• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

• The Journal of Korean Society of Virology
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• v.28 no.3
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• pp.247-265
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• 1998

To determine the sequence and expression of the VP7 gene of Korean isolates (CAU-9), viral RNA was purified and used for cDNA amplification by RT-PCR. The VP7 cDNA was cloned, sequenced, and expressed using baculovirus expression system. The result showed that the sequence homologies CAU-9 compared with foreign isolated strains Wa, 417, TMC-II, 95B and SA11 were ranged from 74.0% to 95.1 % of nucleotide sequence and 35% to 43% of amino acid sequence, respectively. High homology of CAU-9 was observed in Japanease isolates 417 (nucleotide sequence homology was 95.1% and amino acid sequence homology was 43%). To express VP7 gene, the VP7 cDNA was cloned into pCR-Bac vector and inserted into the genome of baculovirus adjacent to the polyhedrin promoter by cotransfection of Spodoptera frugiperda (Sf9) insect cells with wild type baculovirus DNA. In antigenic analysis of Sf9 cells inoculated with the recombinant VP7, immunofluorescence assay revealed positive for viral antigens. In metabolic labeling of Sf9 cell lysates infected with recombinant baculoviruses, it was revealed that the protein of 34 kDa was expressed. The limited study of expressed VP7 protein inoculated with guinea pigs failed to elicit neutalizing antibody. As a results, the sequence analysis and expression of VP7 protein of rotavirus CAU-9 isolated from an infant in Korea could permit the conformation and development of virus like particles which may be useful in designing vaccine strategy.

• Korean Journal of Head & Neck Oncology
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• v.28 no.1
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• pp.3-7
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• 2012

Background and Objectives : Nasopharyngeal carcinoma(NPC) shows a distinct geographic and demographic distribution with high incidences in Chinese and Southeast Asians. Current WHO classification divides NPC into nonkeratinizing carcinoma(NKC)(differentiated and undifferentiated subtypes), keratinizing squamous cell carcinoma(KSCC), and basaloid squamous cell carcinoma(BSCC). Relative frequency of histologic subtypes of NPC is known to vary according to the incidence of NPC. Korea is one of the low-incidence countries according to the GLOBOCAN 2008 database by IARC. The aim of this study is to assess the histopathologic types and EBV status of NPC of Koreans. Materials and Methods : We reviewed and reclassified 168 cases of NPC(132 males and 36 females) diagnosed from January 1996 through July 2006. In situ hybridization for EBV-encoded early RNA(EBER) was performed on 146 cases and the results were compared among different histologic types, genders, and age and stage groups. Results : NKC, undifferentiated subtype(NKC-U) was identified in 106 cases(63.1%) and differentiated subtype(NKC-D) in 49 cases(29.2%). Remaining 13 cases(7.7%) were classified as KSCC. NKC and NKC-U were more common in females than in males. EBV prevalence was higher in NKC than in KSCC(NKC-U, 90% ;, NKC-D, 84.1% ; KSCC, 7.7%) and more common in younger age(${\leq}40$) than older age(>40) group. Conclusion : Histologic type distribution and EBV prevalence of NPC in Korean patients corresponded to that of intermediate incidence area. Pathogenesis of nasopharyngeal KSCC is assumed to be different from that of NKC.

• Journal of Yeungnam Medical Science
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• v.10 no.1
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• pp.190-196
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• 1993

The prognosis of chronic hepatitis C is very variable. In some, the disease is progressive and cirrhosis can develop from chronic hepatitis C. Hepatitis C virus(HCV) may act as a trigger towards hepatocellular carcinoma in patients with cirrhosis. Interferon has been used for the treatment of chronic hepatitis C in abroad, 16 patients with chronic C liver disease were treated with ${\alpha}$-interferon (alfa-2b; "Intron A" Schering Corp. Kenilworth. NJ). All patients were given ${\alpha}$-interferon in subcutaneous doses of 3 million units three times weekly for 1 to 9 months. During therapy, CBC and ALT levels were checked weakly to monthly. After therapy, patients were followed for 1 to 8 months. Among 16 patients treated with ${\alpha}$-interferon, progressive decrease of ALT levels was observed in 14(87.5%). In 11 patients(68.8%), ALT levels fell into the normal range during therapy, and in 9 of 11, within one month after therapy, 6 months after the completion of therapy in 4 of 9 patients(44.4%) whose ALT levels were in the normal range, ${\alpha}$-interferon seems to have effect in controlling disease activity in patients with chronic hepatitis C. But the changes in the usage of ${\alpha}$-interferon, dose and duration, long term follow up and more convenient and simple tests for HCV detection are recommended for the better effect and the exact evaluation on the effect of ${\alpha}$-interferon therapy in patients with chronic hepatitis C.

• Clinical and Experimental Pediatrics
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• v.49 no.4
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• pp.401-409
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• 2006

Purpose : Human metapneumovirus(hMPV) is a respiratory viral pathogen that causes a wide spectrum of illnesses, ranging from asymptomatic infection to severe bronchiolitis. The virus has been identified world widely, but so far it has not been published in Korea. Methods : We obtained clinical samples by nasopharyngeal aspiration from 218 children hospitalized due to acute lower respiratory tract infections at Soonchunhyang University Hospital in Cheonan from October, 2004 to April, 2005. We designed specific primers from conserved region of fusion glycoprotein of hMPV. Total RNA was extracted and RT-PCR was performed, and single specific 423 bp product was obtained. The PCR product was confirmed to be fusion glycoprotein RNA by sequencing. Results : We detected hMPV in 15(6.9 percent) of the 218 hospitalized children. The infected children comprised nine boys and six girls; their mean age was 2.8 years(5 mo-12 yrs) and they were diagnosed with pneumonia(60 percent), bronchiolitis(33.3 percent), croup(6.6 percent). The number of cases of detected hMPV in Korea increased dramatically during the period from March to May 2005. Conclusion : hMPV is circulating in Korean children and is associated with respiratory tract infection. Additional studies are required to define the epidemiology and the extent of diseases in the general population caused by hMPV.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.26 no.3
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• pp.254-261
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• 2000

The present study has attempted to look into the mechanism of ras-induced carcinogenesis in a human epithelial cell system. Human epithelial cells immortalized with Ad12-SV40 hybrid virus were used to assess carcinogenic potential of the ras-oncogene. Cells transfected with pSV2-ras showed characteristics of cellular transformation. The transformation parameters such as cell density, soft-agar colony formation, and cell aggregation were significantly increased in the cells expressing ras oncoprotein. In addition, the duration required for the appearance of foci was shortened in the ras-transfected cells. Consistent with other reports, our results demonstrated an evidence that the ras-oncogene induced the cellular transformation of human epithelial cell system. When a high concentration of glucocorticoid was added into the media, transformation process was accelerated. It is speculated that glucocorticoid may provide an advantageous environment for the proliferation of the transformed cells. The induction of the intracellular free calcium concentrations following agonist treatment was significantly lower in the transformed cells than in the control cells. These effects were more manifested in the presence of extracellular cacium, indicating that the transformation process may alter the influx pathway of extracellular calcium. The induction of $IP_3$ following agonist treatment was also lower in the transformed cells than in the control cells. Thus, it is suggested that phospholipase C-coupled pathway was down-regulated in the process of the ras-induced transformation. While the levels of $TGF-{\beta}_1$ and PAI-2 mRNAs were decreased, the level of fibronectin mRNA was increased. The results indicate that mechanism of the ras-induced transformation may be associated with the altered expressions of growth regulatory factors. The present study demonstrates an evidence that the ras-induced cellular transformation may be associated with alteration of signal transduction and growth regulatory factors. The study will contribute to improve the understanding of molecular mechanism of epithelium-derived cancers including oral cancer.

• Clinical and Experimental Pediatrics
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• v.49 no.7
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• pp.745-750
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• 2006

Purpose : Enteroviruses are the most common cause of aseptic meningitis in patients of all ages. A definite diagnosis of enteroviral meningitis can be established by detection of virus directly in CSF specimens. But this is time-consuming and lacks sensitivity, so polymerase chain reaction(PCR) detecting of viral RNA in patient specimens such as CSF, stool has been demonstrated. But little is known about the influence of sampling time on the results of CSF PCR and stool PCR. We investigated diagnostic utility of PCR of CSF and stool according to sampling time after the onset of symptoms. Methods : PCR results were analyzed according to sampling time for 42 patients diagnosed aseptic meningits in our hospital from $11^{th}$ January to $30^{th}$ August, 2005. Results : The diagnostic yield of the test was higher of CSF specimens obtained ${\leq_-}2$ days after clinical onset(positive PCR results 9/18, 50 percent), compared with CSF collected >2 days after onset(positive PCR results 1/24, 4.2 percent)(P=0.001). Instead, positive PCR results of fecal specimens maintained highly(average 90.5 percent), 10 cases had also positive PCR results even 5-6 days after onset. 10 cases of CSF specimens had positive enterovirus PCR results containing coxsackievirus B5 (n=6), coxsackievirus B3(n=3). 38 cases of stool specimens had positive enterovirus PCR results containing echovirus 18(n=7), echovirus 9(n=3), coxsackievirus B5(n=8), coxsackievirus B3(n=3). 6 cases(coxackie B5) had positive CSF PCR and stool PCR, both. Conclusion : Stool PCR was clinically sensitive for detecting enterovirus during enteroviral meningits and could give a presumptive diagnosis throughout the disease course. A definite diagnosis was obtained by CSF PCR, but its utility was clearly lower for samples obtained >2 days after clinical onset. Therefore, it is recommended that, in addition to performance of CSF PCR, fecal samples obtained from patients with suspected enteroviral meningitis should be tested by PCR, especially when the duration of symptoms is >2 days.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Journal of Plant Biotechnology
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• v.41 no.2
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• pp.81-88
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• 2014

Salt cress (Thellungiella halophila or Thellungiella parvula), species closely related to Arabidopsis thaliana, represents an extremophile adapted to harsh saline environments. To isolate salt-tolerance genes from this species, we constructed a cDNA library from roots and leaves of salt cress plants treated with 200 mM NaCl. This cDNA library was subsequently shuttled into the destination binary vector [driven by the cauliflower mosaic virus (CaMV) 35S promoter] designed for plant transformation and expression via recombination- assisted cloning. In total, 305,400 pools of transgenic BASTA-resistant lines were generated in Arabidopsis using either T. halophila or T. parvula cDNA libraries. These were used for functional screening of genes involved in salt tolerance. Among these pools, 168,500 pools were used for primary screening to date from which 7,157 lines showed apparent salt tolerant-phenotypes in the initial screen. A secondary screen has now identified 165 salt tolerant transgenic lines using 1,551 (10.6%) lines that emerged in the first screen. The prevalent phenotype in these lines includes accelerated seed germination often accompanied by faster root growth compared to WT Arabidopsis under salt stress condition. In addition, other lines showed non-typical development of stems and flowers compared to WT Arabidopsis. Based on the close relationship of the tolerant species to the target species we suggest this approach as an appropriate method for the large-scale identification of salt tolerance genes from salt cress.

• Pediatric Infection and Vaccine
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• v.14 no.1
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• pp.75-82
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• 2007

Purpose : Human metapneumovirus (hMPV) is a newly identified paramyxovirus that causes a variety of clinical syndromes in children, including upper and lower respiratory tract illnesses. hMPV is considered an ubiquitous virus causing respiratory tract diseases among children especially during late winter and spring seasons. We report clinical features of human metapneumovirus infection in Korean children. Methods : hMPV infection was diagnosed by reverse transcriptase-polymerase chain reaction (RT-PCR) in respiratory specimens obtained from patients with acute respiratory tract infections from October, 2004 to May, 2005. Medical records of all hMPV-positive patients were reviewed, retrospectively. Results : A total of 15 hMPV were identified from 443 nasopharyngeal aspirations by RT-PCR (3.4%). The range of age of the patients with hMPV infection was from 1 month to 62 months (median age, 31.5 months), with similar numbers of females (8/15) and males (7/15). Among hMPV-positive children, 53.3% (8/15) were aged less than 24 months. Fever, cough, rhinorrhea, vomiting, diarrhea, tachypnea, and chest wall retractions were common findings. Most common clinical diagnosis was pneumonia (60%). Two of the 15 hMPV-positive patients were also positive for adenovirus. Fever persisted from 0 to 10 days (mean 4.9 days). The duration of hospitalization ranged from 4 to 7 days (mean 5.6 days). Conclusion : hMPV accounted for a small but significant proportion of respiratory tract infection in infants and children. Future development and application of diagnostic tools will determine the burden of disease caused by this newly discovered pathogen.