• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.3
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• pp.193-197
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• 2009

Background: Though it is clear that many types of viruses can infect the oral mucosa, its condition for infection is unclear. The purpose of this study was to analyze the conditions for viral infection of normal oral mucosa and explore the possibility of topical gene therapy to oral mucosa using a viral vector. Methods: Freshly taken fragments of the palate and the tongue of mice were used for organ culture. The specimens were exposed to green fluorescent protein (GFP)-adenoviral vector for 1 hour except for the control. Initial viral titer was $6.3{\times}10^{11}\;pfu/ml$ and the virus was diluted to working concentrations. The dilution ratio was 1:1,000 ($6.3{\times}10^8\;pfu/ml$), 1:10,000 ($6.3{\times}10^7\;pfu/ml$), and 1:100,000 ($6.3{\times}10^6\;pfu/ml$). They were then cultured on a stainless steel wire mesh in an organ culture dish. The specimens were stereoscopically examined every 24 hours for 6 days, after which they were fixed and analyzed through immunohistochemical methods Results: There was no visible expression in the control, $6.3{\times}10^6\;pfu/ml$, and $6.3{\times}10^7\;pfu/ml$ groups. Initial expression was observed at 24 hours after infection in both the palate and the tongue in $6.3{\times}10^8\;pfu/ml$ and the expression significantly increased until 3 days in the palate and 2 days in the tongue after infection (P<0.05). In both groups, the expression was mostly observed at the resection margin. Immunohistochemical studies showed that the epithelial cells were positive to GFP. Conclusion: The present study showed that topically applied adenovirus containing specific genetic information of GFP could successfully transduce GFP in normal oral epithelial cells at the resection margin in organ culture in terms of dose and exposure time.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.11
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• pp.5551-5556
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• 2012

Cytological examination is widely used as a diagnostic tool because of the ease of collecting cells from the involved area. However, the diagnostic yield of cytological examination is unsatisfactory; the reasons include sampling error, poorly prepared samples, small numbers of malignant cells, and low grades of cellular atypia. In this study, we focused on the high infectivity of adenovirus towards epithelial cells and applied the luciferase-expressing adenoviral vector to a new cancer cell detection tool. In addition, adenoviral infectivity was enhanced by modifying viral fiber proteins. The sensitivity of the diagnostic tool was tested using the NCI-H1299 lung cancer cell line, and validated in body fluid samples from cancer patients with a variety of etiology. Results showed that the adenovirus efficiently transfected NCI-H1299 with high sensitivity. Only 10 cancer cells were sufficient for detection of luciferase signals. In body fluid samples, the adenovirus confirmed the diagnosis for malignant and benign cancer, but not in non-epithelial cell derived samples. This study provides proof-of-concept for a more reliable and sensitive diagnostic tool for epithelium-derived cancer.

• Journal of Life Science
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• v.30 no.3
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• pp.221-229
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• 2020

Distylium racemosum is an evergreen tree growing wild on Jeju Island, which has been reported to exert biological activity. Obesity is induced by genetic, metabolic, environmental, and other factors. Among these, certain bacterial and viral infections have been shown to cause obesity, which is known as infectobesity. Human adenovirus (HAdV)-36 is one of the viruses that are known to cause infectobesity in humans. Unlike extensive research on preventing obesity and developing anti-obesity drugs, little research has been conducted specifically on the prevention and treatment of infectobesity. Therefore, this study aimed to evaluate the effects of phytochemicals from D. racemosum on the replication of HAdV-36. A549 cells infected with HAdV-36 were treated with an ethyl acetate fraction of a D. racemosum leaf extract (DRE), and the virus titer was calculated based on the hemagglutination (HA) titer. The results showed a concentration-dependent inhibitory effect of DRE treatment on HA titers. DRE treatment was also found to inhibit the cytopathic effects of the virus and the expression of viral genes. Quercitrin was identified as the constituent of DRE exerting an inhibitory effect on HAdV-36 replication. This study shows that DRE can be used as a candidate substance for the development of treatment for HAdV-36 infections. In addition, this study provides a basis to further investigate DRE for the development of anti-infectobesity medication.

• Clinical and Experimental Pediatrics
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• v.46 no.7
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• pp.679-686
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• 2003

Purpose : Ataxia telangiectasia mutated(ATM) is involved in DNA damage responses at different cell cycle checkpoints, and signalling pathways associated with regulation of apoptosis in response to ionizing radiation(IR). However, the signaling pathway that underlies IR-induced apoptosis in ATM cells has remained unknown. The purpose of this study was, therefore, to investigate the apoptotic pathway that underlies IR-induced apoptosis in a CT-26 cells expressing dominant negative ATM (DN-ATM). Methods : We generated a replication-deficient recombinant adenovirus encoding the DN-ATM(Ad/DN-ATM) or control adenovirus encoding no transgene(Ad/GFP) and infected adenovirus to CT-26 cells. After infection, we examined apoptosis and apoptotic pathway by [$^3H$]-thymidine assay, DNA fragmentation, and Western immunoblot analysis. Results : DN-ATM gene served as the creation of AT phenotype in a CT-26 cells as revealed by decreased cell proliferations following IR. In addition, IR-induced apoptosis was regulated through the reduced levels of the anti-apoptotic protein Bcl-2, the increased levels of the apoptotic protein Bax, and the activation of caspase-9, caspase-3, and PARP. Conclusion : These results indicate that the pathway of IR-induced apoptosis in CT-26 cells expressing DN-ATM is mediated by mitochondrial signaling pathway involving the activation of caspase 9, caspase 3, and PARP.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7825-7829
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• 2014

Objective: To investigate the effect of high expression of XAF1 in vivo or in vitro on lung cancer cell growth and apoptosis. Methods: 1. The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/F35 - Null at the same multiplicity of infection (MOI); (hereinafter referred to as transient transfected cell strain); XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. 2. Methyl thiazolyl tetrazolium (MTT) and annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis before and after infection of Ad5/F35 - XAF1 with Western blotting for apoptosis related proteins, caspase 3, caspase - 8 and PARP. 3. After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1 (hereinafter referred to as stable expression cell strain). Twenty nude mice were randomly divided into groups A and B, 10 in each group: A549/XAF1 stable expression cell strain was subcutaneously injected in group A, and A549/Ctrl stable cell line stable expression cell strain in group B (control group), to observe transplanted tumor growth in nude mice. Results: The mRNA and protein expression of XAF1 in A549 cells transfected by Ad5/F35 - XAF1 was significantly higher than in the control group. XAF1 mediated by adenovirus vector demonstrated a dose dependent inhibition of lung cancer cell proliferation and induction of apoptosis. This was accompanied by cleavage of caspase -3, -8, -9 and PARP, suggesting activation of intrinsic or extrinsic apoptotic pathways. A cell strain of lung cancer highly expressing XAF1 was established, and this demonstrated delayed tumor growth after transplantation in vivo. Conclusion: Adenovirus mediated XAF1 gene expression could inhibit proliferation and induce apoptosis in lung cancer cells in vitro; highly stable expression of XAF1 could also significantly inhibit the growth of transplanted tumors in nude mouse, with no obvious adverse reactions observed. Therefore, the XAF1 gene could become a new target for lung cancer treatment.

• Journal of Chest Surgery
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• v.36 no.11
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• pp.852-857
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• 2003

Background: Conventional treatment for mesothelioma is largely ineffective. We evaluated the novel approach of adenoviral gene transfection of PTEN gene in mesothelioma cancer cell lines, inflammatory and epithelial subtype, which are sensitive to adenoviral p53. Material and Method: Binary adenoviral PTEN and LacZ (Ad/GT-LacZ and Ad/GV16) vectors were used for transduction of the mesothelioma cell lines, REN (p53 sensitive). Protein levels were determined by Western blotting assay. Apoptosis was assessed by fluorescence-activated cell sorter analysis of subdiploid populations. Cell viability was determined with the XTT assay. Statistical analysis was performed with analysis of variance and the Student t test. Result: 72 hours after the treatment of adenoviral PTEN gene, cell killing were 32.9% for REN compared to control cell (2.5%) at MOI of 20. Also we observed the over-expression of proapoptotic protein, bax and decreased expression of bcl-2 protein in REN cells. But the expression of BCL-xl, Bak, Bad proteins were not altered. Conclusion: Adenovirus Pten-mediated overexpression of the Bax gene induces apoptosis and decreased cellular viability in p53-sensitive mesothelioma cells. These data suggest that the transfection of PTEN gene may represent a alternative gene therapy strategy to treat mesothelioma.

• Journal of Microbiology and Biotechnology
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• v.28 no.6
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• pp.1022-1029
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• 2018

Tuberculosis (TB) remains a serious health issue around the word. Adenovirus (Ad)-based vaccine and modified vaccinia virus Ankara (MVA)-based vaccine have emerged as two of the most promising immunization candidates over the past few years. However, the performance of the homologous and heterologous prime-boost immunization regimens of these two viral vector-based vaccines remains unclear. In the present study, we constructed recombinant Ad and MVA expressing an Ag85B-TB10.4 fusion protein (AdH4 and MVAH4) and evaluated the impact of their different immunization regimens on the humoral and cellular immune responses. We found that the viral vector-based vaccines could generate significantly higher levels of antigen-specific antibodies, $IFN-{\gamma}$-producing splenocytes, $CD69^+CD8^+$ T cells, and $IFN-{\gamma}$ secretion when compared with bacillus Calmette-$Gu{\acute{e}}rin$ (BCG) in a mouse model. AdH4-containing immunization regimens (AdH4-AdH4, AdH4-MVAH4, and MVAH4-AdH4) induced significantly stronger antibody responses, much more $IFN-{\gamma}$-producing splenocytes and $CD69^+CD8^+$ T cells, and higher levels of $IFN-{\gamma}$ secretion when compared with the MVAH4-MVAH4 immunization regimen. The number of $IFN-{\gamma}$-producing splenocytes sensitive to $CD8^+$ T-cell restricted peptides of Ag85B (9-1p and 9-2p) and Th1-related cytokines ($IFN-{\gamma}$ and $TNF-{\alpha}$) in the AdH4-MVAH4 heterologous prime-boost regimen immunization group was significantly higher than that in the other viral vector-based vaccine- and BCG-immunized groups, respectively. These results indicate that an immunization regimen involving AdH4 may have a higher capacity to induce humoral and cellular immune responses against TB in mice than that by regimens containing BCG or MVAH4 alone, and the AdH4-MVAH4 prime-boost regimen may generate an ideal protective effect.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.285-290
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• 1994

About 500 bacterial and fungal strains from a wide variety of natural habitats were screened for a new type II restriction endonuclease. Among the 500 species, we selected one species that produced a new restriction endonuclease. This strain has an optimum temperature of $30^{circ}C$ for growth. Morphological, cultural, and physiological characteristics were examined for identification of the isolated strain J-482. This strain was found to belong to the genus Alcaligenes. The restriction endonuclease was named as AspJI and partially purified from Alcaligenes sp. J-482 by DEAE-Sephadex A-50 column chromatography and gel filtration. Most of other nucleases were removed by the purification steps. The AspJI has a substrate specificity to ${lambda}$ DNA, pBR322 and Adenovirus-2 DNA. For its maximal activity, the isolated enzyme requires $MgCl_2$, which should be at least 12.5 mM and it does not need any other cofactors. It is maximally active in the absence of NaCl and is completely inactivated at 100 mM NaCl. The pH and temperature optima for activity were pH 7.5 and $37^{circ}C$, respectively. The DNA fragments generated by digesting ${lambda}$ DNA, pBR322, and Adenovirus-2 DNA with AspJI were the same as that produced by AatII. This suggests that AspJI is an isoschizomer of AatII.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1326-1334
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• 2008

The prime-boost vaccination with DNA vaccine and recombinant viral vector has emerged as an effective prophylactic strategy to control infectious diseases. Here, we compared the protective immunities induced by multiple alternating immunizations with DNA vaccine (pCIgB) and replication-incompetent adenovirus (Ad-gB) expressing glycoprotein gB of pseudorabies virus (PrV). The platform of pCIgB-prime and Ad-gB-boost induced the most effective immune responses and provided protection against virulent PrV infection. However, priming with pCIgB prior to vaccinating animals by the DNA vaccine-prime and Ad-boost protocol provided neither effective immune responses nor protection against PrV. Similarly, boosting with Ad-gB following immunization with DNA vaccine-prime and Ad-boost showed no significant responses. Moreover, whereas the administration of Ad-gB for primary immunization induced Th2-type-biased immunity, priming with pCIgB induced Th1-type-biased immunity, as judged by the production of PrV-specific IgG isotypes and cytokine IFN-$\gamma$. These results indicate that the order and injection frequency of vaccine vehicles used for heterologous prime-boost vaccination affect the magnitude and nature of the immunity. Therefore, our demonstration implies that the prime-boost protocol should be carefully considered and selected to induce the desired immune responses.

• 대한핵의학회:학술대회논문집
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• 2002.11a
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• pp.35.2-35.2
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• 2002