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Objective:To isolate and culture WU polyomavirus (WUPyV), and to analyze the genome-wide evolutionary patterns, homology and population dynamics.Methods:Real-time quantitative PCR was used to detect the nasopharyngeal aspirate samples of hospitalized children with respiratory tract infection in Beijing Friendship Hospital during 2020 to 2022. Primary human airway epithelial cells cultured at the air-liquid interface were used to isolate and culture WUPyV. Whole genome sequence of the isolated strain was obtained by Sanger sequencing. For phylogenetic and evolutionary dynamics analysis, the whole genome was compared with the published whole genome sequences in GenBank database.Results:The detection rate of WUPyV was 4.7% (31/659) during 2020 to 2022, and a clinical strain BJ0593 of WUPyV type Ⅲc was successfully isolated. The homology of the whole genome and gene fragments of WUPyV was high. The average evolutionary rate of VP2 gene was about 1.256×10 -4 substitution/site every year, and the population dynamics of WUPyV tended to be flat in the last decade. Conclusions:This study successfully isolated a clinical WUPyV type Ⅲ strain for the first time, which provided the basis for further investigation on the molecular evolution and pathogenicity of WUPyV.
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Objective:To investigate the efficacy and prognosis of hypofractionated intensity-modulated radiation therapy combined with hormonal therapy in the treatment of pelvic lymph node metastatic prostate cancer.Methods:Clinical data of 42 IV A prostate cancer patients who received hypofractionated intensity-modulated radiation therapy combined with hormonal therapy in Cancer Hospital of Chinese Academy of Medical Sciences between 2006 and 2018 were retrospectively analyzed. The total irradiation doses to the prostate and seminal vesicles were 67.5 Gy/25f, 2.7 Gy/f. The prophylactic irradiation doses to the pelvic lymph nodes were 45-50 Gy with a daily fraction dose of 1.8-2.0 Gy. Thirty-three patients with residual lymph nodes were boosted to 60.0-67.5 Gy for the residual area, 2.4-2.7 Gy/f. Androgen deprivation therapy included surgical castration or luteinizing hormone-releasing hormone agonists combined with antiandrogens. Survival rate was calculated using Kaplan- Meier method. The differences between two groups were analyzed by log-rank test. Prognostic factors were identified by univariate and multivariate analyses. Results:The median follow-up was 65.5 months (range, 5 to 150 months). The 5-year and 10-year failure-free survival (FFS) rates in the whole group were 67% and 45%, respectively. No clinical recurrence was observed in the irradiation field. The 5-year and 10-year prostate cancer-specific survival/overall survival (PCSS/OS) rates were 85% and 60%, respectively. Gleason score (≥8 and<8) and duration of hormonal therapy impacted the FFS (both P<0.05). The duration of hormonal therapy was an independent prognostic factor for PCSS/OS ( P=0.003). Conclusions:Hypofractionated intensity-modulated radiotherapy combined with hormonal therapy yields optimistic clinical efficacy in the treatment of pelvic lymph node metastatic prostate cancer. Gleason score (≥8 and <8) and duration of hormonal therapy are critical prognostic factors.
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Purpose@#Accumulating evidence has suggested that toll-like receptor 4 (TLR4) is critically involved in the pathogenesis of asthma. The aim of this study was to investigate the role of TLR4 in toluene diisocyanate (TDI)-induced allergic airway inflammation. @*Methods@#TLR4−/− and wild-type (WT) C57BL/10J mice were sensitized and challenged with TDI to generate a TDI-induced asthma model. B-cell lymphoma 2 (Bcl-2) inhibitors, ABT-199 (4 mg/kg) and ABT-737 (4 mg/kg), were intranasally given to TDI-exposed TLR4−/− mice after each challenge. @*Results@#TDI exposure led to increased airway hyperresponsiveness (AHR), granulocyte flux, bronchial epithelial shedding and extensive submucosal collagen deposition, which were unexpectedly aggravated by TLR4 deficiency. Following TDI challenge, TLR4−/− mice exhibited down-regulated interleukin-17A and increased colony-stimulating factor 3 in bronchoalveolar lavage fluid (BALF), while WT mice did not. In addition, TLR4 deficiency robustly suppressed the expression of NOD-like receptor family pyrin domain containing 3 and NLR family CARD domain containing 4, decreased caspase-1 activity in TDI-exposed mice, but had no effect on the level of high mobility group box 1 in BALF. Flow cytometry revealed that TDI hampered both neutrophil and eosinophil apoptosis, of which neutrophil apoptosis was further inhibited in TDI-exposed TLR4−/− mice, with marked up-regulation of Bcl-2. Moreover, inhibition of Bcl-2 with either ABT-199 or ABT-737 significantly alleviated neutrophil recruitment by promoting apoptosis. @*Conclusions@#These data indicated that TLR4 deficiency promoted neutrophil infiltration by impairing its apoptosis via up-regulation of Bcl-2, thereby resulting in deteriorated AHR and airway inflammation, which suggests that TLR4 could be a negative regulator of TDI-induced neutrophilic inflammation.
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Purpose@#Accumulating evidence has suggested that toll-like receptor 4 (TLR4) is critically involved in the pathogenesis of asthma. The aim of this study was to investigate the role of TLR4 in toluene diisocyanate (TDI)-induced allergic airway inflammation. @*Methods@#TLR4−/− and wild-type (WT) C57BL/10J mice were sensitized and challenged with TDI to generate a TDI-induced asthma model. B-cell lymphoma 2 (Bcl-2) inhibitors, ABT-199 (4 mg/kg) and ABT-737 (4 mg/kg), were intranasally given to TDI-exposed TLR4−/− mice after each challenge. @*Results@#TDI exposure led to increased airway hyperresponsiveness (AHR), granulocyte flux, bronchial epithelial shedding and extensive submucosal collagen deposition, which were unexpectedly aggravated by TLR4 deficiency. Following TDI challenge, TLR4−/− mice exhibited down-regulated interleukin-17A and increased colony-stimulating factor 3 in bronchoalveolar lavage fluid (BALF), while WT mice did not. In addition, TLR4 deficiency robustly suppressed the expression of NOD-like receptor family pyrin domain containing 3 and NLR family CARD domain containing 4, decreased caspase-1 activity in TDI-exposed mice, but had no effect on the level of high mobility group box 1 in BALF. Flow cytometry revealed that TDI hampered both neutrophil and eosinophil apoptosis, of which neutrophil apoptosis was further inhibited in TDI-exposed TLR4−/− mice, with marked up-regulation of Bcl-2. Moreover, inhibition of Bcl-2 with either ABT-199 or ABT-737 significantly alleviated neutrophil recruitment by promoting apoptosis. @*Conclusions@#These data indicated that TLR4 deficiency promoted neutrophil infiltration by impairing its apoptosis via up-regulation of Bcl-2, thereby resulting in deteriorated AHR and airway inflammation, which suggests that TLR4 could be a negative regulator of TDI-induced neutrophilic inflammation.
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Objective To compare the dosimetric data between preoperative plans and postoperative verification in computed tomography (CT)-guided and 3D-printing non-coplanar templateassisted 125I seed implantation for pelvic tumor,and to explore the feasibility and accuracy of the personalized template designmethod.Methods A total of 51 patients registered from Dec 2015 to Dec 2016 who were applied with 3D-printing guided template assisted radioactive seed implantations in the hospital were included in this study.A prescribed dose of 110-160 Gy was adopted.3D-printing templates were designed and produced for 51 cases.The dosimetric parameters:Dg0,minimum peripheral dose (mPD),V100,V150,V200,conformal index (CI),external index (EI),and homogeneity index (HI) were compared between pre-and post-plans.Results 51 cases' templates were in place well during the operations.Compared with the preoperative planning,the postoperative D90,V100,V150,V200,CI,EI and HI differences had no statistical difference (P ＞ 0.05);mPD is larger than before (t =-2.96,P ＜ 0.05).Conclusions The main dosimetric parameters of postoperative verification were consistent well with the preoperative planning and have good accuracy,which could meet the clinical requirements.
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Objective@#To express envelope protein of ZIKA virus in baculovirus expression system.@*Methods@#Full-length E gene of ZIKA virus was obtained by DNA synthesis and inserted into vector pFastBac1. The constructed recombinant baculovirus transfer vector pFB1-E was transformed to competent DH10Bac cells. The obtained skeleton plasmid rBacmid-E was transfected to sf9 cells, and the constructed recombinant baculovirus rBac-E was determined for titer, for insertion of E gene by PCR, and for expression of E protein by IFA and Western blotting.@*Results@#PCR proved that skeleton plasmid rBacmid-E was constructed correctly. The titer of rBac-E of passage 3 was 2.58×105pfu/ml. The genome of infected cells virus was extracted, the gene band at length of 3 830 bp was observed after PCR amplification. Indirect immunofluorescence of the infected cells showed the specific green fluorescence, 55×103specific band was determined by Western blotting identification in the cell pellet of the infected recombinant baculovirus rBac-E.@*Conclusions@#The recombinant baculovirus with E gene of ZIKA virus was successfully constructed, which laid a foundation of further study on the function of E protein and the vaccine of ZIKA virus.
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Objective To explore the role of ethyl pyruvate (EP) on E-cadherin of airway epithelium and airway inflammation in a TDI-induced mouse asthma model. Methods 30 male BALB/c mice were randomly divided into control group , asthma group and EP group. On day 1 and 8 , mice in asthma group and EP group were treated with 0.3%TDI on the dorsum of both ears for sensitization. And on day 15 , 18 and 21 the mice underwent an aerosol inhalation of 3% TDI, and saline (100 mg/kg) was injected intraperitoneally 1 hour before inhalation. The control group underwent acetone and olive oil (AOO) sensitization on day 1 and 8, AOO challenge on day 15, 18 and 21. Saline (100 mg/kg) was injected intraperitoneally 1 hour before challenge. One hour before each challenge, mice were given EP (100mg/kg) or vehicle via intraperitoneal injection. On day 22, airway reactivity, IL-4 , IFN-γand IgE in the serum were detected , immunohistochemistry and WB were used to assess E-cadherin levels. Results Airway reactivity, IL-4, IFN-γin and IgE in the serum in asthma group are significantly higher than that in control group (P<0.05). Treatment with EP dramatically decreased airway hyperresponsiveness in TDI-challenged mice, as well as IL-4, IFN-γ and IgE (P < 0.05). E-cadherin in control group was distributed evenly at the connection of epithelial cells. E-cadherinin distribution was chaotic and its expression was decreased in asthma group. EP intervention can ameliorate the damage of E-cadherinin. Conclusions EP can ameliorate the destruction of E-cadherin in airway epithilum by TDI.
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<p><b>OBJECTIVE</b>To investigate the effect of 1,25-dihydroxyvitamin D3 (1,25VD3) on house dust mites (HDM)-induced expression of thymic stromal lymphopoietin (TSLP) in human airway epithelial cells in vitro.</p><p><b>METHODS</b>Human airway epithelial 16HBE cells were incubated with 200, 400, and 800 U/L in the absence or presence of 1,25VD3 (10(-8) mol/L) for 6 h and 24 h, and TSLP mRNA and protein expressions in the cells were assessed using quantitative PCR and ELISA.</p><p><b>RESULTS</b>16HBE cells incubated with HDM at 200, 400, and 800 U/L showed significantly increased TSLP mRNA and protein expressions (P<0.05). Pretreatment of the cells with 1,25VD3 obviously lowered 400 U/L HDM-induced TSLP expressions (P<0.05), but 1,25VD3 added along with HDM in the cells did not produce significant effects on TSLP expressions (P=0.58).</p><p><b>CONCLUSION</b>Both 1,25VD3 and HDM can induce TSLP expression and release in 16HBE cells, but pretreatment with 1,25VD3 can decrease HDM-augmented TSLP expression in the cells.</p>
Subject(s)
Animals , Humans , Bronchi , Cell Biology , Calcitriol , Pharmacology , Cell Line , Cytokines , Metabolism , Epithelial Cells , Metabolism , PyroglyphidaeABSTRACT
Matrix protein 2(M2) is an integral tetrameric membrane protein of influenza A virus, which functions as ion channel. M2 sequence has shown remarkable conservation, so there has been growing interest in it as "universal" vaccine. In order to establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator, M2 gene was obtained by PCR amplification from the plasmid containing the segment 7 of influenza A virus strain A/PR/8/34 firstly. The PCR product was cloned into BamH I/Not I restriction site of pcDNA5/FRT/TO vector, and cotransfected with pOG44 which express Flp recombinase into Flp-In T-REx-293 cell. Integration of pcDNA5/FRT/TO-M2 into the cell genome at the Flp Recombination Target (FRT) site brought the SV40 promoter and the initiation codon in frame with the hygromycin resistance gene. Thus, stable cell lines were selected for hygromycin resistance. The expression of M2 protein from hygromycin-resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay (IFA). 16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator. The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.
Subject(s)
Animals , Humans , Cloning, Molecular , Gene Expression , Genetic Vectors , Genetics , HEK293 Cells , Influenza A virus , Genetics , Metabolism , Influenza Vaccines , Genetics , Operator Regions, Genetic , Recombinant Proteins , Genetics , Tetracycline , Pharmacology , Transfection , Viral Matrix Proteins , GeneticsABSTRACT
In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines.
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Animals , Mice , Adenoviridae , Genetics , Metabolism , Antibodies, Viral , Blood , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Allergy and Immunology , Immunization , Influenza A Virus, H5N1 Subtype , Allergy and Immunology , Influenza Vaccines , Allergy and Immunology , Mice, Inbred BALB C , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Vaccines, DNA , Allergy and Immunology , Viral Matrix Proteins , Genetics , Allergy and ImmunologyABSTRACT
We developed vectors expressing two antigen of H5N1 influenza virus. Based on the human H5N1 avian influenza virus strain A/Anhui/1/2005 isolated in China, we amplified the matrix protein 2 (M2) and Hemagglutinin (HA) genes by PCR and subcloned them into pStar vector to construct two genes co-expressing recombinant DNA vaccine pStar-M2/HA. After transfection of 293 cells with the plasmid, we confirmed with indirect immunofluorescence assay (IFA) that M2 and HA genes cloned on plasmid pStar co-expressed successfully. Using Ad-Easy adenovirus vector system, by homologous recombination in bacteria and packaging in 293 cells, we constructed two recombinant adenoviruses, namely Ad-M2 and Ad-HA. After infection of 293 cells with the recombinant adenoviruses, we confirmed with IFA that M2 and HA genes cloned into adenoviruses expressed successfully. We then combined the recombinant DNA vaccine and adenoviral vector vaccines in immunization of BALB/c mice with a prime-boost regime. On day 0 and day 28, we immunized the mice with DNA vaccine and on day 14 and day 42, with recombinant adenovirus vaccines. We took blood samples before each injection and 14 days after the final injection. On day 56, we collected splenocytes from the mice. ELISA and hemagglutination inhibition (HI) assay showed that the vaccines successfully induced specific IgG antibodies against HA protein in serum of the immunized mice. ELISPOT confirmed that the vaccines successfully induced the special cellular immune response to M2 and HA protein of H5N1 influenza virus. The study on combined immunization with M2 and HA genes provided basis for development of novel influenza vaccine.
Subject(s)
Animals , Female , Mice , Adenoviridae , Genetics , Metabolism , Genetic Vectors , Genetics , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Influenza A Virus, H5N1 Subtype , Genetics , Allergy and Immunology , Influenza Vaccines , Allergy and Immunology , Mice, Inbred BALB C , Recombinant Proteins , Genetics , Allergy and Immunology , Vaccination , Vaccines, DNA , Allergy and Immunology , Viral Matrix Proteins , GeneticsABSTRACT
Aim To study the expression of human VEGF165 cDNA in Pichia pastoris and to obtain high-level expressed recombinant human VEGF165 with good biological activity. Methods Amplifying human VEGF165 cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α secreting signal peptides, the recombinant expression plasmids pPIC9K/hVEGF165 was constructed and transformed into KM71. The multiple insert transformants were screened， fermented in flasks and induced by 10 mL/L methanol. Results After 4 days of methanol induction, the expressed hVEGF165 reached up to 60％ of total proteins in supernatant by SDS-PAGE. Western blot assay proved the expressed hVEGF165 having good antigenicity and high specificity. The recombinant protein was further purified with Heparin-Sepharose CL6B affinity chromatography, and was proved to have good biological activity in stimulating HUVEC proliferation. Conclusion High-level expression of secreted hVEGF165 has been successfully achieved in Pichia pastoris expression system.