[目的]克隆草鱼的PKR基因,为草鱼抗病毒基因研究奠定基础。[方法]依据GenBank上斑马鱼(AJ852023.1)和鲫鱼(AY293929.1)的PKR基因序列,利用Primer Premier 5.0软件设计了3对简并引物; 采用100μg/ml PolyI:C体外分别处理草鱼肾细胞(Cteno...[目的]克隆草鱼的PKR基因,为草鱼抗病毒基因研究奠定基础。[方法]依据GenBank上斑马鱼(AJ852023.1)和鲫鱼(AY293929.1)的PKR基因序列,利用Primer Premier 5.0软件设计了3对简并引物; 采用100μg/ml PolyI:C体外分别处理草鱼肾细胞(Ctenopharyngodon indellus kidney cells,CIK)12、36、48h,并提取处理后细胞的总RNA,逆转录后用降落PCR方法扩增这3个处理时间细胞中 PKR 基因。[结果]处理12h时未扩增出PKR基因,处理36和48h时都扩增到了PKR基因,并且表达量随处理时间的增加有所升高,扩增到的部分序列与鲫鱼和斑马鱼的该段序列同源性分别为100.00%和81.48%。[结论]试验成功获得了草鱼PKR基因的部分序列,PolyI:C高效诱导草鱼PKR蛋白表达将有助于开创治疗草鱼类病毒病的一种新思路。展开更多
Background The well-known <pyrotherapy, of Julius Wagner-Jauregg might be the beginning of the study on the immunological concepts of schizophrenia. As the primary immune effector cells in the brain, microglia play...Background The well-known <pyrotherapy, of Julius Wagner-Jauregg might be the beginning of the study on the immunological concepts of schizophrenia. As the primary immune effector cells in the brain, microglia play a pivotal role in neuroinflammatory processes. Maternal viral infection during pregnancy is associated with an increased risk for psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The present study was to quantify microglia activation in vivo in the mature offspring of rats exposed to polyriboinosinic-polyribocytidilicacid(Poly l:C) during pregnancy using 11 C'PK11195 positron emission tomography(PET) and immunohistochemistry.Objective The study aimed to quantify microglia activation in vivo in the prefrontal cortex and hippocampus in mature offspring of prenatal Poly l:C exposed rats.Methods Offspring of Poly l:C-treated dams were the model group, offspring of saline-treated dams were the control group. Behavioural test for two groups was taken by spontaneous activity, prepulse inhibition(PPI) and latent inhibition(LI) test(including active avoidance conditioning task and passive avoidance conditioning task). Randomly selected successful model rats were assessed by behavioural test in the model group and control group rats.11 C-PK11195 micro-PET/CT and immunohistochemistry were performed on the selected rats to measure microglia activation.Results The treatment group showed hyperlocomotion and deficits in PPI and LI compared with the control group. The treatment group also showed an increased11 C-PK11195 uptake ratio in the prefrontal cortex(f=-3.990, p=0.003) and hippocampus(f=-4.462,p=0.001). The number of activated microglia cells was significantly higher in the treatment group than in the control gro叩(hippocampus: f=8.204, p<0.001; prefrontal:f=6.995, p<0.001). Within the treatment group, there were significant correlations between the behavioural parameters and the activation of microglia as measured by PET and immunohistochemistry.Conclusions The present study demonstrated microglia activation in vivo in the prefrontal cortex and hippocampus in mature offspring of prenatal Poly l:C exposed rats.This study suggests that microglia activation may play a possible or potential role in the pathogenesis of schizophrenia.展开更多
AIM: To study the role of luteolin(LUT) in the expression of toll-like receptors 3(TLR3) ligand poly I:C stimulated inflammatory factors in human corneal fibroblasts(HCFs).METHODS: HCFs cells were cultivated with or w...AIM: To study the role of luteolin(LUT) in the expression of toll-like receptors 3(TLR3) ligand poly I:C stimulated inflammatory factors in human corneal fibroblasts(HCFs).METHODS: HCFs cells were cultivated with or without LUT or poly I:C.The expression levels of interleukin(IL)-6, IL-8, monocyte chemotactic protein-1(MCP-1), vascular cell adhesion molecule(VCAM)-1, as well as intercellular adhesion molecule(ICAM)-1 were measured using enzymelinked immunosorbent assay(ELISA), immunoblotting or reverse transcription-quantitative polymerase chain reaction(PCR) analyses.Immunoblotting was used to assess toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-β(TRIF), TLR3, transforming growth factor-bactivated kinase 1(TAK1), tumor necrosis factor receptor-associated factor 6(TRAF6), the transcription factor AP-1, as well as transcription factor nuclear factor(NF-κB)–inhibitory protein IκB-α degradation and phosphorylation.Immunofluorescence assays were used to localize the cellular location of the p65 subunit of NF-κB.RESULTS: Corneal fibroblasts exposed to poly I:C demonstrated decreased VCAM-1, ICAM-1, MCP-1, IL-6, and IL-8 expression levels upon exposure to LUT in a time-dependent and concentration-dependent manner.LUT was observed to suppress poly I:C-triggered expression of TLR3, the translocation of NF-κB p65 into cell nuclei, as well as the phosphorylation of TAK, c-Jun, and IκB-α, while no impact on the expression levels of TRIF and TRAF6 were observed.CONCLUSION: LUT suppress the expression of proinflammatory adhesion molecules, chemokines, and cytokines in poly I:C exposed HCFs.These effects are likely mediated through TAK/NF-κB signal attenuation.Therefore, LUT is a candidate molecule that can prevent the TLR3-mediated inflammation response associated with corneal viral infection.展开更多
A highly immunosuppressive tumor microenvironment(TME)and the presence of the blood‒brain barrier are the two major obstacles to eliciting an effective immune response in patients with high-grade glioma(HGG).Here,we t...A highly immunosuppressive tumor microenvironment(TME)and the presence of the blood‒brain barrier are the two major obstacles to eliciting an effective immune response in patients with high-grade glioma(HGG).Here,we tried to enhance the local innate immune response in relapsed HGG by intracranially injecting poly(I:C)to establish a robust antitumor immune response in this registered clinical trial(NCT03392545).During the follow-up,12/27(44.4%)patients who achieved tumor control concomitant with survival benefit were regarded as responders in our study.We found that the T-cell receptor(TCR)repertoire in the TME was reshaped after poly(I:C)treatment.Based on the RNA-seq analysis of tumor samples,the expression of annexin A1(ANXA1)was significantly upregulated in the tumor cells of nonresponders,which was further validated at the protein level.In vitro and in vivo experiments showed that ANXA1 could induce the production of M2-like macrophages and microglia via its surface receptor formyl peptide receptor 1(FPR1)to establish a Treg cell-driven immunosuppressive TME and suppress the antitumor immune response facilitated by poly(I:C).The ANXA1/FPR1 signaling axis can inhibit the innate immune response of glioma patients by promoting an anti-inflammatory and Treg-driven TME.Moreover,ANXA1 could serve as a reliable predictor of response to poly(I:C),with a notable predictive accuracy rate of 92.3%.In light of these notable findings,this study unveils a new perspective of immunotherapy for gliomas.展开更多
Background The outbreak of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has greatly threatened public health.Recent studies have revealed that the spike receptor-binding domain(RBD)of SARS-CoV-2 is a...Background The outbreak of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has greatly threatened public health.Recent studies have revealed that the spike receptor-binding domain(RBD)of SARS-CoV-2 is a potent target for vaccine development.However,adjuvants are usually required to strengthen the immunogenicity of recombinant antigens.Different types of adjuvants can elicit different immune responses.Methods We developed an RBD recombinant protein vaccine with a polyriboinosinic acid–polyribocytidylic acid[poly(I:C)]adjuvant to evoke a strong immune response.The delivery of poly(I:C)was optimized in two steps.First,poly(I:C)was complexed with a cationic polymer,poly-l-lysine(PLL),to form poly(I:C)–PLL,a polyplex core.Thereafter,it was loaded into five different lipid shells(group II,III-1,2-distearoyl-sn-glycero-3-phosphocholine[DSPC],III-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine[DOPE],IV-DOPE,and IV-DSPC).We performed an enzyme-linked immunosorbent assay and enzyme-linked immunosorbent spot assay to compare the ability of the five lipopolyplex adjuvants to enhance the immunogenicity of the SARS-CoV-2 RBD protein,including humoral and cellular immune responses.Finally,the adjuvant with the highest immunogenicity was selected to verify the protective immunity of the vaccine through animal challenge experiments.Results Recombinant RBD protein has low immunogenicity.The different adjuvants we developed enhanced the immunogenicity of the RBD protein in different ways.Among the lipopolyplexes,those containing DOPE(III-DOPE and IV-DOPE)elicited RBD-specific immunoglobulin G antibody responses,and adjuvants with four components elicited better RBD-specific immunoglobulin G antibody responses than those containing three components(P<0.05).The IC50 and IC90 titers indicated that the IV-DOPE lipopolyplex had the greatest neutralization ability,with IC50 titers of 1/117,490.Furthermore,in the challenge study,IV-DOPE lipopolyplex protected mice from SARS-CoV-2 infection.On the fourth day after infection,the average animal body weights were reduced by 18.56%(24.164±0.665 g vs.19.678±0.455 g)and 0.06%(24.249±0.683 g vs.24.235±0.681 g)in the MOCK and vaccine groups,respectively.In addition,the relative expression of viral RNA in the vaccinated group was significantly lower than that in the MOCK group(P<0.05).Interstitial inflammatory cell infiltration was observed in the MOCK group,whereas no obvious damage was observed in the vaccinated group.Conclusions The IV-DOPE–adjuvanted SARS-CoV-2 recombinant RBD protein vaccine efficiently protected mice from SARS-CoV-2 in the animal challenge study.Therefore,IV-DOPE is considered an exceptional adjuvant for SARS-CoV-2 recombinant RBD protein-based vaccines and has the potential to be further developed into a SARS-CoV-2 recombinant RBD protein-based vaccine.展开更多
The subunit herpes zoster vaccine Shingrix is superior to attenuated vaccine Zostavax in both safety and efficacy,yet its unlyophilizable liposome delivery system and the limited supply of naturally sourced immunologi...The subunit herpes zoster vaccine Shingrix is superior to attenuated vaccine Zostavax in both safety and efficacy,yet its unlyophilizable liposome delivery system and the limited supply of naturally sourced immunological adjuvant QS-21 still need to be improved.Based on poly(lactic-co-glycolic acid)(PLGA)delivery systems that are stable during the lyophilization and rehydration process and using a double-emulsion(w/o/w)solvent evaporation method,we designed a series of nanoparticles with varicella-zoster virus antigen glycoprotein E(VZV-g E)as an antigen and nucleic acids including polyinosinic-polycytidylic acid(Poly I:C)and phosphodiester Cp G oligodeoxynucleotide(Cp G ODN),encapsulated as immune stimulators.While cationic lipids(DOTAP)have more potential than neutral lipids(DOPC)for activating g E-specific cell-mediated immunity(CMI)in immunized mice,especially when g E is encapsulated in and presented on the surface of nanoparticles,PLGA particles without lipids have the greatest potential to induce not only the highest g Especific Ig G titers but also the strongest g E-specific CMI responses,including the highest proportions of interferon-c(IFNc)-and interleukin-2(IL-2)-producing CD4?/CD8?T cells according to a flow cytometry assay and the greatest numbers of IFN-c-and IL-2-producing splenocytes according to an enzyme-linked immunospot(ELISPOT)assay.These results showed that immune-stimulating nucleic acids together with the PLGA delivery system showed promise as a safe and economical varicella and zoster vaccine candidate.展开更多
基金Supported by National Natural Science Foundation of Zhejiang Province (Y3110432 )Huzhou Teachers College Science ResearchFoundation (2010YZ48)~~
文摘[目的]克隆草鱼的PKR基因,为草鱼抗病毒基因研究奠定基础。[方法]依据GenBank上斑马鱼(AJ852023.1)和鲫鱼(AY293929.1)的PKR基因序列,利用Primer Premier 5.0软件设计了3对简并引物; 采用100μg/ml PolyI:C体外分别处理草鱼肾细胞(Ctenopharyngodon indellus kidney cells,CIK)12、36、48h,并提取处理后细胞的总RNA,逆转录后用降落PCR方法扩增这3个处理时间细胞中 PKR 基因。[结果]处理12h时未扩增出PKR基因,处理36和48h时都扩增到了PKR基因,并且表达量随处理时间的增加有所升高,扩增到的部分序列与鲫鱼和斑马鱼的该段序列同源性分别为100.00%和81.48%。[结论]试验成功获得了草鱼PKR基因的部分序列,PolyI:C高效诱导草鱼PKR蛋白表达将有助于开创治疗草鱼类病毒病的一种新思路。
基金provided by the National Natural Science Foundation of China(No 81571318 to XQSNo 81371472 to LXL+5 种基金No 81401110 to XL)the Science and Technology Planning Project of Health and Family Planning Commission(No 201501015 to XQS)the International Science and Technology Cooperation Program of Henan(No 162102410061 to XQS)the Henan Province Union Fund Project(162300410275)the Zhengzhou University doctor team projectthe Youth Fund of the First Affiliated Hospital of Zhengzhou University(to XL and LJP)
文摘Background The well-known <pyrotherapy, of Julius Wagner-Jauregg might be the beginning of the study on the immunological concepts of schizophrenia. As the primary immune effector cells in the brain, microglia play a pivotal role in neuroinflammatory processes. Maternal viral infection during pregnancy is associated with an increased risk for psychiatric disorders with presumed neurodevelopmental origin, including autism spectrum disorders and schizophrenia. The present study was to quantify microglia activation in vivo in the mature offspring of rats exposed to polyriboinosinic-polyribocytidilicacid(Poly l:C) during pregnancy using 11 C'PK11195 positron emission tomography(PET) and immunohistochemistry.Objective The study aimed to quantify microglia activation in vivo in the prefrontal cortex and hippocampus in mature offspring of prenatal Poly l:C exposed rats.Methods Offspring of Poly l:C-treated dams were the model group, offspring of saline-treated dams were the control group. Behavioural test for two groups was taken by spontaneous activity, prepulse inhibition(PPI) and latent inhibition(LI) test(including active avoidance conditioning task and passive avoidance conditioning task). Randomly selected successful model rats were assessed by behavioural test in the model group and control group rats.11 C-PK11195 micro-PET/CT and immunohistochemistry were performed on the selected rats to measure microglia activation.Results The treatment group showed hyperlocomotion and deficits in PPI and LI compared with the control group. The treatment group also showed an increased11 C-PK11195 uptake ratio in the prefrontal cortex(f=-3.990, p=0.003) and hippocampus(f=-4.462,p=0.001). The number of activated microglia cells was significantly higher in the treatment group than in the control gro叩(hippocampus: f=8.204, p<0.001; prefrontal:f=6.995, p<0.001). Within the treatment group, there were significant correlations between the behavioural parameters and the activation of microglia as measured by PET and immunohistochemistry.Conclusions The present study demonstrated microglia activation in vivo in the prefrontal cortex and hippocampus in mature offspring of prenatal Poly l:C exposed rats.This study suggests that microglia activation may play a possible or potential role in the pathogenesis of schizophrenia.
基金Supported by the National Natural Science Foundation of China (No.81770889)Administration of Traditional Chinese Medicine of Guangdong Province (No.20201070)。
文摘AIM: To study the role of luteolin(LUT) in the expression of toll-like receptors 3(TLR3) ligand poly I:C stimulated inflammatory factors in human corneal fibroblasts(HCFs).METHODS: HCFs cells were cultivated with or without LUT or poly I:C.The expression levels of interleukin(IL)-6, IL-8, monocyte chemotactic protein-1(MCP-1), vascular cell adhesion molecule(VCAM)-1, as well as intercellular adhesion molecule(ICAM)-1 were measured using enzymelinked immunosorbent assay(ELISA), immunoblotting or reverse transcription-quantitative polymerase chain reaction(PCR) analyses.Immunoblotting was used to assess toll-interleukin-1 receptor-domain-containing adapter-inducing interferon-β(TRIF), TLR3, transforming growth factor-bactivated kinase 1(TAK1), tumor necrosis factor receptor-associated factor 6(TRAF6), the transcription factor AP-1, as well as transcription factor nuclear factor(NF-κB)–inhibitory protein IκB-α degradation and phosphorylation.Immunofluorescence assays were used to localize the cellular location of the p65 subunit of NF-κB.RESULTS: Corneal fibroblasts exposed to poly I:C demonstrated decreased VCAM-1, ICAM-1, MCP-1, IL-6, and IL-8 expression levels upon exposure to LUT in a time-dependent and concentration-dependent manner.LUT was observed to suppress poly I:C-triggered expression of TLR3, the translocation of NF-κB p65 into cell nuclei, as well as the phosphorylation of TAK, c-Jun, and IκB-α, while no impact on the expression levels of TRIF and TRAF6 were observed.CONCLUSION: LUT suppress the expression of proinflammatory adhesion molecules, chemokines, and cytokines in poly I:C exposed HCFs.These effects are likely mediated through TAK/NF-κB signal attenuation.Therefore, LUT is a candidate molecule that can prevent the TLR3-mediated inflammation response associated with corneal viral infection.
基金supported by a grant from the National Natural Science Foundation of China(81771309,31930039 and 31821003 to Xin Lin and 82202983 to Haihui Jiang)supported by grants from the Capital’s Funds for Health Improvement and Research(2020-2-1075 to Yong Cui)the National Key Research and Development Program of China(2019YFA0508502 to Xin Lin).
文摘A highly immunosuppressive tumor microenvironment(TME)and the presence of the blood‒brain barrier are the two major obstacles to eliciting an effective immune response in patients with high-grade glioma(HGG).Here,we tried to enhance the local innate immune response in relapsed HGG by intracranially injecting poly(I:C)to establish a robust antitumor immune response in this registered clinical trial(NCT03392545).During the follow-up,12/27(44.4%)patients who achieved tumor control concomitant with survival benefit were regarded as responders in our study.We found that the T-cell receptor(TCR)repertoire in the TME was reshaped after poly(I:C)treatment.Based on the RNA-seq analysis of tumor samples,the expression of annexin A1(ANXA1)was significantly upregulated in the tumor cells of nonresponders,which was further validated at the protein level.In vitro and in vivo experiments showed that ANXA1 could induce the production of M2-like macrophages and microglia via its surface receptor formyl peptide receptor 1(FPR1)to establish a Treg cell-driven immunosuppressive TME and suppress the antitumor immune response facilitated by poly(I:C).The ANXA1/FPR1 signaling axis can inhibit the innate immune response of glioma patients by promoting an anti-inflammatory and Treg-driven TME.Moreover,ANXA1 could serve as a reliable predictor of response to poly(I:C),with a notable predictive accuracy rate of 92.3%.In light of these notable findings,this study unveils a new perspective of immunotherapy for gliomas.
文摘Background The outbreak of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has greatly threatened public health.Recent studies have revealed that the spike receptor-binding domain(RBD)of SARS-CoV-2 is a potent target for vaccine development.However,adjuvants are usually required to strengthen the immunogenicity of recombinant antigens.Different types of adjuvants can elicit different immune responses.Methods We developed an RBD recombinant protein vaccine with a polyriboinosinic acid–polyribocytidylic acid[poly(I:C)]adjuvant to evoke a strong immune response.The delivery of poly(I:C)was optimized in two steps.First,poly(I:C)was complexed with a cationic polymer,poly-l-lysine(PLL),to form poly(I:C)–PLL,a polyplex core.Thereafter,it was loaded into five different lipid shells(group II,III-1,2-distearoyl-sn-glycero-3-phosphocholine[DSPC],III-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine[DOPE],IV-DOPE,and IV-DSPC).We performed an enzyme-linked immunosorbent assay and enzyme-linked immunosorbent spot assay to compare the ability of the five lipopolyplex adjuvants to enhance the immunogenicity of the SARS-CoV-2 RBD protein,including humoral and cellular immune responses.Finally,the adjuvant with the highest immunogenicity was selected to verify the protective immunity of the vaccine through animal challenge experiments.Results Recombinant RBD protein has low immunogenicity.The different adjuvants we developed enhanced the immunogenicity of the RBD protein in different ways.Among the lipopolyplexes,those containing DOPE(III-DOPE and IV-DOPE)elicited RBD-specific immunoglobulin G antibody responses,and adjuvants with four components elicited better RBD-specific immunoglobulin G antibody responses than those containing three components(P<0.05).The IC50 and IC90 titers indicated that the IV-DOPE lipopolyplex had the greatest neutralization ability,with IC50 titers of 1/117,490.Furthermore,in the challenge study,IV-DOPE lipopolyplex protected mice from SARS-CoV-2 infection.On the fourth day after infection,the average animal body weights were reduced by 18.56%(24.164±0.665 g vs.19.678±0.455 g)and 0.06%(24.249±0.683 g vs.24.235±0.681 g)in the MOCK and vaccine groups,respectively.In addition,the relative expression of viral RNA in the vaccinated group was significantly lower than that in the MOCK group(P<0.05).Interstitial inflammatory cell infiltration was observed in the MOCK group,whereas no obvious damage was observed in the vaccinated group.Conclusions The IV-DOPE–adjuvanted SARS-CoV-2 recombinant RBD protein vaccine efficiently protected mice from SARS-CoV-2 in the animal challenge study.Therefore,IV-DOPE is considered an exceptional adjuvant for SARS-CoV-2 recombinant RBD protein-based vaccines and has the potential to be further developed into a SARS-CoV-2 recombinant RBD protein-based vaccine.
基金financially supported by the CAMS Initiative for Innovative Medicine(Grant Number 2017-I2M3-022)Central basic scientific research in colleges and universities(Grant Number 3332019162)+1 种基金the National Natural Science Foundation of China(Grant Number 81503117)the Foundation for Studying Abroad from the China Scholarship Council(Grant Number 201808110121)
文摘The subunit herpes zoster vaccine Shingrix is superior to attenuated vaccine Zostavax in both safety and efficacy,yet its unlyophilizable liposome delivery system and the limited supply of naturally sourced immunological adjuvant QS-21 still need to be improved.Based on poly(lactic-co-glycolic acid)(PLGA)delivery systems that are stable during the lyophilization and rehydration process and using a double-emulsion(w/o/w)solvent evaporation method,we designed a series of nanoparticles with varicella-zoster virus antigen glycoprotein E(VZV-g E)as an antigen and nucleic acids including polyinosinic-polycytidylic acid(Poly I:C)and phosphodiester Cp G oligodeoxynucleotide(Cp G ODN),encapsulated as immune stimulators.While cationic lipids(DOTAP)have more potential than neutral lipids(DOPC)for activating g E-specific cell-mediated immunity(CMI)in immunized mice,especially when g E is encapsulated in and presented on the surface of nanoparticles,PLGA particles without lipids have the greatest potential to induce not only the highest g Especific Ig G titers but also the strongest g E-specific CMI responses,including the highest proportions of interferon-c(IFNc)-and interleukin-2(IL-2)-producing CD4?/CD8?T cells according to a flow cytometry assay and the greatest numbers of IFN-c-and IL-2-producing splenocytes according to an enzyme-linked immunospot(ELISPOT)assay.These results showed that immune-stimulating nucleic acids together with the PLGA delivery system showed promise as a safe and economical varicella and zoster vaccine candidate.
