Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three t...Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.展开更多
Mannan-binding lectin(MBL)plays a key role in the lectin pathway of complement activation and can influence cytokine expression.Toll-like receptor 4(TLR4)is expressed extensively and has been demonstrated to be involv...Mannan-binding lectin(MBL)plays a key role in the lectin pathway of complement activation and can influence cytokine expression.Toll-like receptor 4(TLR4)is expressed extensively and has been demonstrated to be involved in lipopolysaccharide(LPS)-induced signaling.We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-kB(NF-kB)activity by using the monocytoid cell line THP-1.We then investigated the possible mechanisms underlying any observed regulatory effect.Using ELISA and reverse transcriptase polymerase chain reaction(RT-PCR)analysis,we found that at both the protein andmRNAlevels,treatment withMBLsuppresses LPS-induced tumor-necrosis factor(TNF)-a and IL-12 production in THP-1 cells.An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-kB DNA binding and translocation in THP-1 cells.While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations,this binding occurred optimally in response to supraphysiological calcium concentrations.This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody(HTA125).Activation of THP-1 cells by LPS treatment resulted in increased MBL binding.We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner,and this interaction could attenuate the binding of LPS to cell surfaces.Taken together,these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways.These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks.展开更多
Objective:To purify Mannan-binding lectin(MBL)from human serum and detect its binding ability to several kindsof bacteria common in infections diseases of children.Methods:MBL was purifide from human serum by affinity...Objective:To purify Mannan-binding lectin(MBL)from human serum and detect its binding ability to several kindsof bacteria common in infections diseases of children.Methods:MBL was purifide from human serum by affinity chromatographyon mannan-Sepharose 4B column.Its binding ability to eight species,97 stratus of bacteria was detected by enzyme-linked lectinassay(ELLA).Results:MBL has different binding ability to bacteria and shows strong binding ability to Klebsiella ornithinolvticaand Escherichia coli,but shows relatively lower binding ability to Staphylococcus haemolyticus,Enterobacter cloacae andStaphylococcus epidermidis.To different isolates of Klebsiella pneumoniae,Haemophilus influenzae and Staphylococcus aureus,MBL shows quite different binding ability.Conclusions:MBL has different binding ability to different bacteria,and has relativelystronger binding ability to Gram-negative bacteria.Its binding ability to different isolates of certain kinds of bacteria is quitedifferent.展开更多
Objective In this study,mannan-binding lectin-associated serine protease 2(MASP2)gene variant was evaluated to assess the risk of radiation pneumonitis(RP)in patients with pulmonary malignancies.Methods A total of 169...Objective In this study,mannan-binding lectin-associated serine protease 2(MASP2)gene variant was evaluated to assess the risk of radiation pneumonitis(RP)in patients with pulmonary malignancies.Methods A total of 169 lung cancer patients with radiotherapy were included in our prospective study(NCT02490319)and genotyped using the Sanger sequencing method.Multivariate Cox hazards analysis and multiple testing were applied to estimate the hazard ratio(HR)and 95%confidence intervals(CIs)of all factors possibly associated with RP risk.Results Patients with mean lung disease≥15 Gy and V20≥24%had higher risk of RP≥grade 2 compared with their counterparts(HR=1.888,95%CI:1.186-3.004,P=0.007;HR=2.126,95%CI:1.338-3.378,P=0.001,respectively).Importantly,CC+CA genotype of MASP2:rs12711521 was strongly associated with an increased occurrence of RP≥grade 2(HR=1.949,95%CI:1.278-2.971,P=0.002).Conclusion MASP2:rs12711521 was found to be significantly associated with RP≥grade 2 in our cohort and may thus be one of the important predictors of severe RP before radiotherapy,if further validated in larger population.展开更多
基金funded by the National Key R&D Program of China[2021YFC2301102]National Natural Science Foundation of China[82202593]Key R&D Program of Hebei Province[223777100D].
文摘Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.
基金This work was supported by the Natural Science Foundation of China(30972679).
文摘Mannan-binding lectin(MBL)plays a key role in the lectin pathway of complement activation and can influence cytokine expression.Toll-like receptor 4(TLR4)is expressed extensively and has been demonstrated to be involved in lipopolysaccharide(LPS)-induced signaling.We first sought to determine whether MBL exposure could modulate LPS-induced inflammatory cytokine secretion and nuclear factor-kB(NF-kB)activity by using the monocytoid cell line THP-1.We then investigated the possible mechanisms underlying any observed regulatory effect.Using ELISA and reverse transcriptase polymerase chain reaction(RT-PCR)analysis,we found that at both the protein andmRNAlevels,treatment withMBLsuppresses LPS-induced tumor-necrosis factor(TNF)-a and IL-12 production in THP-1 cells.An electrophoretic mobility shift assay and western blot analysis revealed that MBL treatment can inhibit LPS-induced NF-kB DNA binding and translocation in THP-1 cells.While the binding of MBL to THP-1 cells was evident at physiological calcium concentrations,this binding occurred optimally in response to supraphysiological calcium concentrations.This binding can be partly inhibited by treatment with either a soluble form of recombinant TLR4 extracellular domain or anti-TLR4 monoclonal antibody(HTA125).Activation of THP-1 cells by LPS treatment resulted in increased MBL binding.We also observed that MBL could directly bind to the extracellular domain of TLR4 in a dose-dependent manner,and this interaction could attenuate the binding of LPS to cell surfaces.Taken together,these data suggest that MBL may affect cytokine expression through modulation of LPS-/TLR-signaling pathways.These findings suggest that MBL may play an important role in both immune regulation and the signaling pathways involved in cytokine networks.
文摘Objective:To purify Mannan-binding lectin(MBL)from human serum and detect its binding ability to several kindsof bacteria common in infections diseases of children.Methods:MBL was purifide from human serum by affinity chromatographyon mannan-Sepharose 4B column.Its binding ability to eight species,97 stratus of bacteria was detected by enzyme-linked lectinassay(ELLA).Results:MBL has different binding ability to bacteria and shows strong binding ability to Klebsiella ornithinolvticaand Escherichia coli,but shows relatively lower binding ability to Staphylococcus haemolyticus,Enterobacter cloacae andStaphylococcus epidermidis.To different isolates of Klebsiella pneumoniae,Haemophilus influenzae and Staphylococcus aureus,MBL shows quite different binding ability.Conclusions:MBL has different binding ability to different bacteria,and has relativelystronger binding ability to Gram-negative bacteria.Its binding ability to different isolates of certain kinds of bacteria is quitedifferent.
基金Supported by grants from the National Natural Science Foundation of China(No.81773360,81700145).
文摘Objective In this study,mannan-binding lectin-associated serine protease 2(MASP2)gene variant was evaluated to assess the risk of radiation pneumonitis(RP)in patients with pulmonary malignancies.Methods A total of 169 lung cancer patients with radiotherapy were included in our prospective study(NCT02490319)and genotyped using the Sanger sequencing method.Multivariate Cox hazards analysis and multiple testing were applied to estimate the hazard ratio(HR)and 95%confidence intervals(CIs)of all factors possibly associated with RP risk.Results Patients with mean lung disease≥15 Gy and V20≥24%had higher risk of RP≥grade 2 compared with their counterparts(HR=1.888,95%CI:1.186-3.004,P=0.007;HR=2.126,95%CI:1.338-3.378,P=0.001,respectively).Importantly,CC+CA genotype of MASP2:rs12711521 was strongly associated with an increased occurrence of RP≥grade 2(HR=1.949,95%CI:1.278-2.971,P=0.002).Conclusion MASP2:rs12711521 was found to be significantly associated with RP≥grade 2 in our cohort and may thus be one of the important predictors of severe RP before radiotherapy,if further validated in larger population.
