Expression of TLR3 in human adenocarcinoma of the lung cells induced by respiratory syncytial virus and its significance in the diagnosis of pneumonia in children / 中国医师杂志

Objective:To study the expression of Toll like receptor 3 (TLR3) in human adenocarcinoma of the lung cells induced by respiratory syncytial virus (RSV) and its significance in the diagnosis of pneumonia in children.Methods:A549 cells were divided into RSV infection group [added 1 μg/ml Lipopolysaccharide (TLR3 agonist) transfected RSV virus after 150 μl intervention], Lipopolysaccharide stimulation group (added 1 μg/ml Lipopolysaccharide 150 μl intervention) and normal control group (normal culture). The mRNA expressions of tumor necrosis factor-α, interleukin 8, TLR3 protein and TLR3 in A549 Cells of different groups were compared. We prospectively selected 80 children with RSV infectious pneumonia admitted to Baoding Second Central Hospital from August 2019 to October 2021 as the RSV pneumonia group, and sixty children with common pneumonia were taken as the common pneumonia group, and 60 healthy children in our hospital were taken as the control group. The mRNA expression of serum TLR3 in different groups was compared, and the diagnostic efficacy of serum TLR3 in RSV pneumonia was evaluated by receiver operating characteristic.Results:There was a statistically significant difference in the expression of TLR3 protein among different groups of A549 cells ( P<0.001). The expression differences of TLR3 mRNA in different groups of A549 cells at different time points were statistically significant(all P<0.001). There was significant difference in the expression of tumor necrosis factor-α and interleukin 8 of A549 cells at different time points in different groups (all P<0.05). There was a statistically significant difference in the expression of serum TLR3 mRNA among the three groups of subjects ( F=155.237, P<0.001). The critical value for TLR3 gene diagnosis was 66.87, with corresponding sensitivity of 73.75%, specificity of 70.83%, and the area under curve (AUC) of 0.803(95% CI: 0.753-0.855). Conclusions:Respiratory syncytial virus induces human lung cancer cells and promotes disease progression through TLR3 expression; Serum TLR3 can be used for the diagnosis of RSV pneumonia.

Relationship between rs128912 single nucleotide polymorphism in promoter region of Toll-like receptor 3 gene and cataract in Chinese Han population / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To analyze the relationship between rs128912 single nucleotide polymorphism(SNP)in the promoter region of Toll-like receptor 3(TLR3)gene and cataract in Chinese Han population.METHODS: A total of 263 patients with cataract admitted to our hospital from June 2019 to June 2021 were selected as study group, and 150 patients with lens dislocation were included in control group. Western blotting was used to detect the expression of TLR3 protein in the anterior capsular tissues of lens in the two groups, and direct sequencing method was applied to analyze the polymorphism of rs128912 locus in the promoter region of TLR3 gene. The expression of peripheral blood TLR3 mRNA of patients with different genotypes was detected by real-time quantitative polymerase chain reaction(RT-qPCR).RESULTS: The expression level of TLR3 protein in the anterior capsular tissues in the study group was higher than that in the control group(P&#x003C;0.05). The frequencies of genotypes(AA, AT, TT)at rs128912 locus in the TLR3 gene promoter region in the study group and the control group were in accordance with Hardy-Weinberg genetic equilibrium, and there were differences in the frequencies of genotypes(AA, AT, TT)and frequencies of alleles(A, T)at rs128912 locus in the TLR3 gene promoter region between both groups(P&#x003C;0.05). The relative expression level of peripheral blood TLR3 mRNA in patients with TT genotype in the study group was higher than that in patients with AA or AT genotypes(P&#x003C;0.05).CONCLUSION: The expression of TLR3 protein in anterior capsular tissues of lens of patients with cataract is significantly up-regulated, and rs128912 locus polymorphism in the TLR3 gene promoter region is related to the susceptibility of cataract in Chinese Han population, and people with TT genotype are more prone to cataract.

Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Toll-like receptor 3 (TLR3) is a member of the TLR family, mediating the transcriptional induction of type I interferons (IFNs), proinflammatory cytokines, and chemokines, thereby collectively establishing an antiviral host response. Studies have shown that unlike other TLR family members, TLR3 is the only RNA sensor that is utterly dependent on the Toll-interleukin-1 receptor (TIR)‍-domain-containing adaptor-inducing IFN-‍β (TRIF). However, the details of how the TLR3-TRIF signaling pathway works in an antiviral response and how it is regulated are unclear. In this review, we focus on recent advances in understanding the antiviral mechanism of the TRIF pathway and describe the essential characteristics of TLR3 and its antiviral effects. Advancing our understanding of TLR3 may contribute to disease diagnosis and could foster the development of novel treatments for viral diseases.

Evaluation of variants in IL6R, TLR3, and DC-SIGN genes associated with dengue in a sampled Colombian population / Evaluación de las variantes en los genes IL6R, TLR3 y DC-SIGN asociadas con dengue en una muestra de población colombiana

Abstract Introduction: Host genetics is recognized as an influential factor for the development of dengue disease. Objective: This study evaluated the association of dengue with the polymorphisms rs8192284 for gene IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN. Materials and methods: Of the 292 surveyed subjects, 191 were confirmed for dengue fever and the remaining 101 were included as controls. The genotypes were resolved using polymerase chain reaction and restriction fragment length polymorphism (PCR- RFLP). In an attempt to determine the risk (Odds Ratio) of suffering dengue fever, data were analyzed using chi-square for alleles and logistic regression for both genotypes and allelic combinations. Confidence intervals were set to 95% for all tests regardless of the adjustment by either self-identification or ancestry. Results: For Afro-Colombians, the allele rs8192284 C offered protection against dengue [OR=0.425,(0.204-0.887), p=0.020]. The alleles rs7248637 A and rs3775290 A posed, respectively, an increased risk of dengue for Afro-Colombians [OR=2.389, (1.170-4.879), p=0.015] and Mestizos [OR=2.329, (1.283-4.226), p=0.005]. The reproducibility for rs8192284 C/C [OR=2.45, (1.05-5.76), p=0.013] remained after adjustment by Amerindian ancestry [OR=2.52, (1.04-6.09), p=0.013]. The reproducibility for rs3775290 A/A [OR=2.48, (1.09-5.65), p=0.033] remained after adjustment by European [OR=2.34, (1.02-5.35), p=0.048], Amerindian [OR=2.49, (1.09-5.66), p=0.035], and African ancestry [OR=2.37, (1.04-5.41), p=0.046]. Finally, the association of dengue fever with the allelic combination CAG [OR=2.07, (1.06-4.05), p=0.033] remained after adjustment by Amerindian ancestry [OR=2.16, (1.09-4.28), p=0.028]. Conclusions: Polymorphisms rs8192284 for IL6R, rs3775290 for TLR3, and rs7248637 for DC-SIGN were associated with the susceptibility to suffer dengue fever in the sampled Colombian population.

Resumen Introducción. La genética del huésped se reconoce como un factor que influye en el desarrollo del dengue. Objetivo. Este estudio evaluó la asociación del dengue con los polimorfismos rs8192284 del gen IL6R, rs3775290 del TLR3 y rs7248637 del DC-SIGN. Materiales y métodos. De los 292 sujetos encuestados, en 191 se confirmó la presencia de fiebre por dengue y los restantes 101 se incluyeron como controles. Los genotipos se resolvieron mediante reacción en cadena de la polimerasa y polimorfismos en la longitud de los fragmentos de restricción (PCR-RFLP). En un intento por determinar el riesgo de sufrir dengue, los datos se analizaron mediante la prueba de ji al cuadrado para los alelos y la regresión logística para los genotipos y las combinaciones alélicas. Los intervalos de confianza se calcularon a 95 % para todas las pruebas independientemente ajustadas por autoidentificación o componente genético ancestral. Resultados. En los afrocolombianos, el alelo C rs8192284 ofreció protección contra el dengue (OR=0,425; 0,204-0,887, p=0,020). Los alelos A rs7248637 y Ars3775290 plantearon un mayor riesgo de dengue para los afrocolombianos (OR=2,389; 1,170- 4,879; p=0,015) y los mestizos (OR=2,329; 1,283-4,226: p=0,005), respectivamente. La reproducibilidad para rs8192284 C/C (OR=2,45; 1,05-5,76; p=0,013) permaneció después del ajuste por el componente genético ancestral amerindio (OR=2,52; 1,04- 6,09; p=0,013). La reproducibilidad del rs3775290 A/A (OR=2,48; 1,09-5,65; p=0,033) permaneció después del ajuste por el componente europeo (OR=2,34; 1,02-5,35; p=0,048), el amerindio (OR=2,49; 1,09- 5,66; p=0,035), y el africano (OR=2,37; 1,04- 5,41; p=0,046). Por último, la asociación del dengue con la combinación alélica CAG (OR=2,07; 1,06-4,05; p=0,033) permaneció después del ajuste por el componente genético amerindio (OR=2,16; 1,09-4,28;p=0,028). Conclusión. Los polimorfismos rs8192284 en IL6R, rs3775290 en TLR3 y rs7248637 en DC-SIGN, se asociaron con la propensión a sufrir dengue en una muestra de población colombiana.

Sex hormones alter the response of Toll-like receptor 3 to its specific ligand in fallopian tube epithelial cells / 대한생식의학회지

OBJECTIVE: The fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated. METHODS: The functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings. RESULTS: The production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-β estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C). CONCLUSION: These results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OE-E6/E7 cell line.

The role of TLR3/NF-kappa B signaling pathway in paraquat-induced acute lung injury / 中华急诊医学杂志

Objective To investigate the pathogenesis of paraquat (PQ) induced acute lung injury through Toll like receptor 3 (Toll-like receptor-3, TLR3), TLR induced nuclear transcription factors (Nuclear Factor-kappa B, NF-κB) and its downstream pro-inflammatory factors TNF-a, IL-1β, IL-6. Methods The acute lung injury model of mice and the acute injury model of type II alveolar epithelial cells (A549) induced by PQ were established. The PQ mediated pathological changes of lung tissue, the cell count and cytospin of bronchoalveolar lavage fluid (BALF) were evaluated, and the pro-inflammatory factors in the lung tissue of mice were determined by ELISA the viability of A549 cells mediated by PQ was detected by CCK8 assay, and the mRNA expression and protein level of TLR3, Phospho-NF-kBp65, tumor necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in the lung tissues and A549 cells were observed by Real-time PCR and Western-blotting. In control group, the mice received normal saline (NS) instead of PQ. Results Compared with the control group, the mice in PQ group showed difficulty breathing, decreased activity, reduced food intake, and weight lost. The total number of BALF cells in the PQ group was significantly increased [NS: (0.018 8±0.102 1) × 105 vs. PQ: (0.237 4±0.121 7) ×105,t=9.804,P＜0.01] with macrophage [NS: (0.162 8±0.086 5) × 105 vs. PQ: (1.063 3± 0.343 3) × 105,t=8.043,P＜0.01],lymphocyte [NS: (0.006 6±0.005 2) × 105 vs. PQ: (0.171 2±0.099 1) × 105, t=5.243,P＜O.Ol] and neutrophils [NS: (0.000 04±0.000 1) × 105 vs. PQ: (0.901 9±0.652 5) × 105, t=4370, P＜0.01]. In PQ group, the appearance and volume of lung tissue increased with hyperemia and edema. HE slices showed inflammatory cell infiltration and pulmonary interstitial hemorrhage. Moreover, the expressions of TNF-a, IL-1β, IL-6 in BALF of PQ group was significantly higher than those of the control group by the ELISA assay [TNF-a: NS: (2.782 1 ± 3.521 5) vs. PQ: ( 7.512 6±3.459 8) pg/mL,t=3.030, P＜0.05; IL-1β: NS: (22.687 5±14.229 3) vs. PQ: (163.100 4±81.118 3) pg/mL,t=5.391,P＜0.01 ; IL-6: NS: (1.653 3±0.442 7) vs. PQ: (648.565 6 ± 422.606 1) pg/mL, t=4.841,P＜0.01]. CCK8 results indicated that the viability of A549 cells decreased by 25.3％ and 36.4％ at 24h after 200～400 μmol/L PQ treatment (all P＜0.05). The mRNA expressions of TNF-a, IL-1p, IL-6 in the lung tissue and A549 cells in PQ group were higher than those in the control group as well (all P＜ 0.05). Furthermore, Western-blotting results revealed that the protein levels of TLR3 and Phospho-NF-κBp65 in the lung and A549 cells mediated by PQ were significantly higher than those in the control group (all P＜ 0.05). Conclusions PQ may induce acute lung injury by up-regulation of the expressions of inflammatory factors TNF-a, IL-1β, IL-6 through the TLR3/NF-κB signaling pathway.

Correlation of TLR3 with the alteration of stromal cells in tumor microenvironment of hepatocellular carcinoma / 临床与实验病理学杂志

Purpose To analyse the correlation between the Toll-like receptor 3 (TLR3) expression and clinic pathologic factors,stromal microenvironment in hepatocellular carcinoma (HCC).Methods The tissue microarrays of human HCC were prepared with self-owned patent technology.The expression of TLR3 in HCC cells as well as various indexes in HCC stroma was examined with immunohistochemistry of SP.The correlation between TLR3 expression with the clinic pathologic factors of HCC,and the correlation between TLR3 expression with the reaction of stromal cells in HCC microenvironment were analyzed by multi-factor correlation analysis.Results The positive expression rate of TLR3 in HCC was 71.57％.The expression of TLR3 in HCC had negative correlation with vascular invasion (P =0.001),cirrhosis (P =0.007),Edmondson's grades (P =0.001) and staging of TNM (P =0.000).It had positive correlation with hepatitis B surface antigen (HBsAg) (P =0.000).It had positive correlation with T cells (P =0.002) and natural killer (NK) cells (P =0.000).It had negative correlation with carcinoma-associated fibroblasts (CAFs) (P =0.000) and microvessel density (MVD) (P =0.000).Conclusion TLR3 has an important influence on the interstitial microenvironment of HCC.

Serum level changes of Toll-like receptor 3,Toll-like receptor 4,fructosamine and glycosylated hemoglobin and the significance in predicting restenosis and re-occlusion after coronary stenting in aged patients with old myocardial infarction / 中华老年医学杂志

Objectives To study the predictive value of Toll-like receptors 3,4(TLR3,TLR4),fructosamine(FMN)and glycated hemoglobin A1c(HbA1c)in the in-stent restenosis and re-occlusion after primary percutaneous coronary intervention(PCI)in patients aged 70-85 years with old myocardial infarction.Methods 51 patients aged 70-85 years with in-stent restenosis after primary PCI from Jan 2007 to Sep 2016 were selected.Serum level changes in TLR3,TLR4 were detected by flow cytometry.The levels of FMN and HbA1c were tested by colorimetric endpoint reaction and high-pressure liquid chromatography respectively.Results The levels of TLR3,TLR4,FMN and HbA1c were gradually elevated along with the increases of artery numbers(0,1,2,>2)and percentage(0%,70-89%,90-99%,100%)of in-stent restenosis,LVEF(%)decrease and NYHA(Ⅰ,Ⅱ,Ⅲ,Ⅳ)increase(all P2)of in-stent restenosis in TLR3,and group of percentage(0%,70-89%,90-99%,100%)in the in-stent restenosis in TLR4,group of LVEF(%)in FMN,and group of NYHAⅠ,Ⅱ,Ⅲ,Ⅳ in HbA1c(%)(all P2)groups were(7.6±0.5),(18.9±0.6),(32.0±0.9),(51.3±0.8),respectively(all P<0.01).The levels of TLR4(%)in the in-stent restenosis percentage(0%,70-89%,90-99%,100%)groups were(10.5±7.0),(20.1±7.2),(33.3±9.7),(69.0±11.3%)respectively(all P<0.01).The levels of FMN(mmol/L)in LVEF[(49~59%),(37~48%),(25~36%)]groups were(0.6±0.4),(9.4±0.6),(18.1±0.8),respectively(all P<0.01).And the level of HbA1c(%)in groups of NYHA Ⅰ,NYHA Ⅱ,NYHA Ⅲ,NYHA Ⅳ were(6.1±0.4),(5.9±0.6),(8.9±0.9),(12.0±0.8),respectively(all P<0.01).Conclusions Serum level changes in TLR3,TLR4,FMN and HbA1c may become the new indicators to forecast the degree of in-stent restenosis in very old patients with old myocardial infarction after primary coronary intervention.

Expressions and significance of Toll-like receptor 3 and Toll-like receptor 4 in renal tissues and peripheral blood mononuclear cells in childhood primary IgA nephropathy / 中华实用儿科临床杂志

Objective To investigate the expressions and clinical significance of Toll like receptor (TLR) 3 and TLR4 in peripheral blood mononuclear cells and renal tissues of children with primary IgA nephropathy.Methods A total of 34 children with primary IgA nephropathy were selected as the IgA nephropathy group,who were confirmed by renal biopsy,from the Department of Pediatrics,the First Affiliated Hospital of Xinxiang Medical University,from September 2014 to June 2016.In the same period,7 cases of renal tumor who underwent nephrectomy in Pediatric Surgery became control group A,and 10 cases of healthy children became control group B,and the expressions of TLR3 and TLR4 in renal tissue were detected by adopting immunohistochemistry between IgA nephropathy group and control group A,and the positive expression rate of TLR3 and TLR4 in peripheral blood mononuclear cells were detected by using flow cytometry in IgA nephropathy group.The expression of TLR3 and TLR4 in peripheral blood mononuclear cells of IgA nephropathy group and control group B were observed and compared.Results The expressions of TLR3 and TLR4 in renal tissue of IgA nephropathy group were respectively (68.28 ±6.37)％ and 0.048 ±0.018,which were significantly higher than TLR3 [(9.69 ±11.02)％] and TLR4 (0.003 ±0.001) in the control group A,and the differences were statistically significant (t =50.080,14.374,all P ＜ 0.01).The positive expressions of TLR3 and TLR4 in peripheral blood mononuclear cells of IgA nephropathy group were respectively (17.62 ± 8.33)％ and (23.85 ± 11.82)％,while the expressions were (0.31 ± 0.06) ％ and (3.02 ± 0.09) ％ respectively in the control group B,and the differences were statistically significant (t =12.109,11.612,all P ＜ 0.05).Conclusion The expressions of TLR3 and TLR4 in renal tissue and peripheral blood mononuclear cells of IgA nephropathy are increased,suggesting that the abnormal activation of TLR3 and TLR4 may be involved in the pathogenesis of IgA nephropathy.

Expressions and significance of Toll-like receptor 3 and Toll-like receptor 4 in renal tissues and peripheral blood mononuclear cells in childhood primary IgA nephropathy / 中华实用儿科临床杂志

Objective To investigate the expressions and clinical significance of Toll like receptor (TLR) 3 and TLR4 in peripheral blood mononuclear cells and renal tissues of children with primary IgA nephropathy.Methods A total of 34 children with primary IgA nephropathy were selected as the IgA nephropathy group,who were confirmed by renal biopsy,from the Department of Pediatrics,the First Affiliated Hospital of Xinxiang Medical University,from September 2014 to June 2016.In the same period,7 cases of renal tumor who underwent nephrectomy in Pediatric Surgery became control group A,and 10 cases of healthy children became control group B,and the expressions of TLR3 and TLR4 in renal tissue were detected by adopting immunohistochemistry between IgA nephropathy group and control group A,and the positive expression rate of TLR3 and TLR4 in peripheral blood mononuclear cells were detected by using flow cytometry in IgA nephropathy group.The expression of TLR3 and TLR4 in peripheral blood mononuclear cells of IgA nephropathy group and control group B were observed and compared.Results The expressions of TLR3 and TLR4 in renal tissue of IgA nephropathy group were respectively (68.28 ±6.37)％ and 0.048 ±0.018,which were significantly higher than TLR3 [(9.69 ±11.02)％] and TLR4 (0.003 ±0.001) in the control group A,and the differences were statistically significant (t =50.080,14.374,all P ＜ 0.01).The positive expressions of TLR3 and TLR4 in peripheral blood mononuclear cells of IgA nephropathy group were respectively (17.62 ± 8.33)％ and (23.85 ± 11.82)％,while the expressions were (0.31 ± 0.06) ％ and (3.02 ± 0.09) ％ respectively in the control group B,and the differences were statistically significant (t =12.109,11.612,all P ＜ 0.05).Conclusion The expressions of TLR3 and TLR4 in renal tissue and peripheral blood mononuclear cells of IgA nephropathy are increased,suggesting that the abnormal activation of TLR3 and TLR4 may be involved in the pathogenesis of IgA nephropathy.

Association of TLR3 gene polymorphism with IgG subclass deficiency and the severity in patients with aspirin-intolerant asthma

PURPOSE: Toll-like receptor 3 (TLR3) recognizes to viral double-stranded RNA and is involved in antiviral defenses. A probable role of TLR3 gene variants in the pathogenesis of aspirin-intolerant asthma (AIA) has been suggested. AIA patients present more frequent asthma exacerbations in which respiratory viral infections could be an exacerbating factor. IgG subclass deficiency was commonly present with bronchial asthma. Based on previous findings, we investigated whether TLR3 variants could affect IgG3 subclass deficiency in AIA. METHODS: We enrolled 279 AIA patients, 403 aspirin-tolerant asthma (ATA) patients, and 315 normal healthy controls (NC) in this study. TLR3 polymorphism at the promoter region -299698G>T was genotyped. The serum levels of IgG subclasses were determined by the single radial immunodiffusion method. Expressions of IgG3 and TLR3 on Epstein-Barr virus transformed-B cells isolated from asthmatic patients were evaluated by flow cytometry to investigate B-cell functions. RESULTS: The TLR3 -299698 T allele was significantly associated with severity and IgG3 deficiency in the AIA group (P=0.044 and P=0.010, respectively), but not in the ATA group. IgG3 expression on B cells from asthmatics with IgG3 deficiency was significantly lower compared to those without (P=0.025). There was a positive correlation between IgG3 expression levels on B cells and serum IgG3 levels (r 2=0.434, P=0.002). CONCLUSION: These results suggest that the TLR3 -299698G>T polymorphism may be associated with IgG3 subclass deficiency and severity in AIA.

Toll-like receptor 3 (TLR3) and the development of type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is a chronic, progressive autoimmune disease characterized by metabolic decompensation often leading to dehydration and ketoacidosis. Viral agents seem to play an important role in triggering the autoimmune destruction that leads to the development of T1DM. Among several viral strains investigated so far, the enterovirus family has been consistently associated with the onset of T1DM in humans. One of the mediators of viral damage is the double-stranded RNA (dsRNA) generated during replication and transcription of viral RNA and DNA. The Toll-like receptor 3 (TLR3) gene codes for an endoplasmic receptor of the pattern-recognition receptors (PRRs) family that recognizes dsRNA, plays an important role in the innate immune response triggered by viral infection. Binding of dsRNA to the TLR3 triggers the release of proinflammatory cytokines, such as interferons, which exhibit potent antiviral action; thus, protecting uninfected cells and inducing apoptosis of infected ones. Therefore, the TLR3 gene is a good candidate for the development of T1DM. Within this context, the objective of the present review was to address the role of the TLR3 gene in the development of T1DM. Arch Endocrinol Metab. 2015;59(1):4-12.

mRNA expressions of Toll-like receptor 3, 7 and type Ⅰ interferons in peripheral blood of patients with chronic hepatitis C / 中华临床感染病杂志

Objective To investigate mRNA expressions of Toll-like receptors (TLR3 and TLR7)and type Ⅰ interferons (IFN-α and IFN-β) in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis C (CHC).Methods Thirty patients with CHC and 30 healthy controls were collected from Renmin Hospital of Wuhan University during September 2013 and May 2014.Liver function was detected using enzyme-linked immunosorbent assay (ELISA).mRNA expressions of TLR3,TLR7,IFN-α and IFN-β were detected by real-time quantitative PCR,and their relative expression values were obtained by 2-△△Ctmethod.The t test was performed for the comparison of mean differences between CHC patients and healthy controls,and Pearson analysis was used to analyze the correlations between TLR mRNA and IFN mRNA,liver function,HCV RNA load.Results Taking mRNA expressions in healthy control group as 1,the relative mRNA expressions of TLR3,TLR7,IFN-α and IFN-β in CHC group were 0.086,0.633,0.145 and 0.423 respectively (t =4.25,2.39,2.37 and 2.80,all P ＜ 0.01).Pearson correlation analysis showed that mRNA expressions of TLR3 and TLR7 were positively correlated with that of IFN-β (r =0.381 and 0.487,all P ＜ 0.05),but not correlated with IFN-α mRNA expression,HCV RNA load,ALT and AST (all P ＞ 0.05).Conclusion The mRNA expressions of TLR3,TLR7 and IFN-α,IFN-β decrease in CHC patients,and the mRNA expressions of TLR3 and TLR7 are positively correlated with that of IFN-β,indicating that increasing the expression of TLRs might be a new strategy in CHC treatment.

Células dendríticas plasmocitóides, expressão de receptores "Toll-like" 9 e 3 e de podoplanina nas lesões cutâneas do Sarcoma de Kaposi associado à síndrome de imunodeficiência adquirida e esporádico / Plasmacytoid dendritic cells and the expression of toll-like receptors 9 and 3 and podoplaninin in cutaneous lesions of Aids-associated Kaposi's sarcoma and classic Kaposi's sarcoma

INTRODUÇÃO: O Sarcoma de Kaposi (SK) é a neoplasia mais frequente dos doentes com Aids. É causada pelo herpes-vírus 8 (HHV-8). As células dendríticas plasmocitóides (CDp) são especializadas na produção de interferon tipo 1 e participam da resposta imune aos vírus. Os receptores "toll-like" são os principais receptores de reconhecimento de padrão, sendo que os receptores toll-like (TLR) 3 e 9 têm função no reconhecimento de vírus. O D2-40 é o anticorpo que reconhece a podoplanina, uma proteína transmembrana, presente no endotélio linfático e que tem função na imunidade. OBJETIVO: Demonstrar e comparar os componentes da imunidade inata: CDp e TLR 3 e 9, nas lesões cutâneas de SK associado a Aids e esporádico. Identificar a presença do HHV-8 nas CDp. Verificar o componente endotelial linfático na progressão das lesões de SK e comparar a expressão dos elementos da imunidade inata estudados, nas lesões com menor e maior componente endotelial linfático. MÉTODOS: Estudo retrospectivo de 50 biopsias de pacientes com diagnóstico de SK, todos com comprovação pelo exame histopatológico e demonstração do antígeno nuclear associado à latência (LANA) do HHV-8. Foram avaliados 11 biopsias de SK da forma clássica (SKc), 22 lesões de doentes com Aids (SK-Aids) e de 17 de doentes com Aids submetidos a tratamento com terapia antirretroviral altamente eficaz (SK-Aids/HAART). Os espécimes foram submetidos a exame por técnica imuno-histoquímica para evidenciar a presença de CDp (anticorpo CD303/BDCA-2), a expressão de TLR 3 e 9, bem como de podoplanina (anticorpo D2-40). Foi realizada também técnica de dupla marcação com CD303 e LANA, objetivando a identificação de CDp infectadas pelo HHV-8.Vinte e três espécimes de granuloma piogênico constituíram o grupo controle. A população de CDp e expressão de TLR 3 e TLR 9 também foi comparada nas lesões cutâneas de SK de doentes com e sem comprometimento visceral pela neoplasia; lesões não tumorais (máculo-papulares/placas)...

Introduction: Kaposi's sarcoma (KS) is the most common Aids-associated malignancy. It is caused by human herpesvirus-8. Plasmacytoid dendritic cells (pDC) are professional interferon producing cells, and participate in the immune response against viruses. Toll-like receptors (TLR) are the main pattern recognition receptors, and TLR 3 and TLR 9 participate in the recognition of viruses. Podoplanin, recognized by antibody D2-40, is a transmembrane protein identified on lymphatic endothelial cells with functions inimmunity. Objective: Demonstrate and compare some innate immunity components: pDC, TLR 3 and TLR 9, in cutaneous lesions of Aids-associated Kaposi's sarcoma and classic Kaposi's sarcoma. Identify the infection of pDC by HHV-8. Compare the lymphatic endothelial component in the course of tumor progression and compare the expression of innate immunity elements in lesions with a predominance of lymphatic endothelial components or not. Methods: Retrospective study of 50 biopsies diagnosed as Kaposi's sarcoma withpositive staining for latency-associated nuclear antigen (LANA) of HHV-8. Eleven classic KS, 22 Aids-associated KS and 17 Aids-associated KS from patients undergoing highly active antiretroviral therapy (HAART) were assessed. Paraffinembedded tissue was submitted to immunohistochemistry technique in order to demonstrate pDC (CD303/BDCA-2 antibody), expression of TLR 3, TLR 9 and podoplanin (D2-40 antibody). We performed double staining with CD303 and LANA in order to identify pDC infection with HHV-8. Twenty-three pyogenic granuloma(PG) specimens were analyzed as a control group. Plasmacytoid dendritic cells population, TLR 3 and TLR 9 expressions were compared between patients with and without visceral disease, nodular stageandpatch/plaque stage and according to bloodlymphocytes T CD4 count(< and >=350 cells/mm3)...

Intrathecal injection of dexmedetomidine reduces neuropathic pain in rat model of chronic constriction injury / 中国医师杂志

Objective To investigate the effect of intrathecal injection (IT) of dexmedetomidine targeting Toll-like receptor 3 (TLR3) on neuropathic pain and spinal cord levels of TLR3 mRNA,interleukin-1β(IL-1β),tumor necrosis factor-alpha (TNF-α) proteins in rat model of chronic constriction injury (CCI).Methods Male Sprague-Dawley rats were randomly divided into five groups(n =10):the sham group(intrathecal normal saline,IT NS),CCI group (CCI + IT NS),DEX-pre group (CCI + IT DEX pre 2ds),DEX-post group (CCl + IT DEX post 7ds) and DEX + ATIP group (CCI + IT DEX + ATIP).The lumbar intrathecal catheters were implanted in L5_6 of rats and CCI models were established as previously described.The thermal and mechanical nociceptive thresholds were assessed by paw withdrawal latency(PWL) to radiant heat and yon Frey filaments.The DEX was administered intrathecally for 7 days starting from 2 day before surgery or 7day after surgery.The spinal cord expression of TLR3 mRNA was assessed by real-time polymerase chain reaction (PCR).Levels of IL-1β,TNF-α in spinal cord were detected by enzyme-linked immunosorbent assay (ELISA).Results Compared with the sham group,animals in CCI group had significandy lower mechanical (F =12.73,P ＜ 0.05) and thermal pain thresholds (F =14.65,P ＜ 0.05),higher expression of TLR3 mRNA (F =11.03,P ＜ 0.05) and levels of IL-1 β (F =9.67,P ＜ 0.05),TNF-α(F =8.78,P ＜ 0.05) in the spinal cord (P ＜ 0.05).Rats in DEX-pre group had significantly higher mechanical (F =11.03,P ＜ 0.05) and thermal pain thresholds (F =15.03,P ＜ 0.05) and significantly lower expression of TLR3 mRNA (F =14.65,P ＜0.05) and levels of IL-1β (F =12.51,P＜0.05,TNF-α (F =9.01,P ＜0.05) in the spinal cord compared with those in the CCI group (at any observed time points after ligation,but most significantly at 7 d).And the effects of DEX-pre group were abated by IT ATIP at the same time or 7days after surgery alone (P ＜ 0.05).Conclusions Intrathecal injection of DEX can decrease the levels of inflammatory factors by decreasing the TLR3 mRNA in the spinal cord of rats and prophylactic relieve the neuropathic pain induced by CCI.

Growth inhibition and apoptosis of a multiple myeloma cell line induced by TLR3 pathway activation / 中华微生物学和免疫学杂志

Objective To investigate the roles of TLR3 pathway activiated by polyI:C in proliferation and apoptosis of multiple myeloma (MM) RPMI8226 cell line.Methods RPMI8226 cells were cultured in RPMI 1640 with different dose of polyl:C.Cells were collected in different time.Proliferation and apoptosis were detected by CCK-8 kit and flow cytometry,separately.Results The proliferation of RPM18226 was inhibited by polyI:C,and it was dose and time dependent,24 h:12.30％ ±2.04％,22.50％±2.20％,37.90％ ±1.30％ ; 48 h:17.80％ ±1.52％,29.60％ ±0.85％,45.80％ ±1.68％ ;72 h:25.10％±1.01％,34.60％±1.27％,60.50％±2.08％,P＜0.05.RPMI8226 cells were incubated with 50 μg/ml,100 μg/ml and 200 μg/ml polyI:C for 48 h.Apoptotic rate were 5.60％ ±1.06％,8.71％ ±1.06％ and 13.93％ ±1.17％,P＜0.05.TLR3 and TRIF mRNA expression increased obviously and dose dependent,TLR3:1.41±0.10,2.24±0.16,4.08±0.13; TRIF:1.07±0.16,1.97±0.13,3.56±0.19,P＜0.05.Conclusion The proliferation of MM cells were inhibited by TLR3 pathway obviously,and apoptosis was induced by polyI:C.

Effects of Iodine excess,polyinosinic-polycytidylic acid and thyroglobulin induced thyroiditis in mice on Toll-like receptor 3 expression / 中国地方病学杂志

Objective To observe the effect of iodine excess(HI),polyinosinic-polycytidylic acid[Poly(I:C),Poly]and thyroglobulin(TG)on the thyroid of mice by the expression of Toll-like receptor 3(TLR3)to reveal the functional role of TLR3 in autoimmune thyroiditis.Methods Forty-two non-obese diabetic mice,body weight (20±3)g,were divided into six groups:control group,HI group,Poly group,TG group,HI+TG group,HI+Poly group. Fed with deionized water and injected intraperitoneally with physiological saline 0.1 ml each day for a week, the mice in control group were injected with physiological saline every other day at the same dose for 1 week before they were sacrificed; HI group drank 0.05% NaI water and were injected intraperitoneally with physiological saline same as control group; Poly group drank deionized water and were injected intraperitoneally with poly 0.1 ml (1 g/L)each day of the week, then the mice were injected with Poly every other day at the same dose for 1 week before they were sacrificed; TG group drank deionized water and were injected intraperitoneally with physiological saline same as control group, immunized with 0.1 mg TG by subcutaneously injecting and the immunization was enhanced after they were fed half dose for 4 and 8 weeks separately. In HI + Poly group, the treatment was the same as HI group and Poly group; HI + TG group: the treatment was the same as HI group and TG group. Eight weeks later, mice were sacrificed and thyroids were taken to make frozen sections, Hematoxylin-Eosin (HE) staining was employed to observe the morphological change of the thyroids. The expression of TLR3 of thyroids was observed under fluorescence microscope after Immumofluorescence using TLR3 antibody and TR3-positive cells were analyzed in the thyroid density. Results HE staining showed thyroids of Poly group had no inflammation under microscope.There were different degrees of inflammatory cell infiltration in HI group and TG group. The inflammatory cell infiltration and the damage of follicular thyroid of HI + TG group and HI + Poly group were serious, and the degrees of inflammation were higher over "++". Thyroid follicular epithelial cell with TLR3 expression could be seen in Poly group and HI group, meanwhile, there were TLR3 strong positive inflammatory cells in HI group under fluorescent microscope. Using stereological analysis of TLR3-positive cell density in the thyroid, the difference between groups was statistically significant(F=7.870, P<0.01 ). TLR3-positive cell density in the thyroid of HI + Poly group was higher[ (9.287 ± 0.522)mm2] than control group[ (0.062 ± 0.025)mm2, P < 0.01] significantly, meanwhile, the density in HI + Poly group was higher than HI group [ (2.574 ± 0.257 )mm2] and Poly group[ (1.361 ± 0.148 )mm2, all P < 0.01]. The density in HI + TG group[ (4.843±0.405)mm2] was higher than HI group and TG group[(1.601 ±0.268)mm2, all P < 0.01 )]. Conclusions Excessive iodine and thyroglobulin can induce thyroiditis, and stimulate the expression of TLR3 in the thyroid follicular epithelial, Poly aggravated thyroiditis induced by iodine excess in NOD mice; TLR3 positive inflammatory cells also appeared in inflammatory region, suggesting that TLR3 is involved in the pathogenesis of autoimmune thyroiditis

Expression of Toll-like receptors 3 (TLR3) on peripheral blood mononuclear cells and serum IFN-γ,TNF-α levels in children with acute rotavirus diarrhea / 中国小儿急救医学

Objective To explore the relationship between TLR3 mRNA expression on peripheral blood mononuclear cells(PBMCs)and acute rotavirus(RV)diarrhea.Methods Sixty-one children with acute RV diarrhea served as study subject,the expression of TLR3 mRNA on PBMCs was detected by real-time fluorescence quantitative RT-PCR.the concentrations of IFN-γand TNF-α in serum were measured by the method of Enzyrme-linked immunosorbent assay(EUSA).Results The expression of TLR3 on PBMCs and the serum levels of IFN-γ and TNF-α in the serious diarrhea group were 0. 820±0.051,(33.67±12.88)Pg/ml, (62.21±14.65)pg/ml,respectively,while it were 0.717±0.040,(24.01±10.06)pg/ml,(50.99± 12.18)pg/ml in the slight diarrhea group,and 0.525±0.029,(12.52±5.19)pg/ml,(28.65±7.44)pg/ml in the control group.Compared with the control group.the expression of TLR3 on PBMCs and the serum levels of IFN-γ,TNF-α in the serious and slight diarrhea group were significantly higher(P<0.01).There were significant differences between the serious and slight diarrhea group(P<0.01).There were positive relationship between the expression of TLR3 on PBMCs and tHe serum IFN-γ,TNF-α levels(r=0.431,P< 0.05,r=0.372,P<0.05).Conclusion The expression of TLR3 on PBMCs in children with acute rotavirus dialThea iS up-regulated,TLR3 and its mediated immune response are associated with the development of acute rotavirus diarrhea.

Engagement of Toll-Like Receptor 3 Induces Vascular Endothelial Growth Factor and Interleukin-8 in Human Rheumatoid Synovial Fibroblasts

BACKGROUND/AIMS: Angiogenesis, which is a critical step in the initiation and progression of rheumatoid arthritis (RA), involves pro-angiogenic factors, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). We investigated the role of Toll-like receptor 3 (TLR3) in the regulation of pro-angiogenic factors in RA fibroblast-like synoviocytes (FLS). METHODS: FLS were isolated from RA synovial tissues and stimulated with the TLR3 ligand, poly (I:C). The levels of VEGF and IL-8 in the culture supernatants were measured using enzyme-linked immunosorbent assays, and the mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction. The expression patterns of VEGF and IL-8 in the RA synovium and osteoarthritis (OA) synovium were compared using immunohistochemistry. RESULTS: The expression levels of TLR3, VEGF, and IL-8 were significantly higher in the RA synovium than in the OA synovium. VEGF and IL-8 production were increased in the culture supernatants of RA FLS stimulated with poly (I:C), and the genes for these proteins were up-regulated at the transcriptional level after poly (I:C) treatment. Treatment with inhibitors of nuclear factor-kappaB (NF-kappaB), i.e., pyrrolidine dithiocarbamate and parthenolide, abrogated the stimulatory effect of poly (I:C) on the production of VEGF and IL-8 in RA FLS. CONCLUSIONS: Our results suggest that the activation of TLR3 in RA FLS promotes the production of proangiogenic factors, in a process that is mediated by the NF-kappaB signaling pathway. Therefore, targeting the TLR3 pathway may be a promising approach to preventing pathologic angiogenesis in RA.

Correlation of toll-like receptor 3 and tumor necrosis factor-α with idiopathic fetal growth restriction / 中华妇产科杂志

Objective To investigate the expression and the significance of toll-like receptor 3 (TLR-3)in placenta,tumor necrosis factor-α(TNF-α)in maternal and cord blood of idiopathic fetal growth restriction(IFGR),and their correlation with the pathogenesis of symmetric and asymmetric IFGR.Methods From April 2008 to April 2009,42 primiparae of singleton pregnancy and their IFGR babies,who delivered at term through cesarean section, in the Third Affiliated Hospital of Zhengzhou University were enrolled. All subjectects were divided into symmetric IFGR group (n=20) and asymmetric IFGR group (n =22). Another 42 non-IFGR pairs were randomly selected as the control group. The polink-2 plus polymerized horseradish peroxidase (HRP) immunohistochemical method and the enzyme linked immunosorbent assay (ELISA) were applied to detect TLR-3 and TNF-α levels. Results (1) The expression of TLR-3 protein were observed in all maternal placenta of the three groups. TLR-3 essentially expressed in syncytiotrophoblasts and hofbouer cells in the symmetric IFGR and control group, but expressed mostly in hofbouer cells and less in syneytiotrophoblasts in the asymmetric IFGR group. (2) The expression of TLR-3 in the syncytiotrophoblasts of the symmetric and asymmetric IFGR group was significantly lower than in the control group (111±14 and 118±11 vs. 156 ± 9, P<0. 01). The number of TLR-3 positive in Hofbourer cell in the symmetric IFGR group was lower than the control group (8. 9±2. 8 vs 17.5±2. 8, P <0. 01 ), but the number in the asymmetric IFGR group was higher (23.8±3.7) compared with the control group (P <0. 01). (3) The TNF-α levels in the maternal and cord blood of the symmetric and the asymmetric group were higher than that of the control group [maternal : (90±10) μg/L and ( 86±11 ) μg/L vs. (73±9) μg/L;cord blood: (92±12) μg/L and (96±8) μg/L vs. (79±9) μg/L;P<0.01]. (4) Neither symmetric nor the asymmetric IFGR group showed any correlations between the maternal and cord blood levels of TNF-α (P>0. 05). (5) Significant correlation was found between the TNF-α level of the cord blood and TLR-3 expression in the placenta in both the symmetric and asymmetric IFGR group(P<0. 05),but no relationship was found between the maternal blood TNF-α level and TLR-3 expression in the placenta (P>0. 05). Conclusions The variantions of TLR-3 expression in placenta and the increased expression of TNF-α in cord blood are associated with the genesis IFGR. The reduced expression of TLR-3 may related to symmetric IFGR, while the increased TLR-3 level in hofbouer cells may lead to asymmetric IFGR.