BACKGROUNDThe Toll-like receptors (TLRs) represent a group of single-pass transmembrane receptors expressed on sentinel cells that are central to innate immune responses.The aim of this study was to investigate the presence of soluble TLRs in pleural effusions, and the diagnostic values of TLRs for pleural effusion with various etiologies.

METHODSPleural effusion and serum samples were collected from 102 patients (36 with malignant pleural effusion, 36 with tuberculous pleural effusion, 18 with bacterial pleural effusion, and 12 with transudative pleural effusion). The concentrations of TLR1 to TLR10 were determined in effusion and serum samples by enzyme linked immunosorbent assay. Four classical parameters (protein, lactate dehydrogenase, glucose and C-reactive protein (CRP)) in the pleural fluid were also assessed. Receiver-operating characteristic curves were used to assess the sensitivity and specificity of pleural fluid TLRs and biochemical parameters for differentiating bacterial pleural effusion.

RESULTSThe concentrations of TLR1, TLR3, TLR4, TLR7 and TLR9 in bacterial pleural effusion were significantly higher than those in malignant, tuberculous, and transudative groups, respectively. Analysis of receiver operating characteristic curves revealed that the area under the curves of TLR1, TLR3, TLR4, TLR7 and TLR9 were 0.831, 0.843, 0.842, 0.883 and 0.786, respectively, suggesting that these TLRs play a role in the diagnosis of bacterial pleural effusion. Also, the diagnostic value of TLRs for bacterial pleural effusions was much better than that of biochemical parameters (protein, lactate dehydrogenase, glucose and CRP).

CONCLUSIONSThe concentrations of TLR1, TLR3, TLR4, TLR7 and TLR9 appeared to be increased in bacterial pleural effusion compared to non-bacterial pleural effusions. Determination of these pleural TLRs may improve the ability of clinicians to differentiate pleural effusion patients of bacterial origin from those with other etiologies.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bacterial Infections ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Middle Aged ; Pleural Effusion ; metabolism ; microbiology ; Prospective Studies ; Toll-Like Receptor 1 ; metabolism ; Toll-Like Receptor 3 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptor 7 ; metabolism ; Toll-Like Receptor 9 ; metabolism ; Toll-Like Receptors ; metabolism ; Young Adult

Recent findings show that Toll-like receptors (TLRs) expressed in immune cells play a crucial role in the innate immune response and the subsequent induction of adaptive immune responses against microbial infection on tissue injury. Furthermore, expression of TLRs in cancer cells is associated with tumor proliferation and invasion. To explore the role of TLRs expression in cervical carcinogenesis in Uighur women, we detected the expressions of TLR3, TLR4, TLR7, and TLR9 in 25 normal cervical tissues, 64 cervical intraepithelial neoplasia (CIN) tissues, and 63 cervical squamous cell carcinoma (CSCC) tissues using immunohistochemical staining, as well as human papillomavirus type 16 (HPV16) infection using PCR. All samples used in this study were from Xinjiang Uighur women. We found the expression levels of TLR4, TLR7, and TLR9 were significantly higher in CIN and CSCC than in normal controls (P < 0.05). Up-regulation of TLR4 and TLR7 were correlated with tumor differentiation but not FIGO stage or lymph node metastasis (P > 0.05). Up-regulation of TLR9 was correlated with lymph node metastasis (P < 0.05) but not tumor differentiation or FIGO stage (P > 0.05). We also analyzed the correlation between the expressions of TLRs and HPV16 infection and found that the expressions of TLR4 and TLR9 significantly correlated with HPV16 infection in CIN (r = 7.434, P = 0.006; r = 7.123, P = 0.008) and CSCC (r = 6.423, P = 0.001; r = 8.478, P = 0.004), whereas the expression of TLR3 was not significantly different in any of the three groups and had no significant correlation with HPV16 infection. Our results suggest that high expression of TLR4, TLR7, and TLR9 may play important roles in the development and progression of CIN and CSCC in Uighur women, and the expressions of TLR4 and TLR9 can be up-regulated by HPV16 infection.
Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; China ; ethnology ; Female ; Gene Expression Regulation, Neoplastic ; Human papillomavirus 16 ; isolation & purification ; Humans ; Lymphatic Metastasis ; Neoplasm Staging ; Papillomavirus Infections ; genetics ; pathology ; Toll-Like Receptor 3 ; metabolism ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptor 7 ; metabolism ; Toll-Like Receptor 9 ; metabolism ; Toll-Like Receptors ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology

BACKGROUND AND OBJECTIVES: Mammalian Toll-like receptors (TLR) participate in innate immune responses to microbial pathogens. Recent interest has been focused on the concept that TLR-induced innate responses can modulate subsequent adaptive immune responses. The objective of this study is to determine whether TLR 2 stimulation in vivo would modulate subsequent allergic responses in an Aspergillus fumigatus (Af) murine model of allergic rhinitis (AR). MATERIALS AND METHOD: Mice were sensitized via intraperitoneal injection with Af antigen and alum, and received a series of three daily intranasal Af antigen challenge. A TLR 2 agonist, PamCys was administrated intranasally one day before sensitization or one day before the first intranasal allergen challenge. Adaptive immune profiles and response to Af challenge were assessed. RESULTS: PamCys decreased the allergen induced nasal recruitment of eosinophils and interleukin (IL)-5 in nasal lavage fluids compared with mice treated with no PamCys. Serologic data revealed that PamCys downregulated Af-specific IgE in the sera of PamCys-treated mice. In addition, spleen cells from the PamCys-treated mice displayed attenuated Af-specific IL-4 and IL-5, but increased interferon (IFN)- and immunosuppressive IL-10. CONCLUSION: The present study demonstrate that TLR 2 agonist decreases allergic responses in this AR model by shifting T helper 2 (Th2) biased immune parameters towards Th1 dominance.
Animals ; Aspergillus fumigatus ; Bias (Epidemiology) ; Eosinophils ; Immunity, Innate ; Immunoglobulin E ; Injections, Intraperitoneal ; Interferons ; Interleukin-10 ; Interleukin-4 ; Interleukin-5 ; Interleukins ; Mice* ; Models, Animal ; Nasal Lavage Fluid ; Rhinitis* ; Spleen ; Toll-Like Receptor 2* ; Toll-Like Receptors*

OBJECTIVE: The aim of this study was to clarify whether stimulation of recombinant IL-17, TLR2 and TLR4 by their specific ligands induces the production of RANKL and IL-6 in the fibroblast-like synoviocytes (FLSs) from RA patients. METHODS: FLSs were isolated from RA synovial tissues and they were stimulated with the IL-17, TLR2 ligand bacterial peptidoglycan (PGN) and TLR4 ligand lipopolysaccharide (LPS). The RANKL levels were assessed by RT-PCR and western blotting. The expressions of IL-17, TLR2, TLR4, RANKL and IL-6 in the RA synovium were quantified by immunohistochemistry and these values were compared with the values obtained in the osteoarthritis synovium. The increased IL-6 production in the culture supernatants of the RA FLSs was quantified by sandwich ELISA. RESULTS: The mRNA and protein levels of RANKL and IL-6 increased in the RA FLSs stimulated with PGN, LPS and IL-17, or PGN plus IL-17 or LPS plus IL-17. The expressions of IL-17, TLR2, TLR4, RANKL and IL-6 were much higher in the RA synovium than those in the osteoarthritis (OA) synovium. CONCLUSION: We observed synergistic effects of TLR-2, TLR-4 and IL-17 upon the induction of RANKL. In conclusion, our data supports the previous evidence of an important role of TLR-2, TLR-4 and IL-17 in the pathogenesis of RA.
Blotting, Western ; Fibroblasts ; Humans ; Immunohistochemistry ; Interleukin-17 ; Interleukin-6 ; Ligands ; Osteoarthritis ; Peptidoglycan ; RNA, Messenger ; Synovial Membrane ; Toll-Like Receptor 2 ; Toll-Like Receptor 4 ; Toll-Like Receptors

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; 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

BACKGROUNDSepticemia and inflammation-mediated septic shock caused by Vibrio vulnificus (VV) is strongly associated with chronic liver disease. This study examined the effects of antimicrobial therapy on expression of hepatic toll-like receptors and inflammatory cytokines in rats with alcohol-induced liver disease complicated by VV sepsis.

METHODSMale Sprague-Dawley rats were assigned to the following treatment groups: normal control (N), alcoholic liver disease control (A), antimicrobial-treated alcoholic liver disease control (AA), alcoholic liver disease with VV sepsis (AV), and antimicrobial-treated alcoholic liver disease with VV sepsis (AVA). Alcohol-induced liver disease was observed in all groups except N. Expression of mRNAs encoding hepatic toll-like receptors 2 and 4, myeloid differentiation protein-2, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6 and IL-10 was determined by RT-PCR.

RESULTSmRNAs encoding toll-like receptors 2 and 4 and myeloid differentiation protein-2 were significantly up-regulated in group AV as compared to control groups at 2 - 24 hours of sepsis; peak expression occurred at 12 hours. These mRNAs were also up-regulated in group AVA but to lesser degrees than in group AV at comparable time post-infection. mRNAs encoding TNF-alpha, IL-1beta and IL-6 were significantly elevated in group AV as a function of infection. In group AVA as compared to AV, expression of TNF-alpha and IL-1beta mRNAs was lower at 12 - 24 hours post-infection and expression of IL-6 mRNA was lower at 24 hours post-infection. Compared with control groups, IL-10 mRNA expression in group AV was markedly higher at 12 - 24 hours of sepsis. Expression of IL-10 mRNA was lower in group AVA as compared to AV at 24 hours of sepsis.

CONCLUSIONSAntimicrobial therapy reduces expression of toll-like receptors and cytokines in rats with alcohol-induced liver disease complicated by VV sepsis. Monitoring hepatic toll-like receptor and cytokine expression during antibiotic therapy may be valuable for determining the course of VV sepsis in subjects with liver disease.

Adaptor Proteins, Signal Transducing ; genetics ; Animals ; Anti-Infective Agents ; therapeutic use ; Cytokines ; genetics ; Interleukin-10 ; genetics ; Interleukin-1beta ; genetics ; Interleukin-6 ; genetics ; Liver ; drug effects ; metabolism ; Liver Diseases, Alcoholic ; drug therapy ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sepsis ; drug therapy ; genetics ; microbiology ; Toll-Like Receptor 2 ; genetics ; Toll-Like Receptor 4 ; genetics ; Toll-Like Receptors ; genetics ; Tumor Necrosis Factor-alpha ; genetics ; Vibrio Infections ; drug therapy ; Vibrio vulnificus ; physiology

The study investigated the inhibitory effect and mechanism of tectorigenin derivative(SGY) against herpes simplex virus type Ⅰ(HSV-1) by in vitro experiments. The cytotoxicity of SGY and positive drug acyclovir(ACV) on African green monkey kidney(Vero) cells and mouse microglia(BV-2) cells was detected by cell counting kit-8(CCK-8) method, and the maximum non-toxic concentration and median toxic concentration(TC_(50)) of the drugs were calculated. After Vero cells were infected with HSV-1, the virulence was determined by cytopathologic effects(CPE) to calculate viral titers. The inhibitory effect of the tested drugs on HSV-1-induced cytopathy in Vero cells was measured, and their modes of action were initially explored by virus adsorption, replication and inactivation. The effects of the drugs on viral load of BV-2 cells 24 h after HSV-1 infection and the Toll-like receptor(TLR) mRNA expression were detected by real-time fluorescence quantitative PCR(RT-qPCR). The maximum non-toxic concentrations of SGY against Vero and BV-2 cells were 382.804 μg·mL~(-1) and 251.78 μg·mL~(-1), respectively, and TC_(50) was 1 749.98 μg·mL~(-1) and 2 977.50 μg·mL~(-1), respectively. In Vero cell model, the half maximal inhibitory concentration(IC_(50)) of SGY against HSV-1 was 54.49 μg·mL~(-1), and the selection index(SI) was 32.12, with the mode of action of significantly inhibiting replication and directly inactivating HSV-1. RT-qPCR results showed that SGY markedly reduced the viral load in cells. The virus model group had significantly increased relative expression of TLR2, TLR3 and tumor necrosis factor receptor-associated factor 3(TRAF3) and reduced relative expression of TLR9 as compared with normal group, and after SGY intervention, the expression of TLR2, TLR3 and TRAF3 was decreased to different degrees and that of TLR9 was enhanced. The expression of inflammatory factors inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) was remarkably increased in virus model group as compared with that in normal group, and the levels of these inflammatory factors dropped after SGY intervention. In conclusion, SGY significantly inhibited and directly inactivated HSV-1 in vitro. In addition, it modulated the expression of TLR2, TLR3 and TLR9 related pathways, and suppressed the increase of inflammatory factor levels.
Animals ; Antiviral Agents/therapeutic use* ; Chlorocebus aethiops ; Herpes Simplex/pathology* ; Herpesvirus 1, Human/metabolism* ; Isoflavones ; Mice ; TNF Receptor-Associated Factor 3/pharmacology* ; Toll-Like Receptor 2/metabolism* ; Toll-Like Receptor 3/metabolism* ; Toll-Like Receptor 9/metabolism* ; Toll-Like Receptors/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Vero Cells ; Virus Replication

OBJECTIVETo examine interleukin-8 (IL-8) mRNA expression induced by flow shear stress in human umbilical vein endothelial cells (HUVECs) and investigate its transcriptional activation.

METHODSFlow laminar shear stress 4.2 dyne/cm(2) was used for the stimulating experiments. The flow shear stress-induced IL-8 mRNA expression was detected by reverse transcription-polymerase chain reaction (RT-PCR). pEGFP1 was used to construct IL-8 reporter gene pEGFP1-IL8USCS for determining IL-8 gene transcriptional activation through gene transfer and flow cytometric analysis. NF-kappaB nuclear translocation was observed by immunocytofluorescent staining. Western blot was used to examine IkappaB phosphorylation and degradation. RT-PCR, Northern blot, immunocytofluorescent staining and laser confocal microscopy were used to determine Toll-like receptor-4 (TLR-4) expression at mRNA and protein levels in the cells.

RESULTSThere was a marked increase in IL-8 mRNA expression in HUVECs after 120 min of exposure to laminar flow shear stress. When exposed to shear stress for 180 min, there was an increase in enhanced green fluorescent protein expression in pEGFP1-IL8USCS-transfected endothelial cells. NF-kappaB p65 immunocytofluorescent staining of HUVECs showed that when exposed to the same flow shear stress for 30 or 60 min, the cell nuclei became stained; after 90 or 120 min, the staining became much more pronounced. A significant increase in P-IkappaB in the cell lysates occurred after 10 min of exposure while blot density dramatically dropped after 60 min of exposure. The density of the IkappaB blot dropped with increasing exposure time after 30 min. TLR-4 was present on the surface of HUVECs. HUVECs constitutively expressed TLR-2 and TLR-4 mRNA; when exposed to flow shear stress for 60 min, TLR-4 mRNA expression increased.

CONCLUSIONNF-kappaB activation is involved in flow shear stress-induced IL-8 mRNA expression in human umbilical vein endothelial cells. TLR-4 receptor for innate immunity most likely mediate these events.

Blotting, Northern ; Cells, Cultured ; Drosophila Proteins ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Interleukin-8 ; genetics ; Membrane Glycoproteins ; analysis ; NF-kappa B ; metabolism ; RNA, Messenger ; analysis ; Receptors, Cell Surface ; analysis ; Stress, Mechanical ; Toll-Like Receptor 2 ; Toll-Like Receptor 4 ; Toll-Like Receptors ; Transcriptional Activation

OBJECTIVETo evaluate the effect of cluster of differentiation 14 (CD-14) and Toll like receptors (TLR) on the expression of interleukin-6 (IL-6) mRNA induced by Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS).

METHODSMC3T3-E1 cells were treated with 10 mg/L Pe-LPS for different hours, and the cells uninvolved by anything as the blank group. The expression of IL-6 was detected by reverse transcription polymerse chain reaction (RT-PCR) and enzyme-liked immunosorbent assay (ELISA). The expression of CD-14, TLR-2 and TLR-4 mRNA was observed at different time point (0 - 24 h) by RT-PCR. The protein of CD-14, TLR-2 and TLR-4 was analyzed with a flow cytometer. MC3T3-E1 cells were pretreated with anti-CD-14, anti-TLR-2 and anti-TLR-4 antibody for 1 h, and then cells were stimulated with 10 mg/L Pe-LPS for 6 h. The expression of IL-6 mRNA was examined by RT-PCR. Statistical analysis was performed using one-way ANOVA Dunnett-t test with SPSS 11.0 software package.

RESULTSThe IL-6 mRNA and proteins increased significantly after treatment with Pe-LPS. When MC3T3-E1 cells treated by Pe-LPS for 6 h, the expression of proteins soared from (11.696 ± 0.672) ng/L to (36.534 ± 0.574) ng/L (P < 0.01); In the control group, the CD-14 and TLR-4 mRNA are ambly-expression, and the ratios of CD-14 and TLR-4 positive cells were (39.038 ± 3.131)% and (11.438 ± 0.385)% respectively in MC3T3-E1. After treatment by Pe-LPS, the expression of CD-14 and TLR-4 mRNA increased significantly, and the ratios of CD-14 and TLR-4 positive cells markedly increased to (62.407 ± 1.800)% and (21.367 ± 2.271)%. TLR-2 expression did not change apparently after Pe-LPS treatment. The expression of IL-6 mRNA was partly inhibited by anti-CD-14 or anti-TLR-4 antibody, but not by TLR-2.

CONCLUSIONSPe-LPS can induce the expression of IL-6 in osteoblast MC3T3-E1 through CD-14 and TLR-4, but not TLR-2.

3T3 Cells ; Animals ; Antibodies ; immunology ; Interleukin-6 ; genetics ; metabolism ; Lipopolysaccharide Receptors ; genetics ; metabolism ; Lipopolysaccharides ; isolation & purification ; pharmacology ; Mice ; Porphyromonas endodontalis ; RNA, Messenger ; metabolism ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism