TLR4 Thr399Ile Research Articles

O-224 Toll-like Receptors Genotype Polymorphism In Egyptian Children With Chronic Viral Hepatitis C

Background Toll-like receptors (TLRs) are important molecules for both innate and adaptive immune responses. The prevalence of TLRs polymorphism varies in different populations and controversial results were reported in HCV patients. We aimed to assess the frequency of TLR2 Arg753Gln, TLR4 Asp294Gly and TLR4 Thr399Ile polymorphisms among Egyptian children with chronic HCV and to study their relation to clinical data. Methods An observational case control study was conducted in Mansoura University Children’s Hospital, Egypt and included 100 Chronic HCV patients (63% males) with mean age of 13 ± 2.8 years in addition to 100 healthy matched controls. Diagnosis of patients relied upon persistently positive HCV PCR for at least 6 months. Eighty two patients received combined pegylated interferon α2b 60 mcg/m 2 /w and ribavirin 15 mg/kg/d. Treatment was continued for 48 weeks for responders (49 patients). All subjects were exposed to history, examination, liver functions, viral markers, HCV PCR and abdominal ultrasound. Liver biopsy was done only for patients. Gene polymorphisms were assessed by restriction fragment length polymorphism. Results Neither patients nor controls had TLR2 Arg753Gln or TLR4 Asp299Gly polymorphisms. Only 3 patients (3%) were heterozygous for TLR4 Thr399Ile polymorphism without significant difference between patients and controls (p = 0.24). No significant correlation was observed between TLR4 Thr399Ile polymorphism and viral load, histologic activity, grade of fibrosis or treatment response (p = 0.96, 0.21, 0.46, 0.49 respectively). Conclusion TLR2 Arg753Gln, TLR4 Asp294Gly and TLR4 Thr399Ile polymorphisms seem to be absent or rare and probably have no role in HCV among Egyptian population.

Toll-like receptor 4 single-nucleotide polymorphisms Asp299Gly and Thr399Ile in ovarian cancers.

Toll-like receptor (TLR4) 4 is present in numerous cell types and serves as the first point of defense in the innate immune system. Single-nucleotide polymorphisms (SNPs) are present in a number TLR genes and have been associated with various infection and inflammation disorders. Asp299Gly and Thr399Ile, TLR4 SNPs, are associated with tumor progression. In the present study, cases of ovarian cancer were analyzed with regards to Asp299Gly and Thr399Ile of the TLR4 gene. Genotype analysis was performed using DNA from tissue samples from stage I–IV patients with ovarian cancer. DNA from tissue samples was extracted and analyzed by a pyrosequencing method following multiplex polymerase chain reaction. The genotypes of these SNPs were analyzed in the present study in a population of 105 patients, with different types of ovarian cancer, between 2004 and 2012. The allele frequencies for TLR4 Asp299Gly identified in this population were 1.00 (A) and 0.00 (G); for TLR4 Thr399Ile the allele frequencies were; 1.00 (C) and 0.00 (T). For TLR4 Asp299Gly the observed genotype frequency was 1.00 (AA), 0.00 (AG) and 0.0 (GG). In TLR4 Thr399Ile the observed genotype frequencies were 1.00 (CC), 0.00 (CT) and 0.00 (TT). TLR4 Asp299Gly and Thr399Ile alleles were not detected in the patients. These results indicated that the TLR4 299Gly and 399Ile alleles were exhibited at a lower frequency in the ovarian cancer patients that were examined.

Polymorphisms of toll-like receptors 2, 3 and 4 in patients that do and do not enter labour spontaneously at term

To assess the association of polymorphisms of toll-like receptors (TLRs) 2, 3 and 4 with the delay in onset of labour at term pregnancies, patients delivering at > 37 weeks and without pre-eclampsia, IUGR or a history of preterm delivery were prospectively evaluated. TLR2 Arg753Gln, TLR3 (c.1377C/T) and TLR4 Asp299Gly and Thr399Ile polymorphisms were genotyped by using PCR-RFLP. Patients labouring spontaneously before the 41st week were compared with those who did not labour spontaneously until this week in terms of baseline characteristics, TLR 2, 3 and 4 polymorphisms. The same comparisons were also performed by using a 40th week cut-off. Chi-square test, two-sample t-test or Mann–Whitney U tests were used for comparisons, as appropriate. In total, 79 patients delivering after 37 weeks were evaluated. All had CC genotype for TLR2 Arg753Gln and TLR4 Thr399Ile. There were no significant differences for TLR4 Asp299Gly GA and TLR3 (c.1377C/T) polymorphisms between patients spontaneously entering or not entering labour until the 41st week; the same was true when the 40th week cut-off was used. Delay in onset of labour at term pregnant women does not seem to be affected by the presence of TLR 2, 3 or 4 polymorphisms. Further studies are needed.

May TLR4 Asp299Gly and IL17 His161Arg polymorphism be associated with progression of primary measles infection to subacute sclerosing panencephalitis?

SSPE is a progressive neurological disorder of children. Only some of the children who are infected with measles virus develop SSPE, which supports individual variation. TLR-2 and TLR-4 play an important role in innate immunity by recognizing envelope proteins of MV. Another important cytokine that plays an important role in orchestrating innate immune function is IL-17. The purpose of our study is to elucidate whether the TLR2, TLR4, IL17F and IL17A gene polymorphisms are susceptibility genes for the development of SSPE. Using the PCR-RFLP methods, the single nucleotide polymorphisms of TLR2 (Arg753Gln, Arg677Trp, -194 to -174 del), TLR4 (Asp299Gly and Thr399Ile) IL17F (His161Arg, Glu126Gly) and IL17A were studied in 54 patients with SSPE and 81 healthy controls. For Asp299Gly polymorphism of the TLR4 gene we found that there were no control individuals who were homozygous carriers of the Gly/Gly genotype, and the risk for SSPE increased at approximately 4.7 fold for the heterozygous carriers of the Asp/Gly genotype (OR 4.727, 95%-CI 1.192-18.742; P=0.01), when compared to healthy controls. Also our findings demonstrate that homozygosity for the Arg161 variant of the IL17F His161Arg polymorphism is inversely associated with development of SSPE (OR 0.114 95%-CI 0.026-0.494; P<0.001). In conclusion, it is suggested that variation in susceptibility to SSPE disease may be in part due to variations in TLR4 and IL17 function resulting from polymorphisms of TLR4 Asp299Gly and IL17F His161Arg.

Genotypic analysis of Asp299Gly and Thr399Ile polymorphisms of TLR4 in Egyptian patients with rheumatoid arthritis and systemic lupus erythematosus

Toll-like receptors (TLRs) are essential molecules of the innate immune system that stimulate numerous inflammatory pathways and harmonize systemic defense against a wide array of pathogens. TLRs may also recognize a number of self-proteins and endogenous nucleic acids. Inappropriate stimulation of the TLR pathway by endogenous or exogenous ligands or as a consequence of mutation in the TLR genes may lead to induction and/or prolongation of autoimmune response and tissue injury. This study was designed to detect the TLR4 (Asp299Gly and Thr399Ile) gene polymorphism in Egyptian patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) as well as its correlation with disease activity. This study enrolled 50 patients who were attending the internal medicine and immunology out-patient clinic of Beni-Suef University Hospital. They were classified into three groups: 20 patients with RA, 20 patients with SLE, and 10 healthy controls. All studied persons were subjected to complete clinical evaluation, laboratory investigations, and detection of mutation in the TLR4 (Asp299Gly and Thr399Ile) gene by PCR technique. No individual in the RA patients group, the SLE patients group, or control population was identified carrying the Asp299Gly and Thr399Ile polymorphisms; in other words, all RA patients, SLE patients, and controls were with the same wild genotype. The similarity in genotype between the patients group and control population concluded that these two missense polymorphisms do not contribute to RA and SLE in a group of Egyptian population.

TLR4 Gene Polymorphism in Patients with Nonalcoholic Fatty Liver Disease in Comparison to Healthy Controls

Recent studies have suggested that bacterial overgrowth and endotoxemia along with its receptor, Toll-like receptor 4 (TLR-4), play a role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). The present study was designed to test and evaluate the TLR4 gene polymorphism in patients with NAFLD in comparison to healthy controls. A total of 119 patients [mean (standard deviation, SD) age 43.4 (11.5) years, 55.5% were males] with NAFLD and 80 healthy controls [mean (SD) age 40.9 (8.1) years, 67.5% were females)] were evaluated in terms of patient demographics, anthropometrics, blood biochemistry, liver histology, and ultrasonographic (USG) findings. Histological evaluation was performed in 111 patients, and blood samples were collected from 119 patients with NAFLD and 80 healthy persons. Allelic variants of TLR4 (Asp299Gly and Thr399Ile) were assayed by real-time PCR. Genomic DNA was amplified using FAM/VIC primers specific for allelic variants of TLR4 Asp299Gly and Thr399Ile with real-time PCR. Amplicons were analyzed with high-resolution melting on a Light Cycler 480 for detecting different melting patterns of polymorphic and wild-type alleles. The number of the subjects with heterozygous mutation at genotype 299 (Asp299Gly) was significantly lower in the NAFLD than in the control group (23.8 vs. 10.9%, P=0.027). Logistic regression analysis revealed that female gender [odds ratio (OR)=2.984, 95% confidence interval (CI) 1.561-5.360, P=0.001] and heterozygous (Asp299Gly) mutation at codon 299 (OR=2.998, 95% CI 1.325-6.783, P=0.008) were the significant predictors of higher likelihood of TRL4 gene polymorphism-related prevention of NAFLD. As the first-time-in-humans controlled study related to investigation of TLR4 gene polymorphism in NAFLD, our findings contribute to the available data that TLR-4 signaling is pivotal for the pathogenesis of NASH and indicate that the TLR4 codon 299 heterozygous gene mutation (Asp299Gly) in humans may have a preventive role against the genesis of NAFLD.

PReS-FINAL-2078: Single nucleotide polymorphisms of toll like receptors 2 and 4 in enthesitis related arthritis and oligo and polyarticular juvenile idiopathic arthritis

Introduction Single nucleotide polymorphisms of Toll like receptors modify cellular immune response and induce pro-inflammatory cytokine production and therefore could be associated with enthesitis related arthritis (ERA) and/or oligoarticular and polyarticular juvenile idiopathic arthritis (JIA). Objectives To determine whether polymorphisms of TLR2 and TLR4 influence susceptibility to ERA or JIA. Methods DNA was extracted from blood samples of 19 ERA patients, 10 patients with oligoarticular or polyarticular JIA and 40 healthy controls, all diagnosed according to ILAR criteria. Polymorphisms of the TLR2 (Arg753Gln) and TLR4 (Asp299Gly, Thr399Ile) were determined using real time and multiplex PCR. Results All JIA patients were carriers of wild type allele for all three polymorphisms. Regarding Arg753Gln polymorphism of TLR2, only one patient with ERA (5.56%) and 2 healthy controls (5%) were carriers of heterozygous allele. There were no homozygous mutants. All ERA patients had wild type allele for Asp299Gly polymorphism of TLR4. For Thr399Ile polymorphism of TLR4, 21.05% ERA patients were heterozygous (CT variant), and none of the ERA patients was homozygous (TT variant), (CC vs CT variant in ERA OR 3.7500, 95% CI 1.0498-13.3952, p 0.0419, CC vs TT variant OR 31.0000, 95% CI 1.7309-555.1867, p 0.0197). In group of healthy controls, TLR4 polymorphisms Asp299Gly and Thr399Ile were in linkage disequilibrium ; 2 controls were hetrozygous and 6 homozygous variant carriers for both polymorphisms, whereas linkage disequilibrium was not found among patient groups (CC vs CT in controls OR 16.0000, 95% CI 3.5905- 71.2994, p 0.0003, CC vs TT OR 5.3333, 95% CI 2.0099-14.1524, p 0.1944). Conclusion Polymorphisms of TLR2 and TLR4 are not associated with oligoarticular/polyarticular JIA. There was also no evidence that variants of TLR are major risk factors for ERA, however, lack of susceptibility should be confirmed on larger group of patients, since four out of 19 patients (21, 05%) were heterozygous. A study on larger cohort is currently ongoing.

Toll-like receptor 4 gene polymorphisms and acute coronary syndrome: No association in a Mexican population

The objective of this study was to establish the role of the TLR-4 gene polymorphisms in individuals in risk of developing ACS. The study included 457 Mexican patients with ACS and 283 control individuals. The TLR-4 Asp299Gly and TLR-4 Thr399Ile single nucleotide polymorphisms were genotyped using 5' exonuclease TaqMan genotyping assays on an 7900HT Fast real-time PCR system according to manufacturer's instructions (Applied Biosystems, Foster City, USA). The results obtained in this study showed that the frequency of the two polymorphisms (TLR-4 Asp299Gly and TLR-4 Thr399Ile) studied were similar between patients with ACS and healthy controls. Multiple logistic regression analysis showed that the largest risk factor for ACS development was given by smoking (11.88-fold increased risk), hypertension (4.32-fold increased risk), type II diabetes (3.44-fold increased risk), gender (2.32-fold increased risk), and dyslipidemia (1.52-fold increased risk). The Asp299Gly and Thr399Ile polymorphisms were not associated with susceptibility to ACS in the Mexican population.

Single-nucleotide polymorphisms in Toll-like receptor (TLR)-2, TLR4 and heat shock protein 70 genes and susceptibility to scrub typhus

Scrub typhus is a highly prevalent bacterial infection in India and South Asia that is caused by Orientia tsutsugamushi. The innate immune response to infections is modulated by Toll-like receptors (TLRs) and heat shock proteins (HSPs). This study was done to assess the prevalence and possible association of TLR and HSP polymorphisms in scrub typhus. TLR4 Asp299Gly, TLR4 Thr399Ile, TLR2 Arg753Gln and HSP70-2 A1267G are single-nucleotide polymorphisms (SNPs) that may modulate their activities, and these SNPs were assessed in 137 scrub typhus patients and 134 controls by PCR restriction fragment length polymorphism. We found that the two TLR4 mutations, TLR4 D299G and TLR4T399I, were present in 19.5% and 22% of the study population, respectively, and was in significant linkage disequilibrium with a D' of 0.8. The TLR2 mutation was found to be rare, whereas the HSP A>G mutation was very common (77.5%). Compared with the controls, the prevalence of heterozygous genotype of the TLR4D299G SNP, but not any of the other SNPs, was significantly higher among scrub typhus patients. Further studies using a larger sample size and more candidate genes may better enable in determining the role of these associations in susceptibility and severity of scrub typhus.

Positive link between variant Toll-like receptor 4 (Asp299Gly and Thr399Ile) and colorectal cancer patients with advanced stage and lymph node metastasis

Toll-like receptors (TLRs) are considered as major endotoxin-signaling receptor and as crucial sensors of innate immunity. TLRs recognize pathogen-associated molecular patterns; induce effectors genes involving inflammatory cytokines and therefore initiation of adaptative immune responses against pathogens. Recently, it has been shown that TLRs are involved in tumor progression. In fact, increased level of TLR4 is associated with progression of colon malignancies. Even, TLR4 polymorphism has been shown associated with susceptibility to have colorectal cancer. Our study aimed to investigate an association between TLR4 Asp299Gly (D299G) and Thr399Ile (T399I) polymorphisms in Tunisian patients with colorectal cancer. Using a primer extension method (SNaPshot), we genotyped two variants of TLR4 D299G and T399I in 100 patients with colorectal cancer and 140 healthy controls in Tunisian population. Interesting, we noted a significant association between T399I polymorphism and tumor differentiation (p = 0.027) and tumor architecture (p = 0.02) in colorectal cancer (CRC) patients. We also showed a significant association of D299G with an increased risk of advanced stage (p = 0.03). Finally, we observed a positive link between D299G and T399I polymorphisms and CRC patients with lymph node (p = 0.00024; p = 0.0005, respectively) and metastasis (p = 0.001; p = 0.002, respectively). However, we found no evidence to support a significant association between TLR4 D299G and T399I polymorphisms and colorectal cancer susceptibility. Our findings suggest that TLR4 D299G and T399I polymorphisms are significantly associated with clinical features variables. TLR4 polymorphisms may serve as biomarker of disease progression. Therefore, our results need confirmation in even larger studies.

Toll-like receptor (TLR) and matrix metalloproteinase (MMP) polymorphisms and periodontitis susceptibility: a meta-analysis

The aim of this study was to determine whether the toll-like receptor (TLR) and matrix metalloproteinase (MMP) polymorphisms confers susceptibility to periodontitis in ethnically different populations. A literature search using PubMed and Embase provided the data to conduct a meta-analysis on the associations between the TLR2 Arg753Gln, TLR4 Asp299Gly, Thr399Ile, MMP-1 -1607 G1/G2 and MMP-9 -1562 C/T polymorphisms and periodontitis. A total of 32 studies (14 on TLR polymorphisms and 18 on MMP polymorphisms) were considered in the meta-analysis. The meta-analysis showed no association between periodontitis and the TLR2 753Arg allele (Odds ratio [OR]=0.962, 95% confidence interval [CI]=0.662-1.400, p=0.841). Meta-analysis of the TLR4 Asp299Gly and Thr399Ile polymorphisms showed no association between periodontitis and the TLR4 299Asp allele in all study subjects (OR=0.984, 95% CI=0.761-1.271, p=0.900; OR=1.030, 95% CI=0.748-1.418, p=0.854). Meta-analysis showed an association between the MMP-1 -1607 G2G2 genotype and periodontitis in Asians (OR=3.778, 95% CI=1.210-11.80, p=0.022). Meta-analysis containing only studies in Hardy-Weinberg equilibrium revealed no association between chronic periodontitis and the MMP-9 -1562TT genotype (OR=0.638, 95% CI=0.265-1.533, p=0.315). This meta-analysis demonstrates a lack of association between the TLR2 Arg753Gln, TLR4 Asp299Gly, Thr399Ile, MMP-9 -1562 C/T polymorphisms and periodontitis, but shows an association between the MMP-1 -1607 G2G2 genotype and periodontitis in Asians.

TLR-4 polymorphisms and leukocyte TLR-4 expression in febrile UTI and renal scarring

In this study, we aimed to determine the relation of TLR-4 Asp299Gly and Thr399Ile polymorphisms and monocyte/neutrophil TLR-4 expression to febrile urinary tract infection (UTI) and renal scar development in children. The study was performed in children with a history of febrile UTI. Patients with and without renal scarring were classified as group 1 and group 2, respectively, while the control cases in our previous study were used as the control group (group 3). All three groups were compared for the rate of TLR-4 Asp299Gly and Thr399Ile polymorphisms, and for basal and lipopolysaccharide-stimulated monocyte/neutrophil TLR-4 expression levels. There were 168 patients (86 in group 1, 82 in group 2) and 120 control cases. Monocyte/neutrophil TLR-4 expression levels were similar in groups 1 and 2. However, both groups had lower TLR-4 expression than group 3. The rate of TLR-4 Asp299Gly polymorphism was not different in all groups. TLR-4 Thr399Ile polymorphism was higher in groups 1 and 2 than in group 3 (14.0, 12.2, and 2.0 %, respectively), while group 1 and group 2 were not different. Furthermore, monocyte TLR-4 expression level was lower in those having TLR-4 Thr399Ile polymorphism than in those without this polymorphism. Patients with febrile UTI had more frequent TLR-4 Thr399Ile polymorphism and lower monocyte/neutrophil TLR-4 expression. These findings indicate that children carrying TLR-4 Thr399Ile polymorphism and/or having low level of monocyte/neutrophil TLR-4 expression have a tendency to develop febrile UTI. However, we could not show the association of TLR-4 polymorphisms and of TLR-4 expression level to renal scarring.

The Role of TLR2, TLR4 and CD14 Genetic Polymorphisms in Gastric Carcinogenesis: A Case-Control Study and Meta-Analysis

BackgroundIn addition to Helicobacter pylori infection, host genetic factors contribute to gastric cancer (GC). Recognition of H. pylori is known to involve Toll-like receptors (TLR), which subsequently leads to activation of NF-κB. Thus, the overall aim of this study was to estimate for the first time the pooled effect size of polymorphisms in TLR2, TLR4 and CD14 on GC development through a meta-analysis.MethodsA case-control study comprising 284 ethnic Chinese individuals (70 non-cardia GC cases and 214 functional dyspepsia controls) was conducted for the genotyping of TLR2 -196 to -174del, CD14 -260 C/T and TLR4 rs11536889 using PCR, RT-PCR and mass spectrometry. Case-control studies of TLR2, TLR4 and CD14 polymorphisms and GC were searched up to June 2012. Pooled odds ratios and 95% confidence intervals were obtained by means of the random effects model.ResultsIn our ethnic Chinese case-control study, the TLR4 rs11536889 C allele increased the risk of GC (OR: 1.89, 95%CI: 1.23–2.92) while the CD14 -260 T allele was protective (OR: 0.62, 95%CI: 0.42–0.91). TLR2 -196 to -174 increased the risk of GC only in H. pylori-infected individuals (OR: 3.10, 95%CI: 1.27–7.60). In the meta-analysis, TLR4 Asp299Gly showed borderline results in the general analysis (pooled OR: 1.58, 95%CI: 0.98–2.60), nevertheless, stratified analysis by ethnicity showed that the mutant allele was a definitive risk factor for GC in Western populations (pooled OR: 1.87, 95%CI: 1.31–2.65). There was a potential association between the TLR2 -196 to -174 deletion allele and GC in Japanese (pooled OR: 1.18, 95%CI: 0.96–1.45). TLR4 Thr399Ile did not provide significant results.Conclusions TLR4 rs11536889 and CD14 -260 C/T are associated with non-cardia GC in Chinese. Based on our meta-analysis, the TLR signalling pathway is involved in gastric carcinogenesis, TLR4 Asp299Gly and TLR2 -196 to -174del showing associations with GC in an ethnic-specific manner.

Association between TLR4 and TLR9 Gene Polymorphisms with Development of Pulmonary Tuberculosis in Zahedan, Southeastern Iran

Some evidence suggests that a variety of genetic factors contribute to development of the tuberculosis (TB). TLR4 and TLR9 have been proposed as susceptibility genes for TB. This study was performed in 124 newly diagnosed TB cases and 149 healthy controls in a TB-endemic region of Iran. The TLR4 genes Asp299Gly, Thr399Ile, and TLR9 gene T-1486C polymorphisms were amplified by polymerase chain reaction (PCR) and then detected by PCR-restriction fragment length polymorphism (RFLP). The frequencies of the mutant alleles of TLR4 Arg299Gly, Thr399Ile, and TLR9 T-1486C polymorphisms were 0.8 versus 0.1, 5.6 versus 3, and 28.6 versus 25.2 in patients and controls, respectively, that were not significant. The synergic effect of TI,II/CC genotypes for TLR4 Thr399Ile and TLR9 T-1486C polymorphisms showed increased risk of PTB susceptibility. In conclusion, no significant relation was found between TLR4 and TLR9 polymorphisms alone and PTB. However, synergic effects of TLR4 Thr399Ile and TLR9-1486T/C polymorphisms might increase risk of PTB.

Association Between TLR4 Polymorphism and Periodontitis Susceptibility: A Meta-Analysis

TLR4 has been implicated in periodontal disease, but the association between the TLR4 Asp299Gly and Thr399Ile polymorphisms and the risk of periodontal disease remains unclear. Therefore, the aim of this study was to investigate the association between the TLR4 Asp299Gly and Thr399Ile polymorphism and periodontal disease. A search of electronic databases identified previous studies evaluating the association of the polymorphisms of TLR4 and periodontitis risk. The association was evaluated by odds ratio (OR) and its 95% confidence interval (CI). The results showed that TLR4 Asp299Gly and Thr399Ile were not associated with a significant risk of periodontitis (OR = 0.96, 95% CI = 0.80-1.16 for G versus A; OR = 1.39, 95% CI = 0.82-2.36 for AG/GG versus AA; OR = 1.05, 95% CI = 0.52-2.15 for T versus C; OR = 0.76, 95% CI = 0.55-1.04 for CT/TT versus CC). In the stratified analyses, there was no significantly increased risk for the studies of chronic periodontitis and aggressive periodontitis. Our meta-analysis revealed that the two common TLR4 polymorphisms, Asp299Gly and Thr399Ile, have no association with the likelihood of periodontitis. In a subgroup analysis by ethnicity and periodontitis type, the results also did not show any association. However, there was a significant increased risk for periodontitis in recessive models of Asp299Gly. The effect of genetic networks and their mutual interactions in the TLR4 signaling pathway on periodontitis susceptibility needs further study.

5-Fluorouracil Induce the Expression of TLR4 on HCT116 Colorectal Cancer Cell Line Expressing Different Variants of TLR4.

Two common single nucleotide polymorphisms (SNPs) of the human TLR4 gene, namely Asp299Gly (D299G) and Thr399Ile (T399I), have been shown to impair the ability of certain individuals to respond properly to TLR4 ligands. 5-Fluorouracil (5-FU) is widely used for the treatment of patients with advanced colon cancers. The present study examined the impact of two common polymorphisms of the TLR4 genes on the response of the HCT116 colorectal cancer cells to 5-FU. HCT116 was transfected with Flag-CMV1-TLR4 wild-type (WT) and D299G, T399I expression plasmids. The cytotoxic effect of 5-FU on transfected cells was assessed by MTT assay. FACS analysis was performed to show the effect of 5-FU and LPS on the expression of different variants of TLR4. The lowest IC50-value was measured in cells expressing the WT TLR4 and non-transfected cells were more resistance to the drug compared to the other cells. 5-FU significantly induced the expression of TLR4 protein in the presence and absence of LPS. 5-FU also induced HMGB1 secretion, Cas3 and PARP activity and these effects were stronger in cells expressing WT TLR4 than the other cells. In conclusion, 5-FU-induced TLR4 expression and LPS had synergistic effect with 5-FU to induced apoptosis in colorectal cancer cells.

Asp299Gly and Thr399Ile polymorphism of TLR-4 gene in patients with prostate cancer from North India

Background:The etiological factors associated with prostate cancer (CaP) have not been completely understood as yet. Genetic predisposition and inflammation is fast emerging as risk factors for CaP is a key player in the innate immune response and plays role in immune- surveillance and inflammation. The present study was conducted to evaluate TLR-4 gene polymorphism in patients with CaP.Material and Methods:DNA was isolated from blood samples of 198 patients with CaP, 200 cases of Benign Prostatic Hyperplasia (BPH) and 119 controls. TLR-4 gene polymorphisms Asp299Gly and Thr399Ile were determined by Restriction Fragment Length Polymorphism (RFLP) technique using Nco1 and Hinf 1 restriction enzymes. All statistical calculations were performed using SPSS for windows, version 13 (SPSS Inc., Chicago, Illinois, USA)Results:A significantly high proportion of patients with CaP had AG genotype (16.6%) as compared to control (4.2%) [OR-4.4, 95% CI (1.57-13.26), P =0.0013] with respect to Asp299Gly single nucleotide polymorphism (SNP). AA genotype showed a protective effect towards CaP development [OR-0.39, 95% CI (0.18-0.83), P=0.007). A trend was observed towards development of BPH with respect to AG genotype (P=0.06). Thr399Ile SNP was not significantly different among the population groups studied.Conclusions:This finding highlights the genetic predispositions to CaP with respect to TLR-4 gene. Individuals with Asp299Gly polymorphism having AG genotype appear to have four fold higher risk for development of Prostate cancer

No evidence for involvement of the toll-like receptor (TLR) 4 gene Asp299Gly and Thr399Ile polymorphisms in susceptibility to primary gouty arthritis

Previous studies demonstrated that toll-like receptor (TLR) 4 was involved in the development of autoinflammatory disease including gouty arthritis (GA). TLR4 functional gene Asp299Gly and Thr399Ile polymorphisms play a role in some autoinflammatory disease susceptibility. We undertook this study to analyze the association between the genetic polymorphisms within TLR4 gene and the susceptibility to GA in Chinese Han people. Two functional variants, Asp299Gly and Thr399Ile, in the TLR4 gene were genotyped using 5' exonuclease TaqMan technology from 218 male GA patients and 226 ethnically matched controls. None polymorphisms of Asp299Gly and Thr399Ile were detected in all GA cases and controls, which indicates that there is no evidence for involvement of the TLR4 gene Asp299Gly and Thr399Ile polymorphisms in susceptibility to primary GA in the Chinese Han population. Further studies with extended single nucleotide polymorphisms should be performed.

Single-nucleotide Polymorphisms in Genes Encoding Toll-like Receptor -2, -3, -4, and -9 in a Case–Control Study with Bladder Cancer Susceptibility in a North Indian Population

Impairment of the immune system may contribute to the risk for having cancer as Toll-like receptors are important for innate immunity. We examined the association between candidate disease-susceptibility polymorphisms in the single nucleotide polymorphism (SNPs) like TLR2 (-196 to-174del), TLR3 (C1377T), TLR4 (Thr399Ile) and TLR9 (G2848A) genes in patients with bladder cancer in a North Indian population. SNPs were comprised of TLR2 (-196 to -174 Del), TLR3(C1377T), TLR4 (Thr399Ile) and TLR9 (G2848A) genes. Allelic and genotypic frequencies of these TLRs SNP from histopathologically confirmed patients of bladder cancer (n = 200) and unrelated healthy controls of similar ethnicity (n = 200) were genotyped by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. In TLR2 I/D gene polymorphism, the combination of ID+DD showed a significant 3-fold increased risk (p = 0.001). TLR2 with heterozygous genotype ID showed a 3-fold risk and combination of heterozygous and variant genotype (ID+DD) also showed a 5-fold risk with tumor stage/grade of patients with bladder cancer. The other genotypes of TLR3, 4 and 9 did not exhibit any significant association with bladder cancer risk. Our results suggested the involvement of TLR2 (-196 to-174 del) in bladder cancer susceptibility; however, TLR3, 4 and 9 genes were not associated with risk of bladder cancer, implicating that polymorphisms in these tested TLRs genes are not likely to be associated with increased risk for developing bladder cancer. Functional studies in ethnically diverse populations may provide a more comprehensive involvement of innate immunity in identifying the disease-associated variants for the etiology of bladder cancer.

Toll-like receptor polymorphisms and vasculitis susceptibility: meta-analysis and systematic review

The aim of this study was to determine whether toll-like receptor (TLR) polymorphisms confers susceptibility to vasculitis. A literature search was conducted using the PubMed and Embase. A meta-analysis on the associations between the TLR4 Asp299Gly polymorphisms and vasculitis was carried out using allele contrast, dominant, and codominant models and a systematic review of other TLR polymorphisms. Fourteen studies involving 2,064 patients and 2,481 controls were included in this systematic review, which comprised nine on Behcet's disease (BD), three on giant cell arteritis (GCA), and one on Henoch-Schenlein purpura (HSP). Meta-analysis of six studies showed a significant association between the Gly/Gly+Gly/Asp genotype of the TLR4 Asp299Gly polymorphism and vasculitis and GCA (Odds ratio [OR]=1.368, 95% confidence interval [CI]=1.300-1.815, p=0.030; OR=1.523, 95% CI=1.099-2.112, p=0.012). Under a random effects model, the adjusted ORs calculated using the trim and fill technique revealed an association between the Gly/Gly+Gly/Asp genotype of the TLR4 Asp299Gly polymorphism and vasculitis (OR=1.544, 95% CI=1.091-2.185, p<0.05). Stratification by vasculitis type using the codominant model showed the trend for the association with GCA (OR=1.569, 95% CI=0.970-2.538, p=0.066). There were three studies on the TLR2 Arg753Gln polymorphism and two on the TLR4 Thr399Ile polymorphism; no association with vasculitis was evident. Among the TLR2, TLR7, and TLR9 polymorphisms included in this review, one Asian study revealed a significant association between the TLR7 rs5743733 and rs3853839 with BD (p=0.002, 0.036) and one Asian study showed an association of TLR9 rs352140 with BD (p=0.009). This meta-analysis demonstrates that the TLR4 Asp299Gly polymorphism may confer susceptibility to GCA. The review of published data suggests that other TLR polymorphisms such as TLR7 and TLR9 may play a role in vasculitis.