The polymorphisms of toll-like receptor (TLR) have been hypothesized to affect the tuberculosis susceptibility. However, the direct evidence remains controversial. Here we performed a comprehensive meta-analysis to summarize the associations between TLR polymorphisms and tuberculosis susceptibility. We systematically searched the PubMed, Embase, Cochrane library, and Chinese National Knowledge Infrastructure up to April 25, 2014. Case-control studies investigating TLR polymorphisms and tuberculosis susceptibility were included in the meta-analysis. Pooled odds ratios and corresponding 95% confidence intervals were calculated for cases and controls. Stata 11.0 and Review Manager 5.1 were adopted to conduct statistical analysis. We included 29 studies, involving 17 804 individuals. The results revealed an obvious increase of tuberculosis risk in TLR2 2258AA, and decreased risk in TLR6 745TT and TLR8 rs3761624 GA genotypes. Meanwhile, different genetic models were performed. TLR8 rs3764879C, TLR8 rs3761624A and TLR8 rs3764880A alleles were associated with high susceptibility, while TLR6 745T and TLR8 rs3788935C alleles were protective. Other polymorphisms, including TLR9 1486C/T, did not show significant associations with tuberculosis infection. Finally, subgroup analysis in TLR8 rs3764880 according to gender found a slight elevated effect of A allele in males. The meta-analysis suggests significant associations between several TLR polymorphisms and tuberculosis, including TLR2 2258G/A, TLR6 745C/T, TLR8 rs3761624, TLR8 rs3764879, TLR8 rs3761624 and TLR8 rs3764880. This study serves as the framework for additional studies to determine further the role of TLRs in tuberculosis infection.
Female ; Genetic Predisposition to Disease ; Humans ; Male ; Polymorphism, Genetic ; Toll-Like Receptors ; genetics ; Tuberculosis ; genetics

OBJECTIVETo investigate the toll-like receptors (TLR) profile of human epidermal keratinocytes.

METHODSWe cultured the immortalized human epidermal keratinocyte cell line HaCaT cells and normal human epidermal keratinocytes (NHEK) and separated epidermis with dispase from foreskins. TLR 1-10 mRNA expression was detected with reverse transcription polymerase chain reaction (RT-PCR). TLR 2 and 4 protein expressions on surface of HaCaT cells and NHEK were detected using flow cytometry.

RESULTSHaCaT cells, NHEK, and epidermis all expressed TLR 1-10 mRNA with different intensity. TLR 4 protein was detected on the surfaces of HaCaT cells and NHEK, while the expression of TLR 2 protein was few.

CONCLUSIONHuman epidermal keratinocytes constitutively express all TLR 1-10 mRNA, which may enable human keratinocytes to respond to a wide range of pathogenic micro-organisms.

Cell Line ; Cell Line, Tumor ; Epidermis ; cytology ; Flow Cytometry ; Humans ; Keratinocytes ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism

OBJECTIVETo investigate the expression of TLR4/2 mRNA in neonatal cord blood mononuclear cells (MNC).

METHODSForty-six neonates without asphyxia and 40 neonates with asphyxia were divided into groups depending on the gestational age. In the neonates without asphyxia, there were 18 full term infants (the gestational age > or = 37 weeks), 16 preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 12 preterm infants whose gestational age was < 32 weeks. In the neonates with asphyxia, 11 were full term infants, 15 were preterm infants whose gestational age was > or = 32 weeks but < 37 weeks and 14 were preterm infants at gestational age < 32 weeks. MNCs were separated and cultured with LPS (1 microg/ml) for 3 h. Cells were collected for analysis of gene expression of TLR4/2 by RT-PCR technique. Cell supernatants were taken to measure TNF-alpha production following the ELISA protocol. Fifteen healthy adults were enrolled into the control group. In addition, the Pearson correlation analyses were carried out between the levels of TLR4, TLR2 mRNA and the levels of TNF-alpha.

RESULTSIn the neonates without asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.75 +/- 0.12, 0.63 +/- 0.08, 2502.6 +/- 273.1 ng/L, separately, in the full term infants, 0.37 +/- 0.04, 0.32 +/- 0.03, 1218.8 +/- 145.7 ng/L, separately, in the preterm infants whose gestational ages were > or = 32 weeks but < 37 weeks, and 0.26 +/- 0.03, 0.20 +/- 0.03, 811.8 +/- 105.2 ng/L separately, in the preterm infants whose gestational ages were < 32 weeks. In the neonates with asphyxia, TLR4, TLR2 mRNA and TNF-alpha levels were 0.58 +/- 0.07, 0.50 +/- 0.06, 1946.4 +/- 244.2 ng/L, separately, in the full term infants, 0.29 +/- 0.03, 0.26 +/- 0.03, 970.0 +/- 94.3 ng/L, separately, in the preterm infants whose gestational age was > or = 32 weeks but < 37 weeks, and 0.17 +/- 0.02, 0.14 +/- 0.02, 652.6 +/- 60.3 ng/L, separately, in the preterm infants whose gestational age was < 32 weeks. The levels of TLR4, TLR2 mRNA and TNF-alpha in the adults were 2.71 +/- 0.75, 2.61 +/- 0.33, 9270.1 +/- 1098.3 ng/L, separately. In the preterm infants and full term infants, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower in comparison to the adults. The lower the gestational age, the lower the levels of TLR4, TLR2 mRNA and TNF-alpha. There were significant differences between the levels of TLR4, TLR2 mRNA and TNF-alpha of the neonates without asphyxia and those of the neonates with asphyxia. In the neonates with asphyxia, the levels of TLR4, TLR2 mRNA and TNF-alpha were lower than those in the neonates without asphyxia (P < 0.01). Whether the neonates were asphyxic or not, the levels of TLR4, TLR2 were paralleled with the levels of TNF-alpha.

CONCLUSIONSThe expression of TLRs in the neonates, especially in the preterm infants was lower than that in the adults, which probably contributes to the susceptibility of neonates to infections.

Blood Cells ; metabolism ; Gene Expression ; Humans ; Infant ; Infant, Newborn ; RNA, Messenger ; genetics ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptors ; metabolism ; Tumor Necrosis Factor-alpha ; immunology

The innate immune response in patients who develop inflammatory bowel disease (IBD) may be abnormal. However, the exact role of Toll-like receptors (TLRs) / CD14 gene in the pathogenesis of IBD has not been fully elucidated. We aimed to investigate the association between polymorphisms of TLR1, 2, 4, 6, and CD14 gene and susceptibility to IBD in Korean population. A total 144 patients of IBD (99 patients with ulcerative colitis, 45 patients with Crohn's disease) and 178 healthy controls were enrolled. Using a PCR-RFLP, we evaluated mutations of TLR1 (Arg80Thr), TLR2 (Arg753Gln and Arg677Trp), TLR4 (Asp299Gly and Thr399Ile), TLR6 (Ser249Pro) genes and the -159 C/T promoter polymorphism of CD14 gene. No TLR polymorphisms were detected in Korean subjects. T allele and TT genotype frequencies of CD14 gene were significantly higher in IBD patients than in healthy controls. In subgroup analysis, T allelic frequency was higher in pancolitis phenotype of ulcerative colitis. In Korean population, the promoter polymorphism at -159 C/T of the CD14 gene is positively associated with IBD, both ulcerative colitis and Crohn's disease.
Adult ; Aged ; Alleles ; Antigens, CD14/*genetics ; Asian Continental Ancestry Group/*genetics ; Colitis, Ulcerative/genetics ; Crohn Disease/genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Inflammatory Bowel Diseases/*genetics ; Male ; Middle Aged ; Phenotype ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Republic of Korea ; Toll-Like Receptor 1/genetics ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 4/genetics ; Toll-Like Receptor 6/genetics ; Toll-Like Receptors/*genetics

OBJECTIVETo investigate the expression of Toll-like receptors (TLRs) in thymus of myasthenia gravis (MG) patients and the relationship with clinical features.

METHODSThymic specimens of 36 patients received extended thymectomy for MG were divided into three groups by pathological type: 13 thymoma tissues (thymoma group) and 13 thymic tissues adjacent to thymomas (parathymoma group) from 13 cases of MG patients with thymomas, and 23 thymic tissues from MG patients without thymomas (MG nonthymoma group). Twenty-one normal thymic specimens from cardiac surgery were used as controls. The levels of TLR2-4 mRNA were examined by RT-PCR, then the levels of TLR4 mRNA were assayed by real time RT-PCR and their relationship with clinical features were analyzed.

RESULTSThe levels of TLR4 mRNA among the different groups had significant differences, while there was no difference in TLR2 and TLR3 levels. The real time RT-PCR showed that the level of TLR4 mRNA in nonthymoma group was significantly higher than that in control group(0.8544+/- 0.1200 vs 0.6851+/- 0.1524, P=0.018). And so is parathymoma group compared with the thymoma group (0.8214+/- 0.1019 vs 0.7101+/- 0.0916, P=0.005). No significant difference of TLR4 mRNA level was found between the parathymoma and nonthymoma groups. Nevertheless, the expression of TLR4 in both groups was increased compared with control group. The levels of TLR4 mRNA had positive correlation with Osserman type(R=0.609; P=0.004) .

CONCLUSIONTLR4 may play a key role in the pathogenesis of MG. It was the thymic tissues adjacent to thymomas but not thymomas themselves participated in the onset of MG.

Adolescent ; Adult ; Female ; Gene Expression Regulation ; Humans ; Male ; Middle Aged ; Myasthenia Gravis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Thymus Gland ; metabolism ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 3 ; genetics ; Toll-Like Receptor 4 ; genetics ; Toll-Like Receptors ; genetics ; Young Adult

This study is to evaluate the role of TLR-4 in lower laminar shear stress-induced interleukin-8 gene transcription activation in vascular endothelial cells by using gene mutation and transfection techniques. RT-PCR, Northern hybridization and immunocytochemical fluorescent staining showed that TLR-4 was expressed in human umbilical vein endothelial cells(HUVEC). When stimulated with 4.2 dyne/cm2 shear stress for 1 hour, an increase of TLR-4 mRNA expression was observed in HUVECs detected by RT-PCR and Northern hybridization. The intracellular domain deletion mutant TLR-4 cDNA (lacking the 155 COOH terminal amino acids of the wild type (TLR-4) and -102 -61 bp DNA sequence in 5'-flanking region of IL-8 gene (IL-8USCS) were isolated from endothelial cells by RT-PCR and PCR. These two DNA fragments were cloned into pcDNA3 and pEGFP1 respectively to construct TLR-4 dominant negative mutant pcDNA3-mTLR4 and IL-8 reporter gene pEGFP1-IL8USCS. ECV304 cells were transfected with the pEGFP1-IL8USCS or co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 by Dosper liposome transfectional reagent and selected by G418, then stimulated with 4.2 dyne/cm2 shear stress for 3 hours. Flow cytometric analysis showed that when exposed to 4.2 dyne/cm2 shear stress for 3 hours, there was a marked increase in the green fluorescent protein expression in pEGFP1-IL8USCS-transfected ECV304 cells. In contrast, there was almost no change in the green fluorescent protein expression in the cells co-transfected with pEGFP1-IL8USCS and pcDNA3-mTLR4 after the stimulation, suggesting the TLR-4 mutant depressed TLR-4 signaling. These experiments suggest that the inflammatory TLR-4/NF-kappa B signaling pathway would probably be involved in flow shear stress-induced IL-8 gene expression in human vascular endothelial cells.
Cells, Cultured ; Endothelium, Vascular ; cytology ; physiology ; Gene Expression Regulation ; Humans ; Interleukin-8 ; genetics ; Membrane Glycoproteins ; genetics ; physiology ; Mutation ; Receptors, Cell Surface ; genetics ; physiology ; Stress, Mechanical ; Toll-Like Receptor 4 ; Toll-Like Receptors ; Transcription, Genetic ; physiology ; Transfection ; Umbilical Veins ; cytology

Microglial cells were purified from a mixed neuroglia culture prepared from the neonatal chicken brain in vitro, and were infected with the vvMDV YL040920 isolate and an attenuated MDV vaccine strain CVI988/Rispens, respectively. The presence of cytopathic effect (CPE) was examined daily, and the MEQ expression in MDV-infected microglia was detected by immunohistochemistry assay. DNA replication of the MDV meq gene and transcription of the gB gene were determined by real-time quantitative PCR (qPCR) and qRT-PCR, respectively. The transcripts of Toll-like receptor (TLR) mRNA in microglia post MDV infection were quantified by qRT-PCR. The results of this study showed that both vvMDV YL040920 and attenuated vaccine strain CVI988/Rispens could infect microglia and produce characteristic CPE with plaque formation. The plaques were formed due to cells shedding at multi-sites, then quickly expanded and integrated. Furthermore, the MEQ protein was detected in nuclei of YL040920 and CVI988/ Rispens-infected microglia, and MDV meq DNA replication and gB gene transcription in MDV-infected microglia were also confirmed. Although both MDV DNA copies and gB transcripts were increased in the virus-infected microglia, the higher viral DNA load and gB transcript were observed for CVI988/Rispens than for YL040920 in vitro (P < or = 0.05/0.001). The transcriptions of TLR15 and TLR1LB gene were found to be up-regulated in microglia following MDV infection in vitro. Purified microglia infected with YL040920 was observed increased TLR15 and TLR1LB transcripts as early as 1 day post infection (dpi), and reached its peak level at 3 dpi, then decreased mildly at 5 dpi. For CVI988/Rispens, it induced an increase of TLR15 transcript as early as 1 dpi, and rose rapidly at 3 dpi, and then decreased slightly at 5 dpi. At the same time, CVI988/Rispens induced the increase of chTLR1LB transcript at 3 dpi and decreased at 5 dpi. By comparing the TLRs transcription between YL040920 and CVI988/Rispens-infected microglia, it was suggested that vvMDV YL040920 might induce more TLR15 transcript than the attenuated vaccine strain CVI988/Rispens (P < or = 0.01/0.001), while CVI988/Rispens induced more TLR1LB transcript than YL040920 (P < or = 0.001).
Animals ; Brain ; metabolism ; virology ; Chickens ; Gene Expression ; Herpesvirus 2, Gallid ; genetics ; physiology ; Marek Disease ; genetics ; metabolism ; virology ; Microglia ; metabolism ; virology ; Poultry Diseases ; genetics ; metabolism ; virology ; Toll-Like Receptor 1 ; genetics ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Transcription, Genetic

Age Factors ; Animals ; Cytokines ; physiology ; Humans ; RNA, Messenger ; analysis ; Sepsis ; etiology ; Signal Transduction ; Toll-Like Receptors ; genetics ; physiology

The aim of this study was to explore the characteristics of Toll-like receptor expression in mesenchymal stem cells derived from bone marrow of healthy donor (BM-MSCs). BM-MSCs were isolated from bone marrow of healthy donor by Ficoll method. Expressions of CD34, CD45, HLA-DR, CD44 and CD71 in BM-MSCs were detected by flow cytometry. CD71 in BM-MSCs was assayed by immunocytochemistry. The adipocyte and osteoblast induction of BM-MSCs were detected by alizarin red stain and oil red stain respectively. TLR 1 - 10 mRNA levels in BM-MSCs were evaluated by semiquantitative RT-PCR. The results showed that expressions of CD34, CD45 and HLA-DR in BM-MSC were negative while the expressions of CD44 and CD71 were positive. CD71 in BM-MSCs was positive. After induced by osteoblast and adipocyte inductor, BM-MSCs were positive for alizarin red staining and oil red staining respectively. All of TLR 1 - 10 mRNA were found in BM-MSCs with high expression levels of TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and low expression levels of TLR1, TLR5, TLR6, TLR10. In conclusion, different levels of TLR 1 - 10 mRNA were expressed in BM-MSCs of healthy donor.
Bone Marrow Cells ; metabolism ; Cell Differentiation ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; metabolism ; RNA, Messenger ; genetics ; Toll-Like Receptors ; metabolism

Tuberculosis is a chronic infectious disease caused by Mycobacterium (M.) tuberculosis. Innate immunity plays an important role in the response to M. tuberculosis. Toll-like receptors (TLRs) are important pattern recognition receptors in innate immunity. TLRs serve as switches that play decisive roles in identifying pathogens-related components. Previous studies found that TLR1, TLR2, TLR4, TLR9 were essential to promote the development of innate immune responses. The SNPs of rs4833095, rs5743618, rs3923647 of TLR1, rs57473708, rs3804099 of TLR2 and rs352139, rs5743836 of TLR9 were closely related to the susceptibility of tuberculosis in some populations. And there appeared certain relationship between the polymorphisms of TLR3, TLR6, TLR7, TLR8, TLR10 and the susceptibility of tuberculosis. The normal function of TLRs ensures the body's normal immune response to M. tuberculosis. The diversity of TLRs genes allows different individuals to respond differently to the same pathogen. Studies targeting on the relationship between single nucleotide polymorphism in TLRs and susceptibility to tuberculosis can predict the susceptibility to tuberculosis in some populations, as well as discover new drugs targets.
Genetic Predisposition to Disease ; Humans ; Mycobacterium tuberculosis ; Polymorphism, Single Nucleotide ; Research/trends* ; Signal Transduction/immunology* ; Toll-Like Receptors/genetics* ; Tuberculosis/immunology*