Plasma Metabonomics of Human Adenovirus-infected Patients with Pneumonia and Upper Respiratory Tract Infection

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.

Inhibition of Dual Specific Oncolytic Adenovirus on Esophageal Cancer via Activation of Caspases by a Mitochondrial-dependent Pathway

We investigated the anti-tumor effects of dual cancer specific-oncolytic adenovirus Ad-VP on esophageal cancer（EC）. The anti-tumor activity of Ad-VP was compared with that of the control recombinant adenoviruses （Ad-GP, Ad-Apoptin, Ad-EGFP） in human esophageal cancer cell EC-109 and human normal liver cell L02 in vitro. In 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assays, the growth of EC-109 cells was slightly inhibited by Ad-GP, Ad-Apoptin and Ad-EGFE However, Ad-VP induced a significant cytotoxic effect. Infection of EC-109 cells with Ad-VP resulted in a significant induction of apoptosis of them in vitro, detected by 4＇,6-diamidino-2-phenylindole（DAPI） or acridine orange and ethidium bromide staining. The results of Western blot and flow cytometric assay indicate the loss of mitochondrial membrane potential（Aψm）, the release of eytochrome c and the activation of caspase-3, 6 and 7 in Ad-VP infected EC-109 cells. In contrast, all these assays show almost no effects of the recombinant adenoviruses on L02 cells. These results demonstrate that the treatment of tumors with Ad-VP selectively inhibits tumor growth and induces apoptosis of esophageal cancer cells. Ad-VP may provide a novel and powerful strategy for cancer gene therapy.

Protective effect of thodioloside and bone marrow mesenchymal stem cells infected with HIF-1-expressing adenovirus on acute spinal cord injury

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.

Transarterial chemoembolization combined with recombinant human adenovirus type 5 H101 prolongs overall survival of patients with intermediate to advanced hepatocellular carcinoma: a prognostic nomogram study

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.

Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

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 or beta-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 degrees C 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-alpha, MIP-2, ICAM-1 mRNA, and NF-kappa B 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-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)

Oncolytic adenovirus-mediated MDA-7/IL-24 overexpression enhances antitumor activity in hepatocellular carcinoma cell lines

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. (Hepatobiliary Pancreat Dis Int 2010; 9:615-621)

Experimental Study of Adenovirus Vector Mediated-hVEGF_(165) Gene on Prevention of Restenosis after Angioplasty

This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF 165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF 165 cDNA was directly injected into left side of the injured carotid arteries. On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer. The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF 165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (IT max and MT max) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

Inhibition of Metastatic Progression of SSTR2 Gene Transfection Mediated by Adenovirus in Human Pancreatic Carcinoma Cells

The inhibition of metastatic progression of Somatostatin receptor type 2 （SSTR2） gene transfection mediated by adenovirus in human pancreatic carcinoma cells and the mechanisms involved in this effect were studied. The full-length human SSTR2 cDNA was introduced into the pancreatic cancer cell line BXPC-3 by adenovirus-mediated transfection. Stable expression of mRNAs and protein of SSTR2 was detected by RT-PCR and Western-blot. The Matrigel-coated Transwell was used to detect the migratory and invasive ability of SSTR2-expressing cells, Adv-GFP control cells and mock control cells. Furthermore, the expression of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor of metalloproteinase-2 （TIMP-2） was detected by RT-PCR in these cells. The stable expression of SSTR2 was detected in BXPC-3 transfected by Adv-GFP-SSTR2. A dramatic decrease of BXPC-3 expressing sst2 cells migrating through a Matrigel-coated filter was observed, as compared with Adv-GFP control and mock control cells （P〈0. 01）. Moreover, the expression of MMP-2 mRNA was significantly reduced in the SSTR2-expressing cells and converse- ly the expression of TIMP-2 mRNA was significantly increased in the SSTR2-expressing cells when compared with the Adv-GFP control and mock control （P〈0. 01）. The expression of reintroduced human SSTR2 gene in BXPC-3 cells by Adv-GFP-SSTR2 had the anti-migratory and anti-invasive effects, and the mechanisms involved in this effect may be due to the down-regulated expression of MMP-2 and up-regulated expression of TIMP-2.

Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B

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. RESULT S: 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 inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the antiapoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

Recombinant adenovirus containing hyper-interleukin-6 and hepatocyte growth factor ameliorates acute-on-chronic liver failure in rats

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.

Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro

OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus 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-l were 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. Human pancreatic 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 the concentration of viral liquids containing the CD gene was 2×1O^(11) pfu/ml. It was found that significant cytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells. CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.

Construction of the recombinant adenovirus vector carrying antisense multidrug resistance gene

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.

Characteristics of fever and response to antipyretic therapy in military personnel with adenovirus-positive community acquired pneumonia

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.

Growth Suppression of Human Lung Cancer Cells and Implanted Tumors by Adenovirus-mediated Transfer of the PTEN Gene

This study examined the effects of a recombinant adenovirus AdTEN-EGFP on the proliferation of A549 cells, a human lung carcinoma cell line, in vitro and on the growth of the implanted tumors in the nude mice in vivo, explored the underlying mechanisms and evaluated the in vitro transfection efficiency of Ad-PTEN-EGFP into A549 cells. The expression of Ad-PTEN-EGFP in the A549 cells was determined. The proliferation and the apoptosis rates of the A549 cells with Ad-PTEN-EGFP transfection or not was detected by MTT and flow cytometry. Ad-PTEN-EGFP at different doses was injected intratumorally to the tumor-bearing mice induced by the A549 cells. Tumor sizes were measured on an alternate day. After all the mice were sacrificed, the implanted tumors were removed for routine histological examination, weight test, HE staining and immunohistochemical staining. The expressions of Bax, P16 and P53 in the tumor tissues and those of caspase-3, CD34 and VEGF in the mouse sera were detected. Tumor cell apoptosis was measured by TUNEL method. The results showed that the vitality of the A549 cells after transfection with Ad-PTEN-EGFP declined. The expression of green fluorescent protein was observed under fluorescent microscope. The transfection rate was in excess of 50%. The mRNA and protein expression of PTEN in the transfected cells was confirmed. The proliferation rate of the transfected cells was significantly decreased when compared with that of the non-transfected cells （P〈0.05）. The number of the apoptosi＇s cells was increased in the transfected cells （P〈0.05）. The models of implanted tumors were successfully estab- lished by injection of the A549 cells in the flank of Balb/c nude mice. Administration of Ad-PTEN-EGFP to the tumor-bearing nude mice resulted in a suppression of tumor growth. There were statistically significant differences in the tumor weight and tumor volume between the Ad-PTEN-EGFP-treated group and the control groups （P〈0.05）. In contrast to those in the control groups, tumor tissues in the Ad-PTEN-EGFP-treated group were shown to have typical extensive vacuolar degeneration and massive hemorrhagic necrosis. Apoptotic bodies were also observed in the tumor cells. The expressions of Bax, caspase-3 and P16 were increased （P〈0.05） while those of CD34, VEGF and P53 decreased （P〈0.05） in the Ad-PTEN-EGFP-treated group. It is concluded that Ad-PTEN-EGFP could induce the apoptosis of the A549 cells and inhibit their proliferation. And it could also substantially suppress the tumor growth in the tumor-bearing nude mice and induce apoptosis of the tumor cells as well. These findings carry significant implications for adenovirus vector-based PTEN gene therapies for lung cancers.

Construction and Identification of Recombinant Adenovirus Vector Containing the hVEGF_(165) Gene

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.

Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

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.

Molecular Characterization of Human Respiratory Adenovirus Infection in Children from November 2016 to October 2017 in Xining City, China

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].

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells

Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.

Synergistic Antitumor Efficacy of Oncolytic Adenovirus Combined with Chemotherapy

Objective： Chemotherapy is an effective means of treating breast cancer, and cancer-specific replicative adenovirus is also a promising antitumor agent in recent years. Our investigation aims to demonstrate that CNHK300 can mediate selective antitumor efficacy and produce synergistic cytotoxicity with chemotherapy on HER-2 over-expressing breast cancer. Methods： We engineered the telomerase-dependent replicative adenovirus CNHK300 by placing the E1A gene under the control of the human hTERT promoter. By analysis of E1A expression, we proved the fidelity of hTERT promoter in adenovirus genome and the selective expression of E1A in telomerase-positive breast cancer cells but not in normal fibroblast cells. By proliferation test, we further showed efficient replication of CNHK300 in breast cancer cells with apparently attenuated proliferation in normal fibroblast cells. Finally, we demonstrated by MTT methods that CNHK300 virus caused potent cytolysis and produced synergistic cytotoxicity with chemotherapy in breast cancer cells with attenuated cytotoxicity on normal cells. Results： In this virus, the E1A gene is successfully placed under the control of the human hTERT promoter. CNHK300 virus replicated as efficiently as the wild-type adenovirus and caused intensive cell killing in HER-2 over-expressing breast cancer cells in vitro. In contrast, telomerase-negative normal fibroblast cells, which expressed no hTERT activity, were not able to support CNHK300 replication. Combined treatment of CNHK300 with paclitaxel improved cytotoxicity on cancer cells. Conclusion： We conclude that CNHK300 can produce selective antitumor efficacy and enhance the in vitro response of chemotherapy on HER-2 overexpressing breast cancer.

THE BIOLOGICAL CHARACTERISTICS OF ADENOVIRUS-MEDIATED IL-18 GENE-MODIFIED MURINE COLORECTAL ADENOCARCINOMA CELL IN VIVO AND IN VITRO

Objective: Interleukin 18 (IL-18) is a strong activator of NK cells and promotes the generation of IL-2, IFN-γ and GM-CSF. In the present study, we constructed adenovirus encoding IL-18 gene (AdIL-18), and observed the biological characteristics of IL-18 gene-modified murine colorectal adenocarcinoma cell (CT26)in vivo andin vitro. Methods: Gene modification was mediated by adenovirus. The proliferation of the cells was determined by MTT and IL-18 was assayed by ELISA. The cytotoxicity of NK and CTL was detected by four-hour51Cr release assay. Results: IL-18 gene modification had no effect on the proliferation and morphology of CT-26 cellsin vitro, but the growth of IL-18-modified CT26 cells was obviously inhibitedin vivo. In addition, although IL-18-modified CT26 cells could form tumor nodulesin vivo as well as LacZ-modified CT26 cells or wild-type CT26 cells, the mean survival time of the mice inoculated with IL-18-modified CT26 cells was significantly prolonged as compared with that of control groups. Thus, the anti-tumor immune responses were induced in the group of mice inoculated with IL-18-modified CT26 cells, which might be related to the activation of NK cells and CTL. However, all the three groups ultimately died of tumor. Conclusion: IL-18-modified CT26 cells could induce the anti-tumor immune responses incompletely, which required other factors for effective anti-tumor responses.