. There was no evidence for an association of any polymorphism with acute rejection. Thus, we concluded that these genes do not predispose to acute renal allograft rejection.

Human platelet antigens: HPA-1, -2, -3, -4, and -5 polymorphisms in kidney transplantation.

To investigate the association between kidney transplant rejection and polymorphisms of HPA-1, -2, -3, -4, and -5, the genomic DNA of 70 renal transplant recipients and 100 healthy blood donors was analyzed by polymerase chain reaction (PCR)-SSP. The patients were classified into two groups. Group 1 included 33 HLA-identical recipients and group 2, 37 one haplo-identical recipients. Thirty-one recipients experienced an acute rejection episode (ARE): 10 in group 1 and 21 in group 2. Ten group 2 patients developed chronic allograft dysfunction (CAD). Before transplantation, five patients in group 1 were lymphocytocytotoxic antibodies (LCT) positive, among them three developed an ARE. In group 2, seven recipients were LCT positive and four had an ARE. After transplantation, 29 patients were LCT positive: 11 in group 1 and 18 in group 2, among them: 6/11 and 11/18 had an ARE. The allelic frequencies of HPA-1, -2, and -5 among patients and controls did not reveal significant differences, whereas the HPA-3a and HPA-4b alleles were significantly more frequent among patients than controls: 91.4% and 27.8% versus 76.5% and 11.5% respectively (P < .05 and P < .001). The frequency of the HPA-3b allele was increased in patients with an ARE (11.3%) and those who developed CAD (20%) compared with those not affected by these complications (6.6% and 6.4%, respectively), but the difference was not significant. The genotype distribution of HPA-1, -3, and -4 genes of GPIIb/IIIa revealed that the most frequent genotype was HPA-1a1a/3a3a/4a4a (19%) among controls and HPA-1a1a/3a3a/4a4b (31.4%) among patients. This genotype was associated with an ARE in 25.8%, namely 50% of group 1 recipients and 14.28% of group 2. The HPA-4b polymorphism of GPIIb/IIIa receptor seem to be an independent risk factor for acute allograft rejection in kidney transplantation.