Celiac vagus nerve mediates expression of the acetylcholine receptor α7nAChR on monocytes in the spleen.

The spleen is required for the vagal cholinergic anti-inflammatory activity to maintain systemic immune homeostasis, but the underlying mechanism of this function is not fully understood yet. We hypothesized that vagus nerve mediates alpha 7 nicotinic acetylcholine receptor (α7nAChR) expression in monocytes, an essential regulator of cholinergic anti-inflammatory activity, and the spleen is essential site for this process. To verify this hypothesis, mice were subjected to splenectomy or celiac vagotomy. The level of α7nAChR expression in circulating monocytes was analyzed by real-time PCR. Impact of α7nAChR agonist PNU282987 on LPS-evoked release of TNF-α and IL-1ß from circulating monocytes was assessed by ELISA. The effect of norepinephrine (NE), acetylcholine (ACh) and neuregulin-1 (NRG-1) on α7nAChR expression was detected by real-time PCR. We found that splenectomy or celiac vagotomy abrogated α7nAChR expression in circulating monocytes. LPS-induced release of TNF-α and IL-1ß from these monocytes was not alleviated significantly by PNU282987 as compared with that of sham mice. NE and ACh addition fails to stimulate α7nAChR expression, but, NRG-1 treatment can significantly induce α7nAChR expression in these monocytes compared with untreated cells in vitro. Overall, our results reveal that celiac vagus nerve mediates α7nAChR expression in monocytes, and the spleen is indispensable site for this process.

Novel Insight into the Mechanism of Metabolic Surgery Causing the Diversity in Glycemic Status in Type 2 Diabetes.

Metabolic surgery results in diverse glycemic status in patients with type 2 diabetes (T2D), including hyperglycemia without remission, significant amelioration of hyperglycemia with partial remission, complete restoration of euglycemia, or with prolonged remission, hyperglycemia recurrence in relapses after remission, or post-bariatric hypoglycemia. Unfortunately, it is not known how metabolic surgery leads to this diverse consequence. Here, we discuss the diversity of glycemic status associated with metabolic surgery and the potential mechanisms of T2D remission. We also highlight the relationship between the change in low-grade inflammation and T2D remission after metabolic surgery. We hypothesize that the level of inflammatory and anti-inflammatory cytokines controls the efficacy of metabolic surgery in patients with T2D. This hypothesis may provide further insight into the mechanism of the beneficial effects of metabolic surgery patients with T2D.

Mechanism of protection from insulin resistance by toll-like receptor 2 deficiency in high-fat diet fed mice: involvement in macrophage polarization.

BACKGROUND: Toll-like receptor 2 (TLR2) deficiency can increase insulin sensitivity and improves glucose tolerance. However, it is not yet fully understood about its underlying mechanism. The regulation of M1/M2 macrophage polarization has been verified to involve in insulin resistance. Here, we evaluated whether the beneficial effect of TLR2 deficiency is mediated by TLR2-associated macrophage polarization in mice fed with high-fat diet (HFD). METHODS AND RESULTS: Wild-type and TLR2 knockout (TLR2-/-) mice received HFD for two months. Following intraperitoneal glucose tolerance and insulin resistance tests, peripheral monocytes were isolated, and in vitro induced for differentiation into M1 and M2 macrophages, respectively. Macrophages polarization was evaluated using flow cytometry. The expression of macrophage polarization marker genes and cytokine production in visceral adipose tissue were measured by qRT-PCR and ELISA. Compared to wild-type mice, TLR2-/- mice showed higher glucose tolerance and insulin sensitivity, along with significantly reduced the population of M1 and increased M2 count in vitro. Additionally, TLR2-/- mice demonstrated higher expression of M2 marker iNOS mRNA and interleukin-10 level in adipose tissues. CONCLUSIONS: Our results reveal that TLR2 knockout-mediated macrophages M2 polarization is a crucial factor for preventing against diet-induced insulin resistance in mice. These findings deepen our knowledge about the mechanism underlying HFD-induced insulin resistance.

"Dual-functional" strontium titanate nanotubes designed based on fusion peptides simultaneously enhancing anti-infection and osseointegration.

Currently, there is an increasing clinical demand for implants that effectively resist bacterial infections while promoting osseointegration. In this study, the fusion peptide technology was used to linearly fuse the antimicrobial peptide (AMP, HHC36) and the bone-promoting peptide (RGD), so that the titanium (Ti)-based implant modified by the polypeptide had the dual function of "antibacterial-promoting bone". Firstly, self-organized vertically-oriented strontium-doped titanium dioxide nanotubes (STN) were manufactured by anodizing and hydrothermal synthesis methods. Secondly, the fusion peptide (HHC36-RGD) was loaded into the tubular structure by a simple vacuum-assisted physical adsorption method. Finally, STN loaded with HHC36-RGD (H-R-STN) was obtained. The characterization results demonstrated that the surface of the H-R-STN had a roughness and hydrophilicity that promoted cell adhesion. Additionally, electrochemical tests showed that H-R-STN coating can reduce the corrosion rate of pure Ti. The fusion peptide and Sr2+ in H-R-STN were released in the initial fast and subsequent slow kinetic model. Expected, H-R-STN can kill more than 99% of clinically common pathogenic bacteria (Staphylococcus aureus and Escherichia coli), and significantly inhibit the formation of bacterial biofilms. Simultaneously, under the synergistic effect of RGD in the fusion peptide and strontium in STN, H-R-STN markedly promoted the adhesion and proliferation of mouse osteoblasts, and significantly promoted osteogenic markers (alkaline phosphatase, runt-related transcription, collagen, mineralization) expression. In summary, the bifunctional titanium-based implant constructed by H-R-STN in this article can effectively prevent bacterial infections and promote early osseointegration. The main advantage of the titanium surface treatment method of the study was that its simplicity, low cost, especially its versatility made it a promising anti-infective bone repair material.

Generation of Heteroaryl-Substituted Sulfonyl Compounds from Sulfur Dioxide via Remote Heteroaryl ipso-Migration.

The generation of heteroaryl-substituted sulfonyl compounds via a catalyst-, base-, and additive-free three-component reaction of heteroaryl-substituted tertiary alcohols, aryldiazonium tetrafluoroborates, and DABCO·(SO2)2 under mild conditions is developed. Various functional groups are tolerated well in this transformation, and a broad substrate scope is demonstrated. A preliminary mechanistic investigation shows that this reaction undergoes a radical process, including the insertion of sulfur dioxide, sulfonyl radical addition to unactivated alkene, and remote heteroaryl ipso-migration.

Mg/Cu-doped TiO2 nanotube array: A novel dual-function system with self-antibacterial activity and excellent cell compatibility.

Many studies were conducted to change the surface morphology and chemical composition of Ti implants for the improvement of antibacterial ability and osseointegration between medical Ti and surrounding bone tissue. In this study, we successfully prepared a novel dual-function coating on pure Ti surface, i.e. Cu and Mg-co-doped TiO2 nanotube (TN) coating, by combining anodisation and hydrothermal treatment (HT), which could act as a delivery platform for the sustained release of Cu and Mg ions. Results showed that the amounts of Cu and Mg were about 5.43 wt%-6.55 wt% and 0.69 wt%-0.73 wt%, respectively. In addition, the surface morphology of Cu and Mg-co-doped TN (CuMTN) coatings transformed into nanoneedles after HT for 1 h. Compared with TN, CuMTN had no change in roughness and remarkable improved hydrophilicity. Antibacterial tests revealed that CuMTN had an antibacterial rate of more than 93% against Escherichia coli and Staphylococcus aureus, thereby showing excellent antibacterial properties. In addition, CuMTN could induce the formation of apatite well after being immersed in simulated body fluid, showing good biological activity. Preosteoblasts (MC3T3-E1) cultured on CuMTN-coated Ti demonstrated better proliferation and osteogenic differentiation than pristine and as-anodised specimens. To the best of our best knowledge, this study had successfully attempted to combine anodisation and HT, introduce Cu/Mg elements and functionalise Ti-based implant surfaces with enhanced hydrophilicity, osteogenesis and antimicrobial properties that can meet clinical needs for the first time.

[Knockout of TLR2 gene attenuates insulin resistance and promotes M2 polarization of macrophages in mice].

Objective To study the effect of deletion of Toll-like receptor 2 (TLR2) gene on insulin resistance and polarization of macrophages in mice. Methods The wild-type (WT) and TLR2 knockout (TLR2-/-) C57BL/6 male mice, aged 28 days, were selected, with 12 mice in each group. The genotype of each mouse was identified by PCR. After mice were fed with basic diet for 3 months, the glucose tolerance test (GTT) and insulin resistance test (ITT) were performed. The mononuclear cells isolated from peripheral blood were stimulated with GM-CSF/IFN-γ and M-CSF/IL-4/IL-13, respectively, to induce differentiation to M1-like and M2-like macrophages. The CD11b, F4/80, CD11c, CD206 and early growth response 2 (EGR2) were detected by flow cytometry to determine the phenotype of macrophages. The levels of TNF-α, IL-6 and IL-10 in the culture supernatant of macrophages were detected using ELISA. Results The result of PCR identification was consistent with the genotype of mice. Compared to WT mice, TLR2-/- mice exhibited the significantly improved glycemic control at 30 min during GTT and the significantly increased insulin sensitivity at 15 minutes during ITT. The flow cytometry showed that M1 markers decreased and M2 macrophages increased in the TLR2-/- mice. ELISA showed that the levels of IL-6 and TNF-α significantly decreased in the culture supernatant of M1 macrophages, while the level of IL-10 significantly increased in the culture supernatant of M2 macrophages in TLR2-/- mice compared with WT mice. Conclusion TLR2 signal has an effect on the polarization of macrophages and makes macrophages tend to switch to M1 phenotype. A higher number of pro-inflammatory factors secreted by M1 macrophages contribute to a low-grade inflammation state in the body, which leads to a decrease in glucose tolerance and insulin sensitivity.

High-glucose administration induces glucose intolerance in mice: a critical role of toll-like receptor 4.

Glucose converted from a diet has been considered a high-risk factor of type 2 diabetes mellitus (T2DM). However, it is not clear how it increases the risk of T2DM. Here, we investigated the effect of high-glucose administration on glucose tolerence in wild-type and toll-like receptor 4 (TLR4) knockout mice. Mice were intragastrically administered with high-glucose. The level of fasting blood glucose, insulin and intraperitoneal glucose tolerance were measured, and insulinogenic index and HOMA-IR were calculated at 1 week. To understand mechanism of glucose action, we also assessed blood glucose, glucagon-like peptide-1 and inflammatory cytokines levels at different time windows following high-glucose load. Our results show that 20 g/kg glucose load leads to glucose tolerance impairment and insulin resistance in wild-type mice. Following 20 g/kg glucose load, the levels of plasma interlukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) increased significantly in wild-type mice, but not in TLR4 knockout mice. Moreover, 20 g/kg glucose load also impaired glucose-induced GLP-1 secretion in wild-type and TLR4 knockout mice. Our results indicate that high-glucose load leads to glucose intolerance with insulin resistance through impairment of GLP-1 secretion, increase of blood glucose levels via activating TLR4 and increasing levels of IL-6 and TNF-α in mice.

Toll-like receptor 4 is necessary for glucose-dependent glucagon-like peptide-1 secretion in male mice.

Lipopolysaccharide (LPS), a natural toll-like receptor 4 (TLR4) ligand, can induce the secretion of glucagon-like peptide-1 (GLP-1) in both animal models and humans consistent with the notion that TLR4 may influence physiological process of GLP-1 secretion. Here, we explored the possible role of TLR4 in the process of glucose-dependent GLP-1 secretion. Wild-type and TLR4 knockout mice were used to observe GLP-1 expression and secretion, as well as the cytokines tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) levels in plasma and ileum after glucose load. TLR4 deficient mice also were given an injection of TNF-α and IL-6, respectively, to confirm the key role of TLR4-mediated inflammatory cytokines in the process of glucose-induced GLP-1 secretion. We found that the TLR4 deficiency impaired the glucose-induced GLP-1 release and prevented an increase in IL-6 and TNF-α levels in plasma and ileum following glucose stimulation. Importantly, injection of TLR4 deficient mice with either TNF-α and IL-6 partly restored the glucose-induced secretion of GLP-1. In conclusion, the production of pro-inflammatory cytokines downstream of TLR4 promotes glucose-dependent GLP-1 secretion from intestinal L-cells in mice.

Mining the potential therapeutic targets for coronary artery disease by bioinformatics analysis.

The present study aimed to mine therapeutic molecular targets that play an important part in the progression of coronary artery disease (CAD). The gene expression profile GSE28829 dataset and the microRNA (miRNA) expression profile GSE59421 dataset were downloaded from the Gene Expression Omnibus (GEO) database. The GEO2R online analytical tool was used to identify differentially expressed genes (DEGs) and miRNAs (DEMs). The target genes of DEMs were identified using the miRWalk2.0 web­based tool and 2 miRNA­gene regulatory networks were constructed using Cytoscape software. Subsequently, enriched Gene Ontology (GO) terms of miRNA­target DEGs were obtained using the Database for Visualization, Annotation and Integrated Analysis, and locations of these genes in the chromosomes were determined by Map Viewer. In the present study, 350 DEGs and 66 DEMs were screened. A total of 3,588 target genes were identified from the DEMs, and 57 of these target genes and established DEGs were identified to overlap. GO terms associated with 5 processes, and 4 types of composition were identified to be enriched in the miRNA­target DEGs. Furthermore, 26 miRNA­gene regulatory pairs were obtained between the 57 target genes and DEMs. The 26 miRNA­target DEGs were unevenly distributed, and no genes were located on the sex chromosomes. As a result of the present study, potential therapeutic targets for CAD were identified through bioinformatics analysis.

Nanostructured Ag+-substituted fluorhydroxyapatite-TiO2 coatings for enhanced bactericidal effects and osteoinductivity of Ti for biomedical applications.

BACKGROUND: Poor mechanical properties, undesirable fast dissolution rate, and lack of antibacterial activity limit the application of hydroxyapatite (HA) as an implant coating material. To overcome these limitations, a hybrid coating of Ag+-substituted fluorhydroxyapatite and titania nanotube (TNT) was prepared. METHODS: The incorporation of silver into the HA-TiO2 hybrid coating improves its antimicrobial properties. The addition of F as a second binary element increases the structural stability of the coating. The TNT/F-and-Ag-substituted HA (FAgHA) bilayer coating on the Ti substrate was confirmed by X-ray diffraction, scanning electron microscope, energy-dispersive X-ray spectroscopy (EDS), and X-ray photoelectron spectroscopy (XPS). RESULTS: The results indicate that the FAgHA/TNT nanocomposite coating has a dense and uniform morphology with a nano-rod-like structure. The solubility measurement result shows that the substitution of F- ions into the AgHA structure has a positive effect on the dissolution resistance of HA. The adhesion strength of FAgHA/TNT has significantly increased because of the interlocking of the roughened surface with nano-rod-like particles that entered into the voids of the TiO2 nanotubes. Compared with that of the bare Ti, the corrosion current density of FAgHA/TNT-coated Ti substrate decreased from 3.71 to 0.18 µA, and its corrosion resistance increased by almost two orders of magnitude. Moreover, despite pure HA, the FAgHA killed all viable Staphylococcus aureus after 24 hours of incubation. Although the fabricated FAgHA/TNT coating is hydrophobic, it induced deposition of the typical spherical apatite when immersed in a simulated body fluid (SBF); the osteoblasts spread very well on the surface of the coating. In addition, in vitro cell culture tests demonstrated cell viability and alkaline phosphatase (ALP) similar to pure HA, which indicated good cytocompatibility. Interestingly, compared with bare Ti, FAgHA/TNT-coated Ti surface was innocent for cell vitality and even more beneficial for cell osteogenesis in vitro. CONCLUSION: Enhancing the osseointegration and preventing infection in implants, the FAgHA/TNT-coated Ti makes implants more successful.

Copper-catalyzed, stereoconvergent, cis-diastereoselective borylative cyclization of ω-mesylate-α,ß-unsaturated esters and ketones.

The Cu(i)-catalyzed stereoconvergent borylative cyclization of ω-mesylate-α,ß-unsaturated compounds is facilitated by a simple Cu-bisphosphine catalyst. This reaction provides a novel route to cis-ß-boron-substituted five- and six-membered carbocycle and heterocycle esters. Mechanistic studies indicate that stereoconvergence and cis-substitution likely stem from the rapid enolation of the borylcopper adduct with the substrate double bond and the formation of a five-membered intermediate, respectively.

[Effect of FABP2 gene G54A polymorphism on lipid and glucose metabolism in simple obesity children].

OBJECTIVE: To explore the relationship between intestinal fatty acid binding protein (FABP2) gene G54A polymorphism and simple childhood obesity, the effect of mutant 54A FABP2 gene on serum lipids and glucose metabolism. METHODS: The total of 83 subjects with overweight/obesity and 100 subjects with healthy/normal weight were involved in this study. The G54A FABP2 gene allele and genotype frequencies between control group and overweight/obesity group were detected using polymerase chain reaction (PCR) -restriction fragment length polymorphism (RFLP) technology, and DNA sequences were confirmed by DNA sequencing. The automatic biochemical analyzer was used to detect fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) levels. Plasma insulin (Ins) was detected by radiation immune method, free fatty acids (FFA) was tested by ELISA method, insulin resistance index ( HOMA-IR ) was also calculated. The correlation between FABP2 G54A polymorphism and the development of children' obesity was analyzed. The relation between FABP2 G54A polymorphism and abnormal blood lipid and insulin resistance was assessed. RESULTS: The results of study on FABP2 gene polymorphism revealed as followed. In overweight/obese groups, the frequencies of GG, GA, AA genotypes was 33.7%, 49.4% and 16.9%, respectively. In control group, the frequencies of GG, GA, AA genotypes was 51. 0% , 40. 0% and 9. 0% , respectively. The differences between two groups was statistically significant (Χ2 = 6.27, P < 0.05). In overweight/obesity group, the frequencies of alleles were 58.4% for 54G and 41.6% for 54A. In control group, the frequencies of alleles were 71.0% for 54G and 29.0% for 54A. There was significant differences (Χ2 = 6.32, P < 0.05). The plasma biochemical variables results showed that compared with the normal control group, plasma TG (P < 0.01), Ins (P < 0.05), HOMA-IR (P < 0.05) were elevated in overweight/obesity group, the difference between two groups was statistically significant. At the same time, in overweight/obesity group, the carriers of AA homozygous genotypes had significantly higher plasma TG levels than those with GG wild genotypes (P < 0.05). A increased tendency of plasma Ins, FFA levels and HOMA-IR was found in the carriers with AA homozygous genotypes, but no differences compared with those with GG wild genotypes. Compared with those with GG wild genotypes, related plasma biochemical variables in the carriers with GA heterozygous genotypes had no differences (P > 0.05). CONCLUSION: The FABP2 gene G54A polymorphism is related to simple children obesity and lipid metabolism abnormality. The allele encoding in FABP2 gene may be a potential factor contributing to promoting lipid metabolism abnormality of and insulin resistance.

[Identification and bioinformatics analysis of differentially expressed microRNAs in mice liver tissues with a high fat diet-induced insulin resistance].

OBJECTIVE: To identify the expression levels of miR-1897-3p, miR-690 and miR-7a-5p in mice liver tissues with a high fat diet-induced insulin resistance using a stem-loop reverse transcriptional real-time fluorescence quantitative PCR( RT-q PCR)method, and predict microRNAs( miRNAs)-regulated target genes and their functions to investigate the relationship between insulin resistance and differentially expressed miRNAs. METHODS: The total of 30 liver tissue samples were obtained from 15 normal control mice and 15 test mice with a high fat diet-induced insulin resistance. The stemloop reverse transcriptional primers and the primers of real-time PCR were designed to establish a RT-q PCR method, and then the expression levels of miR-1897-3p, miR-690 and miR-7a-5p of 30 liver tissue samples were detected and analyzed. Using bioinformatics methods, the target genes of differentially expressed miRNAs were predicted, and then enriched gene ontology( GO), related signal pathways and target gene protein-target gene protein interactions were analyzed, respectively. RESULTS: Compared with control group, the expression levels of miR-1897-3p, miR-690 and miR-7a-5p detected by RT-q PCR in the group with a high-fat diet-induced insulin resistance were significantly decreased( P < 0. 05), which exhibited the similar pattern of down regulation to the previous microarray results. Bioinformatics analysis results showed that a total of 16 target genes were regulated by two differentially expressed miRNAs. Among of the 16 target gene protein, 8 proteins had interactions with ≥4 proteins. Rac1, Rhoa, Prkcz, Tgfbr2, Itch and Ube2d3 protein were located in the central node of the network, and they were associated or cross-linking with insulin signaling pathway. CONCLUSION: miR-1897-3p, miR-690 and miR-7a-5p in liver tissues may be involved in the physiopathologic process of insulin resistance, which may be affect the normal insulin signaling pathway cascade by regulating the expression of target genes.

[Roles of Toll-like receptor 2 (TLR2) and TLR4 in immune responses to Chlamydia trachomatis genital tract infection in mice].

OBJECTIVE: To evaluate the roles of Toll-like receptor 2 (TLR2) and TLR4 in immune responses to Chlamydia trachomatis (Ct) genital tract infection in mice. METHODS: The wild type (WT, n=11), TLR2(-/-) (n=14) and TLR4(-/-) (n=11) mice were inoculated intravaginally with 1×10(4) inclusion forming units (IFUs) of live C.muridarum (strain MoPn/Nigg) to establish the models of Ct genital tract infection. The vaginal swabs were taken at the 3rd, 7th, 10th, 17th, 24th, 31st, 38th, 45th day after the infection. Immunofluorescence assay was used to quantify live organisms from each swab. The inflammatory cytokines interleukin-1α (IL-1α), IL-6 and macrophage inflammatory protein 2 (MIP-2) were measured using ELISA. The 70th day post infection, the titers of mouse serum Ct-specific antibody isotypes were determined using an immunofluorescence technique; after the macrophages harvested from peritoneal cavity were infected with MoPn strain for 24 hours, the culture supernatants were examined for the contents of cytokines IL-1α, IL-6 and MIP-2 using ELISA. The harvested splenocytes were stimulated with UV-inactivated chlamydial antigens for 72 hours, and the culture supernatants were measured for the levels of cytokines interferon γ (IFN-γ), IL-17, IL-4 and IL-5 using ELISA. RESULTS: No significant difference was observed in the number of IFUs from the vaginal swabs at any time points post infection among WT, TLR2(-/-) and TLR4(-/-) mice. All mice displayed similar time course of live organism shedding. Until the 38th day, all mice cleared live organism. Macrophages lacking TLR2 produced significantly decreased amounts of IL-1α, IL-6 and MIP-2 compared with those in WT mice; however, there was no significant difference between WT and TLR4(-/-) mice. The vaginal swab samples from TLR2(-/-) mice also had lower levels of inflammatory cytokines. Splenocytes from the three groups of mice all produced high levels of both IFN-γ and IL-17, and low levels of both IL-4 and IL-5, and no significant difference was found among the three groups. The ratio of serum IgG2a/IgG1 in the three groups of mice was greater than 1, and no significant difference was found among the three groups. CONCLUSION: TLR2, rather than TLR4 mediates early immune response following Ct genital tract infection in mice. However, neither TLR4 nor TLR2 is required for adaptive immune responses induced by Ct genital tract infection.

[Effects of high-fat diet on FABP2 mRNA expression in small intestinal tissue of young rats].

OBJECTIVE: To measured FABP2 mRNA expression of small intestinal rats in response to high-fat diet; and to explore the correlation between expression and child obesity in rats. METHODS: The young obese rat models high-fat diet-induced method. Then the expression levels of FABP2 mRNA jejunum and ileum tissues of young rats were detected and analyzed using real time fluorescence quantitative PCR method. The serum biochemical assessed with an automatic biochemical Analyzer. RESULTS: In both rnd obesity group, the expression levels of FABP2 mRNA were highest in ermediate in the jejunum, and lowest in the duodenum both the control obesity group, there' were significant differences among them ( P <0. 05). Compated with control group, the expression levels of FABP2 mRNA in obesity group were significantly decreased (P <0.05); and the levels of blood glucose and serum TG in the obesity group were significantly higher (P < 0.05 ). CONCLUSION: FABP2 mRNA expression was different in diverse segments of intestinal tissue. Feeding high-fat diets allowed rats to reduce the expression levels of FABP2 mRNA in small intestinal tissue, meanwhile, the rats gained weight and elevate serum TG levels. It suggests that FABP2 involves in lipid metabolism, and is associated with the development of childhood obesity.

Signaling via tumor necrosis factor receptor 1 but not Toll-like receptor 2 contributes significantly to hydrosalpinx development following Chlamydia muridarum infection.

Chlamydial infection in the lower genital tract can lead to hydrosalpinx, which is accompanied by activation of both pattern recognition receptor TLR2- and inflammatory cytokine receptor TNFR1-mediated signaling pathways. In the current study, we compared the relative contributions of these two receptors to chlamydial induction of hydrosalpinx in mice. We found that mice with or without deficiencies in TLR2 or TNFR1 displayed similar time courses of live organism shedding from vaginal swabs, suggesting that these receptor-mediated signaling pathways are not required for controlling chlamydial lower genital infection. However, mice deficient in TNFR1 but not TLR2 developed significantly reduced hydrosalpinx. The decreased pathogenicity correlated with a significant reduction in interleukin-17 by in vitro-restimulated splenocytes of TNFR1-deficient mice. Although TLR2-deficient mice developed hydrosalpinx as severe as that of wild-type mice, peritoneal macrophages from mice deficient in TLR2 but not TNFR1 produced significantly reduced cytokines upon chlamydial stimulation, suggesting that reduced macrophage responses to chlamydial infection do not always lead to a reduction in hydrosalpinx. Thus, we have demonstrated that the signaling pathways triggered by the cytokine receptor TNFR1 play a more significant role in chlamydial induction of hydrosalpinx than those mediated by the pattern recognition receptor TLR2, which has laid a foundation for further revealing the chlamydial pathogenic mechanisms.

Chlamydia trachomatis secretion of hypothetical protein CT622 into host cell cytoplasm via a secretion pathway that can be inhibited by the type III secretion system inhibitor compound 1.

Using antibodies raised with C. trachomatis fusion proteins, we localized a hypothetical protein encoded by the ORF ct622 in the cytoplasm of C. trachomatis-infected mammalian cells. The detection was specific since the antibody labelling of CT622 protein was removed by preabsorption with CT622 but not other fusion proteins. We similarly confirmed that CT621, a known secretion protein encoded by a hypothetical ORF downstream of ct622, was secreted into host cell cytosol. Proteins CT622 and CT621 displayed a similar secretion pattern, with both intra-inclusion and host cell cytosol localization, that was distinct from that of CPAF (chlamydial protease/proteasome-like activity factor). However, the expression and secretion kinetics differed significantly between CT622 and CT621: CT622 mRNA was detected at 2 h, protein at 6 h and secretion of protein into host cell cytoplasm at 36 h post-infection, while CT621 mRNA was detected at 8 h, protein at 16 h and secretion at 24 h. The secretion of both CT622 and CT621 was blocked by N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide (compound 1), an inhibitor known to target the type III secretion system of bacteria. These results suggest that CT621 and CT622 may fulfil different functions during chlamydial intracellular growth. Further characterization of these proteins may generate important information for understanding chlamydial pathogenesis.

[Progress of research on TLRs-mediated signaling pathway].

TLRs belong to a family of pattern recognition receptors (PRRs) that recognize highly conserved microbial antigens termed pathogen associated molecular patterns (PAMPs). So far, ten TLRs have been identified in human genome. Each TLR senses a different set of microbial stimuli, and recruits various of adaptors and activates a series of distinct signaling cascades, and drives specific responses against the pathogens. TLRs bridged innate and adaptive immunity. The discoveries of Toll-like receptors guided the field of innate immunity to its present era of accelerated advancement. In this review, we will focus on the recent progresses of TLRs-mediated signaling. A better understanding of the immunological and molecular mechanisms mediated by TLRs will obviously facilitate the exploiting molecular targets of immunotherapy to control TLR-mediated diseases.

Mice deficient in MyD88 Develop a Th2-dominant response and severe pathology in the upper genital tract following Chlamydia muridarum infection.

MyD88, a key adaptor molecule required for many innate immunity receptor-activated signaling pathways, was evaluated in a Chlamydia muridarum urogenital tract infection model. Compared with wild-type mice, MyD88 knockout (KO) mice failed to produce significant levels of inflammatory cytokines in the genital tract during the first week of chlamydial infection. MyD88 KO mice developed a Th2-dominant whereas wild-type mice developed a Th1/Th17-dominant immune response after chlamydial infection. Despite the insufficient production of early inflammatory cytokines and lack of Th1/Th17-dominant adaptive immunity, MyD88 KO mice appeared to be as resistant to chlamydial intravaginal infection as wild-type mice based on the number of live organisms recovered from vaginal samples. However, significantly high numbers of chlamydial organisms were detected in the upper genital tract tissues of MyD88 KO mice. Consequently, MyD88 KO mice developed more severe pathology in the upper genital tract. These results together have demonstrated that MyD88-dependent signaling pathway is not only required for inflammatory cytokine production in the early phase of host response to chlamydial infection but also plays a critical role in the development of Th1/Th17 adaptive immunity, both of which may be essential for limiting ascending infection and reducing pathology of the upper genital tract by chlamydial organisms.