Objective Human adenovirus(HAdV)infection is common and can develop to serious conditions with high mortality,yet the mechanism of HAdV infection remains unclear.In the present study,the serum metabolite profiles of H...Objective Human adenovirus(HAdV)infection is common and can develop to serious conditions with high mortality,yet the mechanism of HAdV infection remains unclear.In the present study,the serum metabolite profiles of HAdV-7-infected patients with pneumonia or upper respiratory tract infection(URTI)were explored.Methods In total,35 patients were enrolled in the study following an outbreak of HAdV-7 in the army,of whom 14 had pneumonia and 21 had URTI.Blood samples were collected at the acute stage and at the recovery stage and were analyzed by untargeted metabolomics.Results Over 90% of the differential metabolites identified between the pneumonia patients and URTI patients were lipids and lipid-like molecules,including glycerophospholipids,fatty acyls,and sphingolipids.The metabolic pathways that were significantly enriched were primarily the lipid metabolism pathways,including sphingolipid metabolism,glycerophospholipid metabolism,and linoleic acid metabolism.The sphingolipid metabolism was identified as a significantly differential pathway between the pneumonia patients and URTI patients and between the acute and recovery stages for the pneumonia patients,but not between the acute and recovery stages for the URTI patients.Ceramide and lactosylceramide,involved in sphingolipid metabolism,were significantly higher in the pneumonia patients than in the URTI patients with good discrimination abilities[area under curve(AUC)0.742 and 0.716,respectively;combination AUC 0.801].Conclusion Our results suggested that HAdV modulated lipid metabolism for both the patients with URTI and pneumonia,especially the sphingolipid metabolism involving ceramide and lactosylceramide,which might thus be a potential intervention target in the treatment of HAdV infection.展开更多
Rhodioloside has been shown to protect cells from hypoxia injury,and bone marrow mesenchymal stem cells have a good effect on tissue repair.To study the effects of rhodioloside and bone marrow mesenchymal stem cells o...Rhodioloside has been shown to protect cells from hypoxia injury,and bone marrow mesenchymal stem cells have a good effect on tissue repair.To study the effects of rhodioloside and bone marrow mesenchymal stem cells on spinal cord injury,a rat model of spinal cord injury was established using the Infinite Horizons method.After establishing the model,the rats were randomly divided into five groups.Rats in the control group were intragastrically injected with phosphate buffered saline(PBS)(5μL).PBS was injected at 6 equidistant points around 5 mm from the injury site and at a depth of 5 mm.Rats in the rhodioloside group were intragastrically injected with rhodioloside(5 g/kg)and intramuscularly injected with PBS.Rats in the mesenchymal stem cell(MSC)group were intramuscularly injected with PBS and intramuscularly with MSCs(8×10^6/mL in a 50-μL cell suspension).Rats in the Ad-HIF-MSC group were intragastrically injected with PBS and intramuscularly injected with HIF-1 adenovirus-infected MSCs.Rats in the rhodioloside+Ad-HIF-MSC group were intramuscularly injected with MSCs infected with the HIF-1 adenovirus and intragastrically injected with rhodioloside.One week after treatment,exercise recovery was evaluated with a modified combined behavioral score scale.Hematoxylin-eosin staining and Pischingert’s methylene blue staining were used to detect any histological or pathological changes in spinal cord tissue.Levels of adenovirus IX and Sry mRNA were detected by real-time quantitative polymerase chain reaction and used to determine the number of adenovirus and mesenchymal stem cells that were transfected into the spinal cord.Immunohistochemical staining was applied to detect HIF-1 protein levels in the spinal cord.The results showed that:(1)compared with the other groups,the rhodioloside+Ad-HIF-MSC group exhibited the highest combined behavioral score(P<0.05),the most recovered tissue,and the greatest number of neurons,as indicated by Pischingert’s methylene blue staining.(2)Compared with the PBS group,HIF-1 protein expression was greater in the rhodioloside group(P<0.05).(3)Compared with the Ad-HIF-MSC group,Sry mRNA levels were higher in the rhodioloside+Ad-HIF-MSC group(P<0.05).These results confirm that rhodioloside combined with bone marrow mesenchymal stem cells can promote the recovery of spinal cord injury and activate the HIF-1 pathway to promote the survival of bone marrow mesenchymal stem cells and repair damaged neurons within spinal cord tissue.This experiment was approved by the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine,China(approval No.2015KYLL029)in June 2015.展开更多
BACKGROUND:Interleukin 10(IL-10),a Th2 type cytokine,modulates inflammatory responses by inhibiting the production of proinflammatory cytokines.This study was designed to investigate the protective effects of adenovir...BACKGROUND:Interleukin 10(IL-10),a Th2 type cytokine,modulates inflammatory responses by inhibiting the production of proinflammatory cytokines.This study was designed to investigate the protective effects of adenovirus- mediated human IL-10(Ad-hIL-10)gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS:Adenoviruses encoding hIL-10 orβ-galactosidase (Ad-lacZ)were injected via the superior mesenteric vein into prospective donor animals.The donor liver was harvested 48 hours after transduction,and stored for 12 hours at 4℃ in lactated Ringer's solution prior to transplantation.The rats were divided into saline,Ad-lacZ,and Ad-hIL-10 groups.Liver function test,histopathological examination,reverse transcriptase-polymerase chain reaction(RT-PCR),and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS:Liver function(ALT and AST)was significantly improved,and the Suzuki score was significantly decreased in the Ad-hIL-10 group.The levels of hepatic TNF-α,MIP-2,ICAM-1 mRNA,and NF-κB protein in the Ad-hIL-10 group were significantly decreased.The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS:Donor pretreatment with Ad-hIL-10 down- regulates the expression of proinflammatory cytokines TNF-α,MIP-2,and ICAM-1 mRNA.hIL-10 protects against hepatic cold ischemia-reperfusion injury,at least in part,by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.展开更多
BACKGROUND:Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24(IL-24)is a novel tumor suppressor gene,which has suppressor activity in a broad spectrum of human cancer cells.We investigated the effect of...BACKGROUND:Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24(IL-24)is a novel tumor suppressor gene,which has suppressor activity in a broad spectrum of human cancer cells.We investigated the effect of the replication-competent oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24,both expressing human MDA-7/IL-24 on the hepatocellular carcinoma cell lines HepG2,Hep3B,SMMC-7721,HCCLM3,and the normal liver cell line L02. METHODS:Hepatocellular carcinoma cell lines and the normal liver cell line were infected with SG600-IL24 and Ad.IL-24.The mRNA and protein expression of MDA-7/IL-24 in infected cells was confirmed by RT-PCR,ELISA,and Western blotting.MTT assay was used to investigate the proliferation effect.Hoechst staining and Annexin-V and PI staining were performed to study the MDA-7/IL-24 gene expressed in HCC cell lines and the normal liver cell line.Flow cytometry was used to analyse the cell cycle. RESULTS:RT-PCR,ELISA and Western blotting confirmed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24.MTT and apoptosis detection indicated that SG600-IL24 induced growth suppression promoted apoptosis,and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line. CONCLUSIONS:SG600-IL24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma cell lines in vitro but not in the normal liver cell line L02. Compared with Ad.IL-24,SG600-IL24 dramatically enhances antitumor activity in hepatocellular carcinoma cell lines.展开更多
Background: Patients with intermediate to advanced hepatocellular carcinoma(HCC) are most commonly treated with transarterial chemoembolization(TACE). Previous studies showed that TACE combined with recombinant human ...Background: Patients with intermediate to advanced hepatocellular carcinoma(HCC) are most commonly treated with transarterial chemoembolization(TACE). Previous studies showed that TACE combined with recombinant human adenovirus type 5(H101) may provide a clinical survival benefit. In the present study, we aimed to determine the survival benefit of TACE with or without H101 for patients with intermediate to advanced HCC and to develop an e ective nomogram for predicting individual survival outcomes of these patients.Methods: We retrospectively collected data from 590 patients with intermediate to advanced HCC who were treated at Sun Yat?sen University Cancer Center between January 2007 and July 2015. After propensity score matching, 238 patients who received TACE with H101(TACE with H101 group) and 238 patients who received TACE without H101(TACE group) were analyzed. Overall survival(OS) was evaluated using the Kaplan–Meier method; the nomogram was developed based on Cox regression analysis. Discrimination and calibration were measured using the concordance index(c?index) and calibration plots.Results: Clinical and radiologic features were similar between the two groups. OS rates were significantly lower in the TACE group than in the TACE with H101 group(1?year OS rate, 53.8% vs. 61.3%; 2?year OS rate, 33.4% vs. 44.2%; 3?year OS rate, 22.4% vs. 40.5%; all P < 0.05). Multivariate Cox regression analysis for the entire cohort showed that alpha?fetoprotein level, alkaline phosphatase level, tumor size, metastasis, vascular invasion, and TACE with or without H101 were independent factors for OS, all of which were included in the nomogram. Calibration curves showed good agreement between nomogram?predicted survival and observed survival. The c?index of the nomogram for predict?ing OS was 0.716(95% confidence interval 0.686–0.746).Conclusions: TACE plus H101 extends the survival of patients with intermediate to advanced HCC. Our proposed nomogram provides individual survival prediction and stratification for patients with intermediate to advanced HCC who receive TACE with or without H101.展开更多
BACKGROUND:Melanoma differentiation associated gene-7(mda-7)is a novel tumor suppressor gene,which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of vari...BACKGROUND:Melanoma differentiation associated gene-7(mda-7)is a novel tumor suppressor gene,which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways.In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma(HCC)in vitro. METHODS:Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum(control).The others were transfected with adenovirus expressing the mda-7 gene(Ad.mda-7)or adenovirus vector serving as negative control(Ad.vec).The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle.Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells.The expression of MDA-7,Bcl-2 and Bax proteins were detected by Western blotting.RESULTS:The mda-7 gene was expressed in Hep3B and L-02 cells.The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml,respectively.mda-7 induced Hep3B growth suppression and apoptosis,compared with Ad.mda-7 and control(P<0.01).In addition,cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein,but this was not found in L-02.The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS:mda-7 selectively induces growth inhibi- tion and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti- apoptosis protein Bcl-2.It could be an ideal gene for gene therapy in HCC.展开更多
BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment o...BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.展开更多
OBJECTIVE: To evaluate the killing effects of the cytosine deaminase （CD） gene mediated byadenovirus vector on human pancreatic cancer cell lines in vitro.METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pA...OBJECTIVE: To evaluate the killing effects of the cytosine deaminase （CD） gene mediated byadenovirus vector on human pancreatic cancer cell lines in vitro.METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-lwere recombinated in bacteria. The newly recombinated Ad-CD containing green fluoreseent protein（GFP） was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Humanpancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added.XTT assay was used to estimate relative numbers of viable cells.RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and theconcentration of viral liquids containing the CD gene was 2×1O<sup>11</sup> pfu/ml. It was found that significantcytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but littleeffects exerted on the nontransduced pancreatic carcinoma cells.CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for genetherapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzymeprodrug strategy in experimental pancreatic cancer.展开更多
To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF 165 ), the cDNA for hVEGF 165 was subcloned into pACCMV·pLpA. Subsequently, this recombinant ...To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF 165 ), the cDNA for hVEGF 165 was subcloned into pACCMV·pLpA. Subsequently, this recombinant pACCMV·hVEGF was co transfected into 293 cells together with pJM17 to obtain the replication deficient recombinant adenovirus containing hVEGF gene-AdCMV·hVEGF. The VEGF gene expression was detected by using RT PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSMC) infected with AdCMV· hVEGF. Cultured human umbilical vein endothelial cells (HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV·hVEGF to observe the effect of VEGF on proliferation of HUVEC. 48 h after the infection with AdCMV·hVEGF, VSMC demonstrated VEGF expression, and the expressed VEGF could stimulate the proliferation of HUVEC in vitro . Successfully prepared AdCMV·hVEGF 165 could express biologically active VEGF in infected VSMC, and stimulate proliferation of HUVEC.展开更多
AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6(HyperIL-6, HIL-6) and hepatocyte growth factor(HGF)(AdHGF-HIL-6) compared to that of recombinant adenovirus containi...AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6(HyperIL-6, HIL-6) and hepatocyte growth factor(HGF)(AdHGF-HIL-6) compared to that of recombinant adenovirus containing either HIL-6 or HGF(Ad-HIL-6 or Ad-HGF) in rats with acute-on-chronic liver failure(ACLF).METHODS: The recombinant adenoviruses containing HIL-6 and/or HGF were constructed. We established an ACLF model, and rats were randomly assigned to control, model, Ad-GFP, Ad-HIL-6, Ad-HGF or AdHGF-HIL-6 group. We collected serum and liver tissue samples to test pathological changes, biochemical indexes and molecular biological indexes.RESULTS: Attenuated alanine aminotransferase, prothrombin time, high-mobility group box 1(HMGB1), endotoxin, tumour necrosis factor(TNF)-α and interferon-γ were observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGFHIL-6-treated rats with ACLF. Likewise, reduced hepaticdamage and apoptotic activity, as well as reduced HMGB1 and Bax proteins, but raised expression of Ki67 and Bcl-2 proteins and Bcl-2/Bax ratio were also observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. More significant changes were observed in the Ad-HGF-HIL-6 treatment group without obvious side effects. Furthermore, caspase-3 at the protein level decreased in the Ad-HIL-6 and Ad-HGFHIL-6 treatment groups, more predominantly in the latter group.CONCLUSION: This study identifies that the protective efficacy of Ad-HGF-HIL-6 is more potent than that of Ad-HGF or Ad-HIL-6 in ACLF rats, with no significant side effects.展开更多
Background:In 2014,an outbreak of adenoviral pneumonia occurred in the Korean military training center.However,there are limited data on the characteristics of the fever and its response to antipyretic therapy in immu...Background:In 2014,an outbreak of adenoviral pneumonia occurred in the Korean military training center.However,there are limited data on the characteristics of the fever and its response to antipyretic therapy in immunocompetent adults with adenovirus-positive community-acquired pneumonia(CAP).Methods:The medical records of the patients who were admitted to the Armed Forces Chuncheon Hospital for the treatment of CAP between January 2014 and December 2016 were retrospectively analyzed.The patients were divided into three groups,namely,the adenovirus-positive(Adv)group,the adenovirus-negative(Non-Adv)group and the unknown pathogen group,according to the results of a polymerase chain reaction(PCR)test and sputum culture used to measure adenovirus and other bacteria or viruses in respiratory specimens.We evaluated and compared the demographics,clinicolaboratory findings and radiological findings upon admission between the two groups.Results:Out of the 251 military personnel with CAP during the study periods,67 were classified into the Adv group,while 134 were classified into the Non-Adv group and 50 were classified into the unknown pathogen group.The patients in the Adv group had a longer duration of fever after admission((3.2±1.6)d vs.(1.9±1.2)d vs.(2.2±1.5)d,P=0.018)and symptom onset((5.8±2.2)d vs.(3.9±2.5)d vs.(3.7±2.0)d,P=0.006)than patients in the Non-Adv and unknown pathogen groups,respectively.The patients in the Adv group had a higher mean temperature at admission(37.8±0.3 vs.37.3±0.3 vs.37.3±0.3,P=0.005),and more patients were observed over 40 and 39 to 40(14.9%vs.2.2%vs.4.0%,35.8%vs.3.7%vs.6.0%,P<0.001)than those in the Non-Adv and unknown pathogen groups,respectively.The Adv group more commonly had no response or exhibited adverse events after antipyretic treatment compared to the Non-Adv group(17.9%vs.1.5%,35.0%vs.4.3%,P<0.001,P=0.05,respectively).In addition,the time from admission to overall clinical stabilization was significantly longer in the patients in the Adv group than in those in the Non-Adv group((4.3±2.8)d vs.(2.9±1.8)d,P=0.034,respectively).Furthermore,no significant difference in the length of hospital stay was observed between the two groups,and no patient died in either group.Conclusions:In this study,Adv-positive CAP in immunocompetent military personnel patients had distinct fever characteristics and responses to antipyretic treatment.展开更多
AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2...AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), enzymelinked immunosorbent assay (ELISA), and Western blotting, respectively. Apoptosis of HCC cells and normal liver cells was detected by cytometric assay with Hoechst33258 staining. 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to investigate proliferation of HCC cells and normal liver cells, and cell cycle was assayed by flow cytometry. RESULTS: RT-PCR, ELISA and Western blotting showed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT indicated that SG600-IL24 could suppress the growth of HepG2, Hep3B, MHCC97L, with an inhibition rate of 75% ± 2.5%, 85% ± 2.0%, 72% ± 1.8%, respectively (P < 0.01), promote the apoptosis of HepG2, Hep3B, MHCC97L, with an apoptosis rate of 56.59% ± 4.0%, 78.36% ± 3.5%, 43.39% ± 2.5%, respectively (P < 0.01), and block the HCC cell lines in the G2/M phase with a blocking rate of 35.4% ± 4.2%, 47.3% ± 6.2%, 42% ± 5.0%, respectively (P < 0.01) but not the normal liver cell line in a p53-independent manner. CONCLUSION: SG600-IL24 can selectively suppress the proliferation and apoptosis of HCC cell lines in vitro but not normal liver cell line L02 in a p53-independent manner. Compared with Ad.IL-24, SG600-IL24 can significantly enhance the antitumor activity in HCC cell lines.展开更多
BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte i...BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation.METHODS:A normal human liver cell line(L02) was transfected with adenovirus vector containing the CTLA4Ig gene(Ad-CTLA4Ig-EGFP) in vitro,and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining.Transfected cells then were injected into the spleen of Sprague-Dawley rats,the survival of cells was determined by immunohistochemistry,and the immune status was examined through CD4+ and CD69+ T cellcounts and ELISA detection of IL-2 in peripheral blood.RESULTS:L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks.Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation,while none was detected in the control group.Furthermore,the percentages of CD4+ and CD4+ CD69+T cells in the CTLA4-transfected group were 24.5% and 45.1%,markedly lower than those in the control group at 4 weeks post-transplantation(P<0.01).Furthermore,the IL-2 level was also lower in the CTLA4transfected group than in the control group.CONCLUSION:Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.展开更多
Human adenoviruses (HAdVs) belong to the genus Mastadenovirus, family Adenoviridae, and can cause respiratory tract and gastrointestinal tract infections, as well as ocular infections in humans[1]. Till date, a total ...Human adenoviruses (HAdVs) belong to the genus Mastadenovirus, family Adenoviridae, and can cause respiratory tract and gastrointestinal tract infections, as well as ocular infections in humans[1]. Till date, a total of 84 unique genotypes of AdVs have been identified and classified as human Mastadenovirus species A to G, and specific types are often associated with certain clinical symptoms, epidemiological settings, and demographic risk groups[2]. Among these species, members of the species HAdV-B (HAdV-3, HAdV-7, HAdV-11, HAdV-16, HAdV-21, HAdV-34, HAdV-35, HAdV-50, etc), HAdV-C (HAdV-1, HAdV-2, HAdV-5, and HAdV-6), and HAdV-E (HAdV-4) have been generally associated with respiratory infections[3]. HAdV infections are mild and self-limited in healthy individuals, whereas they can result in high mortality rates in immunocompetent and immunocompromized patients[4].展开更多
Forty seven clinical samples of Fowl adenovirus (FAdV) associated with Inclusion Body Hepatitis (IBH) from Peruvian broilers received between July 2006 and April 2013 were genotyped using sequencing of L1 Loop of Hexo...Forty seven clinical samples of Fowl adenovirus (FAdV) associated with Inclusion Body Hepatitis (IBH) from Peruvian broilers received between July 2006 and April 2013 were genotyped using sequencing of L1 Loop of Hexon gene. All 47 clinical samples presented macroscopic and histopathology lesions consistent with IBH, and amplified a specific fragment of Hexon gene by Polymerase Chain Reaction (PCR). A unique nucleotide sequence of 789 base pairs of Hexon gene (position 273 to 1061) was obtained in all 47 clinical samples analyzed. This sequence showed a high level of conservation in amino acid and nucleotide sequence (>99%) with a Fowl Adenovirus C serotype 4 previously identified. Sequence and phylogenetic analysis indicate no genotypic variation in Peruvian isolates. The presence of a unique genotype very closely related with genotype C1 previously reported in Peru and Ecuador (>99%), suggests the presence of FAdV C serotype 4 genotype C1 in clinical cases of IBH from Peruvian broilers.展开更多
AIM:To observe effect of adenovirus-mediated brain derived neurotrophic factor in early retinal neuropathy of diabetes in rats. METHODS:Adult male Wistar rats,9 weeks of age,were injected intraperitoneally with STZ to...AIM:To observe effect of adenovirus-mediated brain derived neurotrophic factor in early retinal neuropathy of diabetes in rats. METHODS:Adult male Wistar rats,9 weeks of age,were injected intraperitoneally with STZ to induce diabetes.Two weeks after the models were established,Ad.BDNF was administered into the vitreous cavities of rats.Four weeks after the models were set up,the rats were killed and the retina was removed for Western blotting and whole-mount immunohistochemistry for tyrosine hydroxylase(TH)to observe the changes of TH and dopaminergic amacrine cells in retina. RESULTS:The protein levels of TH and the number of positive staining dopaminergic amacrine cells and the staining gray scale of experimental group without Ad.BDNF were lower statistically.But there was no statistically significant difference between the experimental group with Ad.BDNF and control group. CONCLUSION:In the early stage of STZ diabetic,the administration of Ad.BDNF into the vitreous cavities can increase TH protein levels and the density of dopaminergic amacrine cells in the STZ rats’ retina.展开更多
obesity and liver steatosis are usually described as related diseases.obesity is regarded as exclusive consequence of an imbalance between food intake and physical exercise,modulated by endocrine and genetic factors.N...obesity and liver steatosis are usually described as related diseases.obesity is regarded as exclusive consequence of an imbalance between food intake and physical exercise,modulated by endocrine and genetic factors.Non-alcoholic fatty liver disease(NAFLD),is a condition whose natural history is related to,but not completely explained by over-nutrition,obesity and insulin resistance.There is evidence that environmental infections,and notably adipogenic adenoviruses(ADV)infections in humans,are associated not only with obesity,which is sufficiently established,but also with allied conditions,such as fatty liver.In order to elucidate the role,if any,of previous ADV36 infection in humans,we investigated association of ADV36-ADV37 seropositivity with obesity and fatty liver in humans.Moreover,the possibility that lifestyle-nutritional intervention in patients with NAFLD and different ADV36 seropositive status,achieves different clinical outcomes on ultrasound bright liver imaging,insulin resistance and obesity was challenged.ADV36 seropositive patientshave a more consistent decrease in insulin resistance,fatty liver severity and body weight in comparison with ADV36 seronegative patients,indicating a greater responsiveness to nutritional intervention.These effects were not dependent on a greater pre-interventional body weight and older age.These results imply that no obvious disadvantage-and,seemingly,that some benefit-is linked to ADV36 seropositivity,at least in NAFLD.ADV36 previous infection can boost weight loss and recovery of insulin sensitivity under interventional treatment.展开更多
Objective: To determine the prevalence of astrovirus, norovirus, adenovirus in children below five years old with diarrhea by multiplex reverse transcriptase polymerase chain reaction(RT-PCR) along with rotavirus anti...Objective: To determine the prevalence of astrovirus, norovirus, adenovirus in children below five years old with diarrhea by multiplex reverse transcriptase polymerase chain reaction(RT-PCR) along with rotavirus antigen detection by enzyme linked immunosorbant assay.Methods: The study was conducted on children below five years old complaining of acute diarrhea. The study included stool examination by molecular method for detection of norovirus, adenovirus and astrovirus by multiplex RT-PCR. Rotavirus antigen was detected in the stool by enzyme linked immunosorbant assay.Results: The study included 100 children below 5 years old with acute diarrhea.Multiplex RT-PCR was positive in 34% of the children. The most frequently detected virus was rotavirus(44%), followed by norovirus(30%), adenovirus(20%) and astrovirus(14%). The clinical symptoms were more significantly associated with viral diarrhea such as fever(P = 0.03), bloody diarrhea(P = 0.025), vomiting(P = 0.000 1) and watery diarrheas(P = 0.05). The frequency of diarrhea with viral pathogen was significantly presented in winter season(39.7%). There were significant frequencies of norovirus and adenovirus in age ranging 1–2 years old(P = 0.04, P = 0.01 respectively).Conclusions: The present study spotlights on the prevalence of viral pathogens as an important etiology in diarrhea in children below five years old. Astrovirus, norovirus and adenovirus are common along with rotavirus in this group of patients. Multiplex PCR leads to improve the laboratory diagnosis of these viruses along with antigen detection method. Further longitudinal studies are required to evaluate the epidemiological data associated with these viruses and for proper management of such drastic infection.展开更多
AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HB...AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.展开更多
AIM:To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia.METHODS:Gateway recombinant cloning technology was used to constru...AIM:To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia.METHODS:Gateway recombinant cloning technology was used to construct adenovirus vectors.The wild-type(wt) and mutant(mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction(PCR).The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDownmultiple cloning site(MCS)-/internal ribozyme entry site(IRES)/enhanced green fluorescent protein(EGFP).Then the desired DNA fragments were integrated into the destination vector pAV.Des1 d yielding the final expression constructs pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-cytomegalovirus(CMV)>mutlumican/IRES/EGFP,respectively.RESULTS:The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mutlumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology.Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells.CONCLUSION:We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology,which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia.展开更多
基金supported by the National Natural Science Foundation of China(No.82073617)Joint Research Fund for Beijing Natural Science Foundation and Haidian Original Innovation(No.L202007)+1 种基金Fundamental Research Funds for the Central Universities and Peking University Health Science Center(No.BMU2021YJ041)Peking University Medicine Fund of Fostering Young Scholars'Scientific&Technological Innovation(No.BMU2021PY005).
文摘Objective Human adenovirus(HAdV)infection is common and can develop to serious conditions with high mortality,yet the mechanism of HAdV infection remains unclear.In the present study,the serum metabolite profiles of HAdV-7-infected patients with pneumonia or upper respiratory tract infection(URTI)were explored.Methods In total,35 patients were enrolled in the study following an outbreak of HAdV-7 in the army,of whom 14 had pneumonia and 21 had URTI.Blood samples were collected at the acute stage and at the recovery stage and were analyzed by untargeted metabolomics.Results Over 90% of the differential metabolites identified between the pneumonia patients and URTI patients were lipids and lipid-like molecules,including glycerophospholipids,fatty acyls,and sphingolipids.The metabolic pathways that were significantly enriched were primarily the lipid metabolism pathways,including sphingolipid metabolism,glycerophospholipid metabolism,and linoleic acid metabolism.The sphingolipid metabolism was identified as a significantly differential pathway between the pneumonia patients and URTI patients and between the acute and recovery stages for the pneumonia patients,but not between the acute and recovery stages for the URTI patients.Ceramide and lactosylceramide,involved in sphingolipid metabolism,were significantly higher in the pneumonia patients than in the URTI patients with good discrimination abilities[area under curve(AUC)0.742 and 0.716,respectively;combination AUC 0.801].Conclusion Our results suggested that HAdV modulated lipid metabolism for both the patients with URTI and pneumonia,especially the sphingolipid metabolism involving ceramide and lactosylceramide,which might thus be a potential intervention target in the treatment of HAdV infection.
基金supported by the National High Technology Research and Development Program of China (863 Program), No. 2015CB755400 (to XQH)
文摘Rhodioloside has been shown to protect cells from hypoxia injury,and bone marrow mesenchymal stem cells have a good effect on tissue repair.To study the effects of rhodioloside and bone marrow mesenchymal stem cells on spinal cord injury,a rat model of spinal cord injury was established using the Infinite Horizons method.After establishing the model,the rats were randomly divided into five groups.Rats in the control group were intragastrically injected with phosphate buffered saline(PBS)(5μL).PBS was injected at 6 equidistant points around 5 mm from the injury site and at a depth of 5 mm.Rats in the rhodioloside group were intragastrically injected with rhodioloside(5 g/kg)and intramuscularly injected with PBS.Rats in the mesenchymal stem cell(MSC)group were intramuscularly injected with PBS and intramuscularly with MSCs(8×10^6/mL in a 50-μL cell suspension).Rats in the Ad-HIF-MSC group were intragastrically injected with PBS and intramuscularly injected with HIF-1 adenovirus-infected MSCs.Rats in the rhodioloside+Ad-HIF-MSC group were intramuscularly injected with MSCs infected with the HIF-1 adenovirus and intragastrically injected with rhodioloside.One week after treatment,exercise recovery was evaluated with a modified combined behavioral score scale.Hematoxylin-eosin staining and Pischingert’s methylene blue staining were used to detect any histological or pathological changes in spinal cord tissue.Levels of adenovirus IX and Sry mRNA were detected by real-time quantitative polymerase chain reaction and used to determine the number of adenovirus and mesenchymal stem cells that were transfected into the spinal cord.Immunohistochemical staining was applied to detect HIF-1 protein levels in the spinal cord.The results showed that:(1)compared with the other groups,the rhodioloside+Ad-HIF-MSC group exhibited the highest combined behavioral score(P<0.05),the most recovered tissue,and the greatest number of neurons,as indicated by Pischingert’s methylene blue staining.(2)Compared with the PBS group,HIF-1 protein expression was greater in the rhodioloside group(P<0.05).(3)Compared with the Ad-HIF-MSC group,Sry mRNA levels were higher in the rhodioloside+Ad-HIF-MSC group(P<0.05).These results confirm that rhodioloside combined with bone marrow mesenchymal stem cells can promote the recovery of spinal cord injury and activate the HIF-1 pathway to promote the survival of bone marrow mesenchymal stem cells and repair damaged neurons within spinal cord tissue.This experiment was approved by the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine,China(approval No.2015KYLL029)in June 2015.
文摘BACKGROUND:Interleukin 10(IL-10),a Th2 type cytokine,modulates inflammatory responses by inhibiting the production of proinflammatory cytokines.This study was designed to investigate the protective effects of adenovirus- mediated human IL-10(Ad-hIL-10)gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS:Adenoviruses encoding hIL-10 orβ-galactosidase (Ad-lacZ)were injected via the superior mesenteric vein into prospective donor animals.The donor liver was harvested 48 hours after transduction,and stored for 12 hours at 4℃ in lactated Ringer's solution prior to transplantation.The rats were divided into saline,Ad-lacZ,and Ad-hIL-10 groups.Liver function test,histopathological examination,reverse transcriptase-polymerase chain reaction(RT-PCR),and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS:Liver function(ALT and AST)was significantly improved,and the Suzuki score was significantly decreased in the Ad-hIL-10 group.The levels of hepatic TNF-α,MIP-2,ICAM-1 mRNA,and NF-κB protein in the Ad-hIL-10 group were significantly decreased.The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS:Donor pretreatment with Ad-hIL-10 down- regulates the expression of proinflammatory cytokines TNF-α,MIP-2,and ICAM-1 mRNA.hIL-10 protects against hepatic cold ischemia-reperfusion injury,at least in part,by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.
基金supported by a grant from the National Natural Science Foundation of China(30872510)
文摘BACKGROUND:Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24(IL-24)is a novel tumor suppressor gene,which has suppressor activity in a broad spectrum of human cancer cells.We investigated the effect of the replication-competent oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24,both expressing human MDA-7/IL-24 on the hepatocellular carcinoma cell lines HepG2,Hep3B,SMMC-7721,HCCLM3,and the normal liver cell line L02. METHODS:Hepatocellular carcinoma cell lines and the normal liver cell line were infected with SG600-IL24 and Ad.IL-24.The mRNA and protein expression of MDA-7/IL-24 in infected cells was confirmed by RT-PCR,ELISA,and Western blotting.MTT assay was used to investigate the proliferation effect.Hoechst staining and Annexin-V and PI staining were performed to study the MDA-7/IL-24 gene expressed in HCC cell lines and the normal liver cell line.Flow cytometry was used to analyse the cell cycle. RESULTS:RT-PCR,ELISA and Western blotting confirmed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24.MTT and apoptosis detection indicated that SG600-IL24 induced growth suppression promoted apoptosis,and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line. CONCLUSIONS:SG600-IL24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma cell lines in vitro but not in the normal liver cell line L02. Compared with Ad.IL-24,SG600-IL24 dramatically enhances antitumor activity in hepatocellular carcinoma cell lines.
文摘Background: Patients with intermediate to advanced hepatocellular carcinoma(HCC) are most commonly treated with transarterial chemoembolization(TACE). Previous studies showed that TACE combined with recombinant human adenovirus type 5(H101) may provide a clinical survival benefit. In the present study, we aimed to determine the survival benefit of TACE with or without H101 for patients with intermediate to advanced HCC and to develop an e ective nomogram for predicting individual survival outcomes of these patients.Methods: We retrospectively collected data from 590 patients with intermediate to advanced HCC who were treated at Sun Yat?sen University Cancer Center between January 2007 and July 2015. After propensity score matching, 238 patients who received TACE with H101(TACE with H101 group) and 238 patients who received TACE without H101(TACE group) were analyzed. Overall survival(OS) was evaluated using the Kaplan–Meier method; the nomogram was developed based on Cox regression analysis. Discrimination and calibration were measured using the concordance index(c?index) and calibration plots.Results: Clinical and radiologic features were similar between the two groups. OS rates were significantly lower in the TACE group than in the TACE with H101 group(1?year OS rate, 53.8% vs. 61.3%; 2?year OS rate, 33.4% vs. 44.2%; 3?year OS rate, 22.4% vs. 40.5%; all P < 0.05). Multivariate Cox regression analysis for the entire cohort showed that alpha?fetoprotein level, alkaline phosphatase level, tumor size, metastasis, vascular invasion, and TACE with or without H101 were independent factors for OS, all of which were included in the nomogram. Calibration curves showed good agreement between nomogram?predicted survival and observed survival. The c?index of the nomogram for predict?ing OS was 0.716(95% confidence interval 0.686–0.746).Conclusions: TACE plus H101 extends the survival of patients with intermediate to advanced HCC. Our proposed nomogram provides individual survival prediction and stratification for patients with intermediate to advanced HCC who receive TACE with or without H101.
基金a grant from the KeyProject of the China Hubei Provincial Science and Technology Department(2006AA304B52-4).
文摘BACKGROUND:Melanoma differentiation associated gene-7(mda-7)is a novel tumor suppressor gene,which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways.In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma(HCC)in vitro. METHODS:Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum(control).The others were transfected with adenovirus expressing the mda-7 gene(Ad.mda-7)or adenovirus vector serving as negative control(Ad.vec).The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle.Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells.The expression of MDA-7,Bcl-2 and Bax proteins were detected by Western blotting.RESULTS:The mda-7 gene was expressed in Hep3B and L-02 cells.The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml,respectively.mda-7 induced Hep3B growth suppression and apoptosis,compared with Ad.mda-7 and control(P<0.01).In addition,cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein,but this was not found in L-02.The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS:mda-7 selectively induces growth inhibi- tion and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti- apoptosis protein Bcl-2.It could be an ideal gene for gene therapy in HCC.
文摘BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.
基金This work was supported by a grant from Shanghai Science Foundation (No. 994119044).
文摘OBJECTIVE: To evaluate the killing effects of the cytosine deaminase （CD） gene mediated byadenovirus vector on human pancreatic cancer cell lines in vitro.METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-lwere recombinated in bacteria. The newly recombinated Ad-CD containing green fluoreseent protein（GFP） was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Humanpancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added.XTT assay was used to estimate relative numbers of viable cells.RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and theconcentration of viral liquids containing the CD gene was 2×1O<sup>11</sup> pfu/ml. It was found that significantcytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but littleeffects exerted on the nontransduced pancreatic carcinoma cells.CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for genetherapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzymeprodrug strategy in experimental pancreatic cancer.
文摘To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF 165 ), the cDNA for hVEGF 165 was subcloned into pACCMV·pLpA. Subsequently, this recombinant pACCMV·hVEGF was co transfected into 293 cells together with pJM17 to obtain the replication deficient recombinant adenovirus containing hVEGF gene-AdCMV·hVEGF. The VEGF gene expression was detected by using RT PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSMC) infected with AdCMV· hVEGF. Cultured human umbilical vein endothelial cells (HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV·hVEGF to observe the effect of VEGF on proliferation of HUVEC. 48 h after the infection with AdCMV·hVEGF, VSMC demonstrated VEGF expression, and the expressed VEGF could stimulate the proliferation of HUVEC in vitro . Successfully prepared AdCMV·hVEGF 165 could express biologically active VEGF in infected VSMC, and stimulate proliferation of HUVEC.
基金Supported by Natural Science Foundation of Chongqing,No.cstc2012jj A10052Young High-End Medical Reserve Personnel Training Plan Foundation of Chongqing,China
文摘AIM: To investigate the protective efficacy of recombinant adenovirus containing hyper-interleukin-6(HyperIL-6, HIL-6) and hepatocyte growth factor(HGF)(AdHGF-HIL-6) compared to that of recombinant adenovirus containing either HIL-6 or HGF(Ad-HIL-6 or Ad-HGF) in rats with acute-on-chronic liver failure(ACLF).METHODS: The recombinant adenoviruses containing HIL-6 and/or HGF were constructed. We established an ACLF model, and rats were randomly assigned to control, model, Ad-GFP, Ad-HIL-6, Ad-HGF or AdHGF-HIL-6 group. We collected serum and liver tissue samples to test pathological changes, biochemical indexes and molecular biological indexes.RESULTS: Attenuated alanine aminotransferase, prothrombin time, high-mobility group box 1(HMGB1), endotoxin, tumour necrosis factor(TNF)-α and interferon-γ were observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGFHIL-6-treated rats with ACLF. Likewise, reduced hepaticdamage and apoptotic activity, as well as reduced HMGB1 and Bax proteins, but raised expression of Ki67 and Bcl-2 proteins and Bcl-2/Bax ratio were also observed in the Ad-HGF-, Ad-HIL-6- and Ad-HGF-HIL-6-treated rats with ACLF. More significant changes were observed in the Ad-HGF-HIL-6 treatment group without obvious side effects. Furthermore, caspase-3 at the protein level decreased in the Ad-HIL-6 and Ad-HGFHIL-6 treatment groups, more predominantly in the latter group.CONCLUSION: This study identifies that the protective efficacy of Ad-HGF-HIL-6 is more potent than that of Ad-HGF or Ad-HIL-6 in ACLF rats, with no significant side effects.
文摘Background:In 2014,an outbreak of adenoviral pneumonia occurred in the Korean military training center.However,there are limited data on the characteristics of the fever and its response to antipyretic therapy in immunocompetent adults with adenovirus-positive community-acquired pneumonia(CAP).Methods:The medical records of the patients who were admitted to the Armed Forces Chuncheon Hospital for the treatment of CAP between January 2014 and December 2016 were retrospectively analyzed.The patients were divided into three groups,namely,the adenovirus-positive(Adv)group,the adenovirus-negative(Non-Adv)group and the unknown pathogen group,according to the results of a polymerase chain reaction(PCR)test and sputum culture used to measure adenovirus and other bacteria or viruses in respiratory specimens.We evaluated and compared the demographics,clinicolaboratory findings and radiological findings upon admission between the two groups.Results:Out of the 251 military personnel with CAP during the study periods,67 were classified into the Adv group,while 134 were classified into the Non-Adv group and 50 were classified into the unknown pathogen group.The patients in the Adv group had a longer duration of fever after admission((3.2±1.6)d vs.(1.9±1.2)d vs.(2.2±1.5)d,P=0.018)and symptom onset((5.8±2.2)d vs.(3.9±2.5)d vs.(3.7±2.0)d,P=0.006)than patients in the Non-Adv and unknown pathogen groups,respectively.The patients in the Adv group had a higher mean temperature at admission(37.8±0.3 vs.37.3±0.3 vs.37.3±0.3,P=0.005),and more patients were observed over 40 and 39 to 40(14.9%vs.2.2%vs.4.0%,35.8%vs.3.7%vs.6.0%,P<0.001)than those in the Non-Adv and unknown pathogen groups,respectively.The Adv group more commonly had no response or exhibited adverse events after antipyretic treatment compared to the Non-Adv group(17.9%vs.1.5%,35.0%vs.4.3%,P<0.001,P=0.05,respectively).In addition,the time from admission to overall clinical stabilization was significantly longer in the patients in the Adv group than in those in the Non-Adv group((4.3±2.8)d vs.(2.9±1.8)d,P=0.034,respectively).Furthermore,no significant difference in the length of hospital stay was observed between the two groups,and no patient died in either group.Conclusions:In this study,Adv-positive CAP in immunocompetent military personnel patients had distinct fever characteristics and responses to antipyretic treatment.
基金Supported by National Natural Science Foundation of China,No. 30872510Natural Science Foundation of Hubei Province,No. 2008CDB127
文摘AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), enzymelinked immunosorbent assay (ELISA), and Western blotting, respectively. Apoptosis of HCC cells and normal liver cells was detected by cytometric assay with Hoechst33258 staining. 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to investigate proliferation of HCC cells and normal liver cells, and cell cycle was assayed by flow cytometry. RESULTS: RT-PCR, ELISA and Western blotting showed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT indicated that SG600-IL24 could suppress the growth of HepG2, Hep3B, MHCC97L, with an inhibition rate of 75% ± 2.5%, 85% ± 2.0%, 72% ± 1.8%, respectively (P < 0.01), promote the apoptosis of HepG2, Hep3B, MHCC97L, with an apoptosis rate of 56.59% ± 4.0%, 78.36% ± 3.5%, 43.39% ± 2.5%, respectively (P < 0.01), and block the HCC cell lines in the G2/M phase with a blocking rate of 35.4% ± 4.2%, 47.3% ± 6.2%, 42% ± 5.0%, respectively (P < 0.01) but not the normal liver cell line in a p53-independent manner. CONCLUSION: SG600-IL24 can selectively suppress the proliferation and apoptosis of HCC cell lines in vitro but not normal liver cell line L02 in a p53-independent manner. Compared with Ad.IL-24, SG600-IL24 can significantly enhance the antitumor activity in HCC cell lines.
基金supported by a grant from the National Basic Research of China(973 Program 2007CB512903)
文摘BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation.METHODS:A normal human liver cell line(L02) was transfected with adenovirus vector containing the CTLA4Ig gene(Ad-CTLA4Ig-EGFP) in vitro,and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining.Transfected cells then were injected into the spleen of Sprague-Dawley rats,the survival of cells was determined by immunohistochemistry,and the immune status was examined through CD4+ and CD69+ T cellcounts and ELISA detection of IL-2 in peripheral blood.RESULTS:L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks.Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation,while none was detected in the control group.Furthermore,the percentages of CD4+ and CD4+ CD69+T cells in the CTLA4-transfected group were 24.5% and 45.1%,markedly lower than those in the control group at 4 weeks post-transplantation(P<0.01).Furthermore,the IL-2 level was also lower in the CTLA4transfected group than in the control group.CONCLUSION:Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.
基金supported by Key Project of Qinghai Health and Family Planning Commission [2017-wjzd-08]Qinghai Thousand People Plan,Qinghai High-level Talents Plan in Public Health and National Science and Technology Major Project of the Ministry of Science and Technology of China during ‘13th Five-Year Plan’[2017zx10004-208]
文摘Human adenoviruses (HAdVs) belong to the genus Mastadenovirus, family Adenoviridae, and can cause respiratory tract and gastrointestinal tract infections, as well as ocular infections in humans[1]. Till date, a total of 84 unique genotypes of AdVs have been identified and classified as human Mastadenovirus species A to G, and specific types are often associated with certain clinical symptoms, epidemiological settings, and demographic risk groups[2]. Among these species, members of the species HAdV-B (HAdV-3, HAdV-7, HAdV-11, HAdV-16, HAdV-21, HAdV-34, HAdV-35, HAdV-50, etc), HAdV-C (HAdV-1, HAdV-2, HAdV-5, and HAdV-6), and HAdV-E (HAdV-4) have been generally associated with respiratory infections[3]. HAdV infections are mild and self-limited in healthy individuals, whereas they can result in high mortality rates in immunocompetent and immunocompromized patients[4].
文摘Forty seven clinical samples of Fowl adenovirus (FAdV) associated with Inclusion Body Hepatitis (IBH) from Peruvian broilers received between July 2006 and April 2013 were genotyped using sequencing of L1 Loop of Hexon gene. All 47 clinical samples presented macroscopic and histopathology lesions consistent with IBH, and amplified a specific fragment of Hexon gene by Polymerase Chain Reaction (PCR). A unique nucleotide sequence of 789 base pairs of Hexon gene (position 273 to 1061) was obtained in all 47 clinical samples analyzed. This sequence showed a high level of conservation in amino acid and nucleotide sequence (>99%) with a Fowl Adenovirus C serotype 4 previously identified. Sequence and phylogenetic analysis indicate no genotypic variation in Peruvian isolates. The presence of a unique genotype very closely related with genotype C1 previously reported in Peru and Ecuador (>99%), suggests the presence of FAdV C serotype 4 genotype C1 in clinical cases of IBH from Peruvian broilers.
文摘AIM:To observe effect of adenovirus-mediated brain derived neurotrophic factor in early retinal neuropathy of diabetes in rats. METHODS:Adult male Wistar rats,9 weeks of age,were injected intraperitoneally with STZ to induce diabetes.Two weeks after the models were established,Ad.BDNF was administered into the vitreous cavities of rats.Four weeks after the models were set up,the rats were killed and the retina was removed for Western blotting and whole-mount immunohistochemistry for tyrosine hydroxylase(TH)to observe the changes of TH and dopaminergic amacrine cells in retina. RESULTS:The protein levels of TH and the number of positive staining dopaminergic amacrine cells and the staining gray scale of experimental group without Ad.BDNF were lower statistically.But there was no statistically significant difference between the experimental group with Ad.BDNF and control group. CONCLUSION:In the early stage of STZ diabetic,the administration of Ad.BDNF into the vitreous cavities can increase TH protein levels and the density of dopaminergic amacrine cells in the STZ rats’ retina.
文摘obesity and liver steatosis are usually described as related diseases.obesity is regarded as exclusive consequence of an imbalance between food intake and physical exercise,modulated by endocrine and genetic factors.Non-alcoholic fatty liver disease(NAFLD),is a condition whose natural history is related to,but not completely explained by over-nutrition,obesity and insulin resistance.There is evidence that environmental infections,and notably adipogenic adenoviruses(ADV)infections in humans,are associated not only with obesity,which is sufficiently established,but also with allied conditions,such as fatty liver.In order to elucidate the role,if any,of previous ADV36 infection in humans,we investigated association of ADV36-ADV37 seropositivity with obesity and fatty liver in humans.Moreover,the possibility that lifestyle-nutritional intervention in patients with NAFLD and different ADV36 seropositive status,achieves different clinical outcomes on ultrasound bright liver imaging,insulin resistance and obesity was challenged.ADV36 seropositive patientshave a more consistent decrease in insulin resistance,fatty liver severity and body weight in comparison with ADV36 seronegative patients,indicating a greater responsiveness to nutritional intervention.These effects were not dependent on a greater pre-interventional body weight and older age.These results imply that no obvious disadvantage-and,seemingly,that some benefit-is linked to ADV36 seropositivity,at least in NAFLD.ADV36 previous infection can boost weight loss and recovery of insulin sensitivity under interventional treatment.
文摘Objective: To determine the prevalence of astrovirus, norovirus, adenovirus in children below five years old with diarrhea by multiplex reverse transcriptase polymerase chain reaction(RT-PCR) along with rotavirus antigen detection by enzyme linked immunosorbant assay.Methods: The study was conducted on children below five years old complaining of acute diarrhea. The study included stool examination by molecular method for detection of norovirus, adenovirus and astrovirus by multiplex RT-PCR. Rotavirus antigen was detected in the stool by enzyme linked immunosorbant assay.Results: The study included 100 children below 5 years old with acute diarrhea.Multiplex RT-PCR was positive in 34% of the children. The most frequently detected virus was rotavirus(44%), followed by norovirus(30%), adenovirus(20%) and astrovirus(14%). The clinical symptoms were more significantly associated with viral diarrhea such as fever(P = 0.03), bloody diarrhea(P = 0.025), vomiting(P = 0.000 1) and watery diarrheas(P = 0.05). The frequency of diarrhea with viral pathogen was significantly presented in winter season(39.7%). There were significant frequencies of norovirus and adenovirus in age ranging 1–2 years old(P = 0.04, P = 0.01 respectively).Conclusions: The present study spotlights on the prevalence of viral pathogens as an important etiology in diarrhea in children below five years old. Astrovirus, norovirus and adenovirus are common along with rotavirus in this group of patients. Multiplex PCR leads to improve the laboratory diagnosis of these viruses along with antigen detection method. Further longitudinal studies are required to evaluate the epidemiological data associated with these viruses and for proper management of such drastic infection.
基金Supported by Grants from National Natural Science Foundation of China, No. 30901344
文摘AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.
基金Supported by the Natural Science Foundation of Guangdong Province(No.2015A030310158No.2014A030313359)+1 种基金the Fundamental Research Funds for the Central Universities(No.21611446)the Scientific and Cultivation Foundation of the First Affiliated Hospital of Jinan University(No.2015201)
文摘AIM:To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia.METHODS:Gateway recombinant cloning technology was used to construct adenovirus vectors.The wild-type(wt) and mutant(mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction(PCR).The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDownmultiple cloning site(MCS)-/internal ribozyme entry site(IRES)/enhanced green fluorescent protein(EGFP).Then the desired DNA fragments were integrated into the destination vector pAV.Des1 d yielding the final expression constructs pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-cytomegalovirus(CMV)>mutlumican/IRES/EGFP,respectively.RESULTS:The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mutlumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology.Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells.CONCLUSION:We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology,which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia.
