Tarak Dhaouadi

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.

Polymorphisms of Toll-like receptor-4 and CD14 in systemic lupus erythematosus and rheumatoid arthritis.

BackgroundToll-like receptor 4 (TLR4) and its co-receptor CD14 play a major role in innate immunity by recognizing PAMPs and signal the activation of adaptive responses. These receptors can recognize endogenous ligands mainly auto-antigens. In addition, TLR4 (Asp299Gly) and CD14 (C/T -159) polymorphisms (SNPs) may modify qualitatively and/or quantitatively their expression. Therefore, they could be implied in autoimmune diseases and can influence both susceptibility and severity of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).Patients and methodsTLR4 (Asp299Gly) and CD14 (C/T -159) SNPs were genotyped using polymerase chain reaction (PCR)-RFLP in 127 SLE patients, 100 RA patients, and 114 healthy controls matched in age and gender.ResultsCD14*T allele was significantly more frequent in SLE patients (0.456) comparatively to controls (0.355), p = 0.02 OR (95% CI) = 1.53 [1.04-2.24]. In RA patients, the higher frequency of CD14*T allele (0.405) failed to reach significance, p = 0.28. Investigation of the TLR4 (Asp299Gly) SNP showed no significant association neither with SLE nor with RA.Analysis of these SNPs according to clinical and biological features showed a significant higher frequency of arthritis in SLE patients carrying CD14*T/T genotype (92%) comparatively to those with C/C and C/T genotypes (72.5%), p = 0.04. Moreover, SLE patients carrying CD14*T/T/TLR4*A/A haplotype had significantly more arthritis (91.3%) than the rest of SLE group (73%), p = 0,044 and confirmed by multivariable analysis after adjustment according to age and gender, p = 0.01.ConclusionThe CD14 (-159)*T allele seems to be associated with susceptibility to SLE and arthritis occurrence.

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.

Comparative Study of Human and Automated Screening for Antinuclear Antibodies by Immunofluorescence on HEp-2 Cells

Background : Several automated systems had been developed in order to reduce inter-observer variability in indirect immunofluorescence (IIF) interpretation. We aimed to evaluate the performance of a processing system in antinuclear antibodies (ANA) screening on HEp-2 cells. Patients and Methods : This study included 64 ANA-positive sera and 107 ANA-negative sera that underwent IIF on two commercial kits of HEp-2 cells (BioSystems® and Euroimmun®). IIF results were compared with a novel automated interpretation system, the “ Cyclopus CADImmuno®” (CAD). Results : All ANA-positive sera images were recognized as positive by CAD (sensitivity = 100%), while 17 (15.9%) of the ANA-negative sera images were interpreted as positive (specificity = 84.1%), κ=0.799 (SD=0.045). Comparison of IIF pattern determination between human and CAD system revealed on HEp-2 (BioSystems®), a complete concordance in 6 (9.37%) sera, a partial concordance (sharing of at least 1 pattern) in 42 (65.6%) cases and in 16 (25%) sera the pattern interpretation was discordant. Similarly, on HEp-2 (Euroimmun®) the concordance in pattern interpretation was total in 5 (7.8%) sera, partial in 39 (60.9%) and absent in 20 (31.25%). For both tested HEp-2 cells kits agreement was enhanced for the most common patterns, homogenous, fine speckled and coarse speckled. While there was an issue in identification of nucleolar, dots and nuclear membranous patterns by CAD. Conclusion : Assessment of ANA by IIF on HEp-2 cells using the automated interpretation system, the “ Cyclopus CADImmuno®” is a reliable method for positive/negative differentiation. Continuous integration of IIF images would improve the pattern identification by the CAD.

BAFF Promoter Polymorphisms and Serum levels in Tunisian Patients with Systemic Lupus Erythematosus

Objectives: To investigate any associations between regulatory genetic polymorphisms of the B-lymphocyte activating factor (BAFF) gene, disease susceptibility and serum soluble BAFF (s-BAFF) levels in Tunisian systemic lupus (SLE) patients. Patients and methods: This case-control study included 124 SLE patients and 152 healthy controls. Three single nucleotide polymorphisms (SNPs) (-2841 T>C, -2701 A>T and -871 C>T) in the 5’ regulatory region of the BAFF gene were explored by PCR-RFLP. Serum BAFF levels were measured by ELISA (R&D Systems®). Results: s-BAFF levels were elevated in SLE patients (1717.08 pg/ml) and in anti-dsDNA positive antibodies patients (1948.28 pg/ml) compared to both controls (665.82 pg/ml, p<10-8) and patients without anti-dsDNA antibodies (1281.51 pg/ml, p=0.007). In contrast, no correlation was found between global disease activity registered in SLEDAI and s-BAFF levels (p=0.7). The -2841*C mutated allele was associated to SLE predisposition and anti-dsDNA antibody occurrence, p=0.03 and p=0.021 respectively. Single allele, genotype and haplotype association analyses showed no significant association with s-BAFF values, clinical features or SLEDAI score. Conclusion: In Tunisian, the rs9514827 (T>C -2841) SNP in the BAFF gene promoter seems to be related to SLE susceptibility and anti-dsDNA antibodies occurrence. Even if serum s-BAFF levels were significantly higher in SLE patients and in anti-dsDNA antibody positive sera, it did not seem to be correlated with disease activity or the occurrence of lupus nephritis. Again, studied genetic markers failed to predict the serum s-BAFF levels as there was no correlation between the two parameters.

IL-17A, IL-17RC polymorphisms and IL17 plasma levels in Tunisian patients with rheumatoid arthritis

Background Interleukin-17 (IL-17), a cytokine mainly secreted by Th17 cells, seems to play a significant role in the pathogenesis of rheumatoid arthritis (RA). Functional genetic polymorphisms in IL-17 and its receptor genes can influence either qualitatively or quantitatively their functions. Therefore, we aimed to study the impact of IL17-A and IL17RC polymorphisms on plasma level of IL-17 and RA susceptibility and severity. Methods In this context, IL-17A*rs2275913 and IL-17RC*rs708567 polymorphisms were investigated together with the quantification of IL17 plasma level in 115 RA patients and 91 healthy control subjects matched in age, sex and ethnic origin. Results There were no statistically significant associations between IL-17A and IL-17RC studied polymorphisms and RA susceptibility. In contrast, IL-17A plasma levels were significantly higher in patients (55.07 pg/ml) comparatively to controls (4.75 pg/ml), p<10E-12. A ROC curve was used to evaluate the performance of plasma IL-17 in detecting RA. Given 100% specificity, the highest sensitivity of plasma IL-17A was 61.7% at a cut-off value of 18.25 pg/ml; p < 10E-21, CI = [0.849–0.939]. Analytic results showed that the IgM-rheumatoid factor and anti-CCP antibodies were significantly less frequent in patients with the IL-17RC*A/A genotype than those carrying *G/G and *G/A genotypes; p = 0.013 and p = 0.015, respectively. Otherwise, IL-17 plasma levels’ analysis showed a significant association with the activity of RA (DAS28≥5.1 = 74.71 pg/ml vs. DAS28<5.1 = 11.96 pg/ml), p<10E-6. Conclusion IL-17A*rs2275913 (G/A) and IL-17RC*rs708567 (G/A) polymorphisms did not seem to influence RA susceptibility in Tunisian population. This result agrees with those reported previously. Plasma IL-17A level seems to be predictive of severe RA occurrence.

The role of IL‐23/IL‐17 axis in human kidney allograft rejection

Th17 cell subset has been implicated in autoimmune diseases, tumor immunity and, transplant rejection. In order to investigate the role of IL‐17/IL‐23 pathway in allograft outcome, intragraft expression of IL‐17 mRNA and single nucleotide polymorphisms (SNPs) of IL‐17A, IL‐17F, IL‐17RC, and IL23R genes were evaluated with a quantification of IL‐17A, IL‐17F, and IL‐23 plasma levels. This study revealed that recipients with acute rejection (AR) had a significant increase in IL‐17A mRNA expression levels after transplantation compared to controls (P = 0.037). Moreover, IL‐17A plasma levels were significantly higher in AR group; pretransplantation (Day–1 [D–1]): P = 0.00022 and posttransplantation (Day 7 [D7]): P < 10–14. IL‐17F and IL‐23 plasma levels were significantly higher in AR at D7 only (47.86 vs. 22.99 pg/ml; and 33.82 vs. 18.811 pg/ml; P = 0.015 and P < 10–17, respectively). Using receiver‐operating characteristic curves, D7 IL‐17A and IL‐23 plasma levels exhibited excellent sensitivities and specificities for predicting AR. Genetic study revealed no association between IL‐17A, IL‐17F, IL‐17RC, and IL23R studied SNPs and AR. Nevertheless, a significant improvement of graft survival was found in kidney transplant recipients carrying IL‐17F‐rs763780*A/A, IL‐17RC*G/G, and *G/A genotypes. Besides, IL‐17A mRNA levels were significantly higher in patients carrying the IL‐23R*G/G genotype comparatively to those with *G/A genotype. Based on these findings, significant increase of IL‐17A mRNA and protein levels in AR recipients that are genetically controlled highlights the role of this cytokine that can be a useful clinical biomarker to predict early acute renal allograft rejection.

BAFF polymorphisms and serum levels of BAFF in Tunisian systemic lupus erythematosus patients

Although different authors suggest that the B-lymphocyte activating factor (BAFF) may be involved in the selective loss of B-cell tolerance in human systemic lupus erythematosus (SLE), the mechanisms responsible for the deregulation of this molecule in SLE remain unclear [1-3].

MCP-1, CCR2 and CCR5 polymorphisms in Tunisian patients with atopic asthma

Chemokines and their receptors play an important role in the late inflammatory stage of asthma. In this study, we aimed to investigate polymorphisms of MCP-1 (CCL2), CCR2 and CCR5 which can affect qualitatively and/or quantitatively their production and thus influence both susceptibility and severity of asthma and its clinical and biological features.MCP-1 (A/G -2518), CCR2 (+/64I), CCR5 (G/A -59029) and CCR5 (∆32) polymorphisms were evaluated by PCR in 107 Tunisian patients with asthma and 169 healthy controls.No significant association was found between the four investigated polymorphisms and asthma. Nevertheless the haplotype MCP1*AG/CCR2*+/+ was significantly l ess frequent in patients (20.5%) compared to controls (32.5%) (p=0.03; OR=0.54; 95% CI: 0.29-0.98). Whereas no difference was observed in CCR2/CCR5 haplotypes between patients and controls. Analysis of polymorphisms with clinical and biological features showed that the concomitant presence of MCP-1*G/CCR2*64I alleles was less frequent in severe forms (4.34%) compared to moderate disease (12%) but the difference was not significant (p=0.27). No association was observed between the four polymorphisms and the presence of atopic rhinitis or atopic conjunctivitis and an elevated rate of serum IgE over 200 IU/ml.Additional effects of MCP-1 and its receptor CCR2 polymorphisms seem to be involved in disease susceptibility to asthma in Tunisian patients; nevertheless they could be protective against its severe forms.