Effects of olanzapine treatment on prepulse inhibition in neurodevelopmental mice model of schizophrenia / 中华行为医学与脑科学杂志

Objective:To establish neurodevelopmental mice model of schizophrenia(SZ) with prepulse inhibition(PPI) deficits and investigate the effectiveness of olanzapine on PPI disruption.Methods:On the 9th day of pregnancy of SPF grade C57BL/6 mice, female mice were injected with polyinosinic acid poly (I∶C) (6 mg/kg) through tail vein for immune stimulation. The stress model was constructed by chronic unpredictable mild stress 30-40 d after birth (PND30-40). The offspring mice were divided into pregnancy immune stimulation + adolescent stress group (P + S + group), pregnancy immune stimulation group (P + S- group), adolescent stress group (P-S+ group) and non stimulation group (P-S-group), with 18 mice in each group. The mice in P+ S+ group were divided into OLZ intervention group (OLZ group) and non-OLZ intervention group (non-OLZ group), with 9 mice in each group. The PPI function of mice was detected by acoustic startle reflex test after modeling and OLZ intervention. Adopt StatView Version 5.0 software for data analysis, and multi factors analysis of variance was used to test the main effect, interactive effect and simple effect of each factor.Results:The main effects of maternal Poly(I: C) immune activation and pubertal chronic unpredictable stress were significant( F(1, 330)=47.72, P<0.01), and there was a significant interaction between the two factors( F(1, 330)=14.80, P<0.01), simple effect analysis showed that average percent prepulse inhibition (PPI%) in P+ S+ group((15.42±6.13)%) was significantly decreased compared with groups of P+ S-((27.33±4.58)%), P-S+ ((31.17±3.97)%) and P-S-((47.14±12.28)%)(all P<0.01). There was significant gender difference in Prepulse inhibition(PPI)score( F(1, 396)=61.94, P<0.01), in male and female mice, average startle reactivity of Pulse under Prepulse+ Pulse influence of distinct intensities was significantly different( F(1, 198)=18.68, 18.44, P<0.01), and the maternal Poly(I∶C) immune activation had a significant main effect( F(1, 198)=32.18, 12.58, P<0.01) and interaction with pubertal chronic unpredictable stress( F(1, 198)=34.54, 11.39, P<0.01), simple effect analysis suggested that the average startle reactivity of Prepulse+ Pulse in P+ S+ group(0.47±0.12) was significantly higher than other three groups(P+ S-: 0.36±0.11, P-S+ : (0.25±0.22), P-S-: (0.31±0.19)) in male mice( P<0.01) and in P-S+ group was significantly higher than the other three groups in female mice ( P<0.01). OLZ treatment could efficiently reverse the deficits on PPI by increasing PPI%( F(1, 165)=18.24, P<0.01), OLZ could reduce PPI score in male "dual-hit" model mice( F(1, 102)=21.81, P<0.01)and raise it in female( F(1, 102)=4.88, P<0.05). Conclusion:OLZ can reverse PPI deficits in schizophrenic neurodevelopmental model mice, and in the evaluation of PPI function in the model mice through PPI of acoustic startle reflex, PPI% has better stability and reactivity to OLZ intervention.

Molecular cloning, expression and mimicking antiviral activity analysis of retinoic acid-inducible gene-I in duck (Anas platyrhynchos)

Intracellular double-stranded RNA (dsRNA) is a chief sign of replication for many viruses. Pattern recognition receptors (PRRs) of the innate immune system detected the dsRNA and initiate the antiviral responses. Retinoic acid-inducible gene I (RIG-I), a member of PRRs, plays an essential regulatory role in dsRNA-induced signalling. In this study, the full-length complementary DNA (cDNA) of duck RIG-I (duRIG-I) was cloned using the reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). The cDNA of duRIG-I contained 97-bp 50 UTR, 141-bp 30 -UTR and 2802 bp complete open-reading frame (ORF) encoding 933 amino acids. Multiple sequence alignments showed that duRIG-I shared high similarity with RIG-I from other vertebrates. Quantitative real-time PCR (qRT-PCR) analysis revealed that duRIG-I mRNA was expressed in all tested tissues, with high levels in the liver, heart, spleen, kidney and thymus, while lower in the duodenum. duRIG-I could be induced by treatment with poly(I:C). Further, overexpression of duRIG-I significantly activated the transcription of poly(I:C)-induced IFN-b, IRF7, TRIF, Mx, STAT1 and STAT2 mRNA, and duRIG-I knockdown showed the opposite results. Overall, our results suggested that duRIG-I could be an important receptor for mimicking antiviral state in duck, which warrant further studies to show the possible mechanism.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Expression of IL-23 and IL-17 in immune injury of the liver of mice sensitized by trichloroethylene and aggravated by Poly I:C / 中国职业医学

OBJECTIVE: To observe the expression of interleukin(IL)-23 and IL-17 in the liver of mice sensitized by trichloroethylene(TCE), and to explore the role of IL-23 and IL-17 in polyinosinic: polycytidylic acid(Poly I:C) exacerbated TCE-sensitized mice with immune injury of the liver. METHODS: Female specific pathogens free BALB/c mice were randomly divided into control group(n=5), solvent control group(n=5), TCE group(n=20), and TCE+Poly I:C group(n=20). A TCE-sensitized mouse model was established in TCE group and TCE+Poly I:C group. Three hours before the last challenge, mice in TCE+Poly I:C group was intraperitoneally injected with a mass concentration of 0.5 g/L poly I:C, 100 μL per mouse. The two groups of mice were divided into sensitized and non-sensitized subgroups according to the results of skin sensitization. After 48 hours of the final challenge, serum alanine aminotransferase(ALT) was detected by colorimetric method. The histopathological changes of mouse liver were observed, and the expression of IL-23 and IL-17 in liver tissues was detected by immunohistochemistry and Western blotting method. RESULTS: The sensitization rates of TCE and TCE+Poly I:C groups were 35.0%(7/20) and 40.0%(8/20) respectively, with no significantly statistical difference(P>0.05). Pathological examination showed that there was cell edema in some areas of the liver tissues of mice in the TCE-sensitized subgroup, while the TCE+Poly I:C sensitized subgroup showed cell vacuolar degeneration and loose cytoplasm. Serum ALT activity and the expression of IL-23 and IL-17 in liver tissues in the TCE-sensitized subgroup were higher than that in the blank control group, the solvent control group and the TCE non-sensitized subgroup(P<0.05). Serum ALT activity and IL-23 and IL-17 expression in the TCE+Poly I:C sensitized subgroup were higher than that in the TCE-sensitized subgroup(P<0.05). The relative expression of IL-23 and IL-17 protein in liver tissues in TCE-sensitized subgroup was higher than that of the blank control group and the solvent control group(P<0.05), while that in TCE+Poly I:C sensitized subgroup was higher than that of TCE-sensitized subgroup(P<0.05). CONCLUSION: IL-23/IL-17 axis may play an important role in the development of immune injury of liver in the TCE-sensitized mice and Poly I:C exacerbated TCE-sensitized mice.

Comparison of polycytidylic acid-induced and dexamethasone-induced thymic atrophy and their thymic expression of RIG-Ⅰ like receptors signaling pathway / 中国免疫学杂志

Objective:To provide experimental evidences for choosing murine models in the pathogenesis research of thymic impairment induced by viral infection,we compared the impacts of polycytidylic acid(Poly(I:C)) and dexamethasone(DEX) on the thymic morphology and thymic output function,and explored the implication of RLR signaling pathway.Methods: 24 male C57BL/6 mice were randomly assigned into three groups and treated with Poly(I:C),DEX,or saline respectively.Thereafter,their thymic morphology,pathological changes,thymic index,and thymic pathology were examined.Their contents of T-cell receptor excision circles (TRECs) and proportions of the naive CD4+T cell in the peripheral blood were determined to evaluate their thymic output function.The expression levels of thymic RLR/MAVS/IFN-α/β signaling pathway and IL-1β were also measured.Results: Both Poly (I:C) and DEX treatment caused thymic atrophy in appearance and structural destruction under the microscope inspection,and DEX treatment did much more severe damage,especially to the thymic cortex.TRECs decreased significantly in both groups.The proportions of na?ve/memory CD4+T cell subsets remained stable,though total CD4+T cell decreased in DEX group,while the proportion of na?ve CD4+T cell in Poly (I:C) group increased significantly.The expression of RIG-Ⅰ,MDA5,LGP2,and IFN-α/β were up-regulated in DEX group, while it remained unchanged in Poly (I:C) group.Conclusion:Both Poly (I:C) and DEX induced thymic atrophy and the impaired thymic output function.Nevertheless,the expression of RLR-IFN signaling pathway up-regulated more significantly in DEX group instead of in Poly (I:C) group.These results implied the existence of different pathological manifestations and mechanisms underlying the impaired thymic function in different animal models,as well as impact on na?ve/memory CD4+T cell proportions.Our research provides references for choosing animal models in the basic research and drug development for viral infection induced thymic atrophy based on the RLR signaling pathway.

Mechanism of Poly(I∶C)-induced apoptosis in lung adenocarcinoma A549 cells / 国际肿瘤学杂志

Objective To study the effects of Poly(I:C) on lung adenocarcinoma A549 cells viability and illuminate the mechanism of Poly (I:C)-induced apoptosis in A549 cells.Methods A549 cells were transfected with the complex of Poly(I:C) and lipofectamine 3000.The viability of A549 cells was tested by methyl thiazolyl tetrazolium (MTF) method.The apoptotic cells were tested by flow cytometry.The caspase proteins were tested by Western blotting and the expressions of interferon-β (IFN-β) and CXCL-10 were assayed by real-time PCR.After employing the pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK,the variation of Poly (I:C) proapoptosis in A549 cells was observed.RNA interfering experiments were employed to knock down melanoma differentiation related antigen 5 (MDA5) or retinoic acidinduced gene Ⅰ (RIG-Ⅰ),and the above indexes were tested.Results The viability of A549 cells was significantly reduced to 74.92％ ±--6.24％ after 200 ng/ml Poly (I:C) transfection compared with that before transfection (95.32％ ± 3.05％,t =2.883,P =0.041).The apoptotic rates induced by 100,200,400 ng/ml Poly(I:C) were 9.97％-± 0.88％,23.63％-± 1.41％,32.57％-± 2.39％,respectively.All of them were higher than that in the control group (0.74％-± 0.15％),with significant differences (t =4.489,P =0.002;t =11.616,P =0.000;t =16.932,P =0.000).Besides,the death receptor pathway proteins such as TNF-related apoptosis inducing ligand (TRAIL),cleaved-caspase-8 and cleaved-caspase-3 increased obviously.MDA5/RIG-Ⅰ pathway was also activated dramatically and the expressions of IFN-β,CXCL-10 were significantly up-regulated.The apoptotic rates reduced to 3.17％ ± 0.66％,5.35％ ± 0.64％ with pancaspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK pretreatment,compared with the control group (15.87％ ±0.93％),and the differences were statistically significant (t =8.643,P =0.001;t =6.824,P =0.002).Moreover,the expressions of TRAIL,IFN-β and CXCL-10 induced by Poly (I:C) were inhibited with MDA5 or RIG-Ⅰ depletion.Conclusion Poly(I:C) can reduce the survival rate of A549 cells and promote the apoptosis mainly by activating the death-receptor pathway mediated by MDA5/RIG-Ⅰ probably,which may involve in IFN-β,CXCL-10.

Expression of intrahepatic chemokine CXCL13 in a mouse model of primary biliary cholangitis / 医学研究生学报

Objective The expression of chemokine C-X-C motif ligand 13 (CXCL13) within liver in primary biliary cholangitis (PBC) patients is significantly increased, but its origin and mechanism is not clear yet.The study aimed to investigate the expression of CXCL13 in the liver of mice through establishing a mouse model of PBC.Methods C57BL/6 mice were randomly divided into experiment group (n=20) control group(n=10).The mice in the experimental group were intraperitoneally injected with polyriboinosinic polyribocytidylic acid (Poly I:C) while the mice in control group were injected with PBS of the same volume.The level of serum AMA was quantified by ELISA and intrahepatic inflammatory cells were assessed by HE staining.Kupffer cells, liver sinusoidal endothelial cells, and infiltrating lymphocytes in the liver of mice were collected by in situ perfusion enzyme digestion and magnetic bead separation methods.The transcriptional level of intrahepatic CXCL13 in liver tissues and cell subpopulations were detected by qPCR.Results The serum AMA titers of the mice in experiment group increased gradually with the prolonging of modeling time and the positive rates at the 4th, 8th, and 12th week after the first injection of Poly I:C were 5.9%, 52.9% and 76.5% respectively.While the serum AMA titers of the mice in control group were at a lower level through the modeling process, with only 2 mice presenting a little higher level above positive cutoff value at the 12th week.The results of HE staining in liver tissues of both groups showed that there were a great amount of intensely infiltrating inflammatory cells in the mice of experimental group while no inflammatory cell infiltration were found in the mice of control group.The separation purity of Kupffer cells and liver sinusoidal endothelial cells in the mice of experiment group tested by flow cytometry were 76%-80%, 68%-72% respectively.Compared with the CXCL13 mRNA level in Kupffer cells [2.34(0.22-8.64)], the expression levels in liver sinusoidal endothelial cells and infiltrating lymphocytes declined[0.27(0.03-1.64), 0.05(0-0.22), P<0.05].Conclusion The chemokine CXCL13 is predominantly produced by Kupffer cells in the liver of PBC mice established by Poly I:C injection.

Galangin Suppresses Pro-Inflammatory Gene Expression in Polyinosinic-Polycytidylic Acid-Stimulated Microglial Cells

Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum. In this study, we investigated the anti-inflammatory effects of galangin under in vitro and in vivo neuroinflammatory conditions caused by polyinosinic-polycytidylic acid (poly(I:C)), a viral mimic dsRNA analog. Galangin suppressed the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in poly(I:C)-stimulated BV2 microglia. On the other hand, galangin enhanced anti-inflammatory interleukin (IL)-10 production. Galangin also suppressed the expression of pro-inflammatory markers in poly(I:C)-injected mouse brains. Further mechanistic studies showed that galangin inhibited poly(I:C)-induced nuclear factor (NF)-κB activity and phosphorylation of Akt without affecting MAP kinases. Interestingly, galangin increased the expression and transcriptional activity of peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. To investigate whether PPAR-γ is involved in the anti-inflammatory function of galangin, BV2 cells were pre-treated with PPAR-γ antagonist before treatment of galangin. We found that PPAR-γ antagonist significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α and IL-6 in poly(I:C)-stimulated microglia. In conclusion, our data suggest that PI3K/Akt, NF-κB, and PPAR-γ play a pivotal role in mediating the anti-inflammatory effects of galangin in poly(I:C)-stimulated microglia.

Galangin Suppresses Pro-Inflammatory Gene Expression in Polyinosinic-Polycytidylic Acid-Stimulated Microglial Cells

Galangin (3,5,7-trihydroxyflavone) is a polyphenolic compound abundant in honey and medicinal herbs, such as Alpinia officinarum. In this study, we investigated the anti-inflammatory effects of galangin under in vitro and in vivo neuroinflammatory conditions caused by polyinosinic-polycytidylic acid (poly(I:C)), a viral mimic dsRNA analog. Galangin suppressed the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines in poly(I:C)-stimulated BV2 microglia. On the other hand, galangin enhanced anti-inflammatory interleukin (IL)-10 production. Galangin also suppressed the expression of pro-inflammatory markers in poly(I:C)-injected mouse brains. Further mechanistic studies showed that galangin inhibited poly(I:C)-induced nuclear factor (NF)-κB activity and phosphorylation of Akt without affecting MAP kinases. Interestingly, galangin increased the expression and transcriptional activity of peroxisome proliferator-activated receptor (PPAR)-γ, known to play an anti-inflammatory role. To investigate whether PPAR-γ is involved in the anti-inflammatory function of galangin, BV2 cells were pre-treated with PPAR-γ antagonist before treatment of galangin. We found that PPAR-γ antagonist significantly blocked galangin-mediated upregulation of IL-10 and attenuated the inhibition of tumor necrosis factor (TNF)-α and IL-6 in poly(I:C)-stimulated microglia. In conclusion, our data suggest that PI3K/Akt, NF-κB, and PPAR-γ play a pivotal role in mediating the anti-inflammatory effects of galangin in poly(I:C)-stimulated microglia.

Induction of Indoleamine 2,3-dioxygenase by Pre-treatment with Poly(I:C) May Enhance the Efficacy of MSC Treatment in DSS-induced Colitis

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly owing to their immunosuppressive properties. The goal of the study was to determine whether TLR ligands can enhance the therapeutic efficacy of bone marrow-derived MSCs for the treatment of inflammatory bowel disease. Mice (C57BL6) were administered with 4% dextran sulfate sodium (DSS) in drinking water for 7 days and injected with MSCs on days 1 and 3 following DSS ingestion. Our results demonstrated that among various TLR ligands, MSCs treated with polyinosinic-polycytidylic acid [poly(I:C)], which is a TLR3 ligand, more profoundly induced IDO, which is a therapeutically relevant immunosuppressive factor, without any observable phenotype change in vitro. The poly(I:C)-treated MSCs attenuated the pathologic severity of DSS-induced murine colitis when injected i.p. but not i.v. In summary, preconditioning MSCs with poly(I:C) might improve their efficacy in treating DSS-induced colitis, and this effect at least partly depends on the enhancement of their immunosuppressive activity through increasing their production of IDO.

Observation of adequate stimulus methods for key initiative factor expression in human keratinocytes of anaphylactic disease / 中国药理学通报

Aim To explore the expressed level of ini-tiative key factors TSLP and IL-33 in a human kerati-nocyte cell line,HaCaT cells were chosen to be stimu-lated by different stimulants,and develop a stable and effective in vitro model to observe allergic sensitization. Methods HaCaT cells were cultured in K-SFM with different stimulants to screen out the stimulants which could significantly improve the expressed level of TSLP and IL-33.Expressed level of TSLP and IL-33 was an-alyzed by ELISA kits and immunofluorescence.Re-sults (1 )The dose-response relationship of single stimulant indicated that both Poly(I:C)and TNF-αcould significantly improve expressed level of TSLP and IL-33 in HaCaT cells,but the rest of stimulants was not observed significant stimulation in concentration range of this experiment.(2)Dose-effect relationship of combined stimulants indicated that poly(I ∶C)1 00 mg·L -1 combined with TNF-α20 μg·L -1 was the most efficient.(3)Time-effect relationship of the a-bove-mentioned combined stimulants showed that 1 2 h was the optimal time of stimulation.Conclusions Different stimulants and different time result in various expressed levels of TSLP and IL-33 in HaCaT cells.1 2 h stimulus duration of Poly(I:C)1 00 mg·L -1 com-bined with TNF-α20 μg · L -1 is the most efficient stimulating way.This result provides an effective in vitro model to study the pathomechanism and drug effi-cacy of allergic sensitization.

Differential microglial and astrocytic response to bacterial and viral infection in the developing hippocampus of neonatal rats.

Polyinosinic:polycytidylic acid (Poly I:C; 5 mg/kg body weight, ip) and lipopolysaccharide (LPS; 0.3 mg/kg body weight, ip) induced microglial and astrocytic activation in Sprague Dawley rats. Higher microglial and astrocytic activities were noticed in Poly I:C infused rats throughout the hippocampus till postnatal day 21 with a comparatively weaker response in LPS group. However, LPS induced inflammation persisted even after postnatal day 21, indicating thereby, that the Poly I:C (viral mimic) produces an acute inflammation, while LPS (bacterial endotoxin) produces chronic inflammation when exposed during early neonatal life.

Animal models of prenatal immune challenge and their contribution to the study of schizophrenia: a systematic review

Prenatal immune challenge (PIC) in pregnant rodents produces offspring with abnormalities in behavior, histology, and gene expression that are reminiscent of schizophrenia and autism. Based on this, the goal of this article was to review the main contributions of PIC models, especially the one using the viral-mimetic particle polyriboinosinic-polyribocytidylic acid (poly-I:C), to the understanding of the etiology, biological basis and treatment of schizophrenia. This systematic review consisted of a search of available web databases (PubMed, SciELO, LILACS, PsycINFO, and ISI Web of Knowledge) for original studies published in the last 10 years (May 2001 to October 2011) concerning animal models of PIC, focusing on those using poly-I:C. The results showed that the PIC model with poly-I:C is able to mimic the prodrome and both the positive and negative/cognitive dimensions of schizophrenia, depending on the specific gestation time window of the immune challenge. The model resembles the neurobiology and etiology of schizophrenia and has good predictive value. In conclusion, this model is a robust tool for the identification of novel molecular targets during prenatal life, adolescence and adulthood that might contribute to the development of preventive and/or treatment strategies (targeting specific symptoms, i.e., positive or negative/cognitive) for this devastating mental disorder, also presenting biosafety as compared to viral infection models. One limitation of this model is the incapacity to model the full spectrum of immune responses normally induced by viral exposure.

Polyinosinic-polycytidylic acid and tumor immunity / 国际肿瘤学杂志

Polyinosinic-polycytidylic acid is a synthetic analog of double-stranded RNA,which plays a vital role in the regulation of immune system.Toll-like receptor 3 ( TLR3 ),melanoma differentiation-associated gene 5 (MAD-5) and retinoic acid inducible gene - Ⅰ ( RIG- Ⅰ ) are the main three intracellular receptors binding to polyinosinic-polycytidylic acid which activates human anti-tumor immune responses including the activation of innate immunity and acquired immunity through TIR-domain-containing adapter-inducing inter feron-β (TRIF) and interferon-β.Thus,polyinosinic-polycytidylic acid plays an important role in regulating anti-tumor immune responses.The immunoregulation mechanisms ofpolyinosinic-polycytidylic acid in melanoma,breast cancer,malignant glioma and lung cancer have been clarified,which will provide new strategies for tumor immunotherapy.

Stimulation of TLR3 inhibits the proliferation of breast cancer cells MDA-MB-231 / 国际生物医学工程杂志

ObjectiveToll-like receptors (TLRs) play important role in the progression and tumor immunity of some types of cancer,some research have demonstrated that agonist of TLR3 can trigger apoptosis of cancers.This study was proposed to investigate if Poly(I:C),the specific agonist of TLR3,could impact proliferation or apoptosis of progressive breast cancer cells MDA-MB-231,and to investigate the primary mechanism of the function.MethodsExpression of TLR1-10 mRNA was detected by quantitative real-time reverse transcription-polymerase chain reaction.Cell Counting Kit-8 was used to determine the inhibitory effect of Poly(I:C) on proliferation of MDA-MB-231 cells.Cell apoptosis was assayed by flow cytometry with V-FITC/PI staining.Results First,the toll-like receptors 1-10 were all expressed on MDA-MB-231 cells,while the expression level of TLR8 was lower than that of others.Second,according to the CCK-8,the proliferation of MDA-MB-231 cells was inhibited,but the apoptosis was not affected on the basis of Apoptosis Kit.At last,the mRNA expression of TNF-α、IFN-β and IFN-γ were elevated approximately 20 times after Poly(I:C) stimulation for 6 hours.ConclusionMDA-MB-231 cells express all toll-like receptors on mRNA level,and TLR8 was expressed lower than others.The stimulation of TLR3 with Poly(I:C) can inhibit the proliferation of MDA-MB-231,but had no effect on apoptosis.TNF-α、IFN-β and IFN-γ maybe participate in this process.

Curative effect observation on the treatment of chronic type B hepatitis with Gankang Granule / 国际中医中药杂志

Objective To observe the clinical effects of treating chronic type B hepatitis with Gankang Granule combined with poly I-C and immune ribonucleic acid. Methods A total of 100 cases of chronic type B hepatitis were randomly recruited into a control group and a treatment group, 50 cases in each group. The control group was treated with poly I-C and immune ribonucleic acid, while the treatment group was treated with Gankang Granule on the basis of the control group. Results Symptoms in both groups were improved. 23 eases in the treatment group showed negative value of HBeAg,contrasting to 13 cases in the control group. 38 cases in the treatment group turned to be negative value of HBV- DNA, higher than the number of 23 cases in the control group. The treatment group demonstrated a significantly better therapeutic results than the control group (χ2 value was 6.267 and 6.345 respectively, both P＜0.05). Conclusion Gankang Granule combined with poly I-C and immune ribonucleic acid can obviously improve hepatic functions and symptoms of chronic type B hepatitis,shorten the course of disease, and increase the rate of negative value of HBeAg and HBV- DNA.

Toll-like Receptor3-mediated Induction of Chemokines in Salivary Epithelial Cells

Toll-like receptors (TLRs) functionally expressed in salivary epithelial cells, but their roles remain elusive. Among TLRs family, TLR3 is activated by dsRNA, a byproduct of viral infection. The aim of this study was to investigate the role of TLR3 in the inflammatory immune responses using HSG cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and ELISA were performed to identify expression of TLRs and TLR3-mediated chemokine inductions. The chemotaxis assay of activated T lymphocytes was also performed. Treatment of HSG cells with polyinosinic: polycytidylic acid (poly(I:C)) significantly increased interferon-gamma-inducible protein 10 (IP-10), interferoninducible T-cell alpha chemoattractant (I-TAC), and regulated on activation, normal T-cells expressed and secreted (RANTES) gene expressions in a concentration-dependent manner. Anti-TLR3 antibody blocked the increases of IP-10 and I-TAC genes. Poly(I:C)-induced increases of IP-10 and I-TAC were also confirmed at protein levels from cell lysates, but their release into extracellular medium was detected only in IP-10. We found that the culture media from HSG cells stimulated with poly(I:C) significantly increases T lymphocyte migration. Our results suggest that TLR3 plays an important role in chemokine induction, particularly IP-10, in salivary epithelial cells.

Expression of Toll-like receptor 3 on the peripheral blood dendritic cells after stimulated by poly I: C in patients with chronic hepatitis B / 中华传染病杂志

Objective To elucidate the expression of Toll-like receptor 3 (TLR3) on dendritic cells(DCs) in patients with chronic hepatitis B (CHB), and to explore the correlation between hepatitis B virus (HBV) persistent infection and TLR3 expression. Methods Sixty CHB patients (CHB group) and 20 healthy controls (control group) were enrolled. The peripheral blood mononuclear cells (PBMCs) were isolated and CD14~+ monocytes were sorted by immunomagnetic beads. Immature DCs (imDC) were induced and proliferated in vitro and mature DCs (mDC) were obtained after the poly I:C stimulation. The expression of intracellular TLR3 mRNA was detected by real-time polymerase chain reaction (PCR), and surface markers [CD80 and human leucocyte antigen (HLA)-DR] were determined by flow cytometry after 48 h of stimulation. The comparison of quantitative data was done using t test. The qualitative data were compared using chi-square test.Results The mean fluorescence intensities (MFI) of intracellular TLR3 of imDC before poly I:C stimulation in CHB group and control group were 1212.05 ± 250.80 and 1192.95 ± 301.40,respectively, which were not significantly different (t = 0. 280, P>0. 05). While after stimulation,those were 1352.98± 313.67 and 1593. 00± 349. 65, respectively, the latter was significantly higher than the former (t = 2. 880, P<0. 05). The levels of TLR3 mRNA inside mDCs in both groups were increased after poly I:C stimulation, which were 0. 1204 ±0.0267 and 0. 1780 ± 0.0664, respectively in CHB group and control group, and that in control group was significantly higher (t = 3. 909, P<0.05). Furtherly, patients in CHB group were divided into HBeAg(+ ) and HBeAg( -) subgroups.After stimulation, the MFI and mRNA of TLR3 inside mDC were greatly elevated in both subgroups,but there were no difference between these two subgroups (t = 0. 366, P>0. 05). Conclusions The intracellular expressions of TLR3 in mDC in CHB group and control group are obviously increased after the poly I:C stimulation, but the increased level in CHB group is lower than that in control group. The results suggest that the insufficiency of TLR3 synthesis may be related to the HBVpersistent infection.

Defective activation induced cell death of CD4+ T cells in primary biliary cirrhosis murine model / 中华微生物学和免疫学杂志

Objective To study the activation induced cell death (AICD) of CD4+ T cells in primary biliary cirrhosis(PBC)murine model induced by poly I∶C. Methods Thirty female C57BL/6 mice were divided into model and control group randomly, and the former were injected with 5 mg/kg of poly I∶C, the later with PBS. PBC mice were detected 16 weeks after injection. CD4+ T cells isolated from spleen were stimulated in vitro by Con A and anti-CD3, and the apoptosis were determined by Annexin-V and PI staining. The expression of Fas, FasL and TRAIL were assayed by relative quantitative real-time PCR. Bcl-2 was detected by Western blot. Results Compared with control group, the portal areas of mice in model group were infiltrated with mononuclear cells obviously. The positive rate of serum antimitochondrial antibody(AMA) and the level of alkali phosphatase (ALP) were higher than that in control group (P<0.001). AICD of splenic CD4+ T cells in model group was lower than that of control group (P<0.001). The mRNA of FasL and TRAIL in model mice was down-regulated. Simultaneously, the anti-apoptosis protein Bcl-2 was up-regulated in model group. Conclusion These observations suggest that a defect in AICD of auto-reactive TH1 cells may contribute to the pathogenesis of PBC model. Furthermore, this defect in AICD may results from the change of Fas/FasL, TRAIL pathway and the up-regulation of Bcl-2.