• Journal of Life Science
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• v.22 no.6
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• pp.703-712
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• 2012

Adeno-associated virus (AAV) has been considered to be a very safe and efficient gene delivery system. However, the major obstacles to therapeutic usage of AAV have been to achieve highly efficient and reproducible production processes, and also to develop a reliable quantifying method of various serotypes with a simple protocol. We compared the efficiency of the conventional production protocol of AAV2 and adenovirus (Ad) co-infection to that of a new method containing AAV2 infection followed by pHelper transfection. We tested HEK293 and 293T, and further examined the time-dependent changes of AAV2 production. The new method of AAV2 and pHelper DNA gave about ten times higher production efficiency than that of the conventional protocol. The highest production efficiency in 293T was achieved as $1.61{\times}10^5$ virus genomes (v.g.)/cell by the new method of 10 MOI of AAV2 infection and 5 days post-infection. This protocol of the highest efficiency was then applied to produce various AAV serotypes and showed the efficiencies higher than $10^5$ v.g./cell. Next, we designed the universal PCR primers of highly conserved regions for various AAV serotypes to develop a simple and reliable titration method. The universal primers could amplify all the tested AAV serotypes with similar sensitivities by ten molecular copies. Therefore, this pair of universal primers can be further utilized to detect AAV contaminants in therapeutic adenoviral vectors.

• Clinical and Experimental Pediatrics
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• v.52 no.6
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• pp.661-666
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• 2009

Purpose : To determine the prevalence and clinical features of codetected respiratory etiological agents for acute respiratory infection in hospitalized children. Methods : Nasopharyngeal aspirates were obtained from hospitalized children with acute respiratory infection at Dankook University Hospital from September 2003 through June 2005. Immunofluorescent staining and culture were used for the detection of respiratory viruses (influenza virus [IFV] types A, B; parainfluenza virus [PIV] types 1, 2, 3; respiratory syncytial virus [RSV]; adenovirus [AdV]). Polymerase chain reaction (PCR) assays were used for Mycoplasma pneumoniae (MP) and Chlamydia trachomatis (CT) detection, and PCR and culture were performed for enterovirus detection. Acid-fast staining and culture were performed for tuberculosis detection. The demographic and clinical characteristics were reviewed retrospectively from the patients medical records. Results : Evidence of two or more microbes was found in 28 children: RSV was detected in 14, PIV 3 in 10, AdV in 10, MP in 8, PIV 2 in 8, CT in 4, and PIV 1 in 3. Codetected agents were found as follows: RSV＋PIV 2, 6 patients; AdV＋MP, 4 patients; AdV＋PIV, 3 patients; RSV＋MP, 3 patients; PIV 1＋PIV 3, 3 patients. Distinct peaks of codetected agents were found in epidemics of MP and each respiratory virus. Conclusion : The codetected infectious agents were RSV, PIV, AdV, and MP, with distinct peaks found in epidemics of MP and each respiratory virus. Although advances in diagnostic methods have increased the prevalence of codetection, its clinical significance should be interpreted cautiously.

• Pediatric Infection and Vaccine
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• v.21 no.1
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• pp.43-52
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• 2014

Purpose: This study aimed to evaluate the disease severity of children suffering from gastroenteritis using different scales. The results are compared and subsequently classified on the basis of the type of virus causing the disease in order to investigate the differences in clinical characteristics and disease severity according to pathogen. Method: This study was conducted prospectively with patients under 5 years of age diagnosed with acute gastroenteritis and hospitalized at 9 medical institutions in 8 regions across the Republic of Korea. Disease severity was evaluated using the Vesikari Scale, the Clark Scale, and the modified Flores Scale. Fecal samples collected from patients were used to detect rotavirus and enteric adenovirus by enzyme immunoassay, and for RT-PCR of norovirus, astrovirus, and sapovirus. Results: There were a total of 214 patients with a male : female ratio of 1.58 : 1, of which 35 were under the age of 6 months (16.4%), 105 were aged 6-23 months (49.1%), and 74 were aged 24-59 months (34.5%). The rate of concordance between the Vesikari and Clark Scales was 0.521 (P<0.001) and, in severe cases, the Vesikari Scale was 60.7% and Clark Scale was 2.3%, indicating that the Clark Scale was stricter in the evaluation of severe cases. Conclusions: In children with gastroenteritis, there were differences in disease severity based on the scale used. Therefore, to achieve consistent results among researchers, either only a single scale or a measure of all scales should be used to determine disease severity.

• Clinical and Experimental Pediatrics
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• v.49 no.1
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• pp.56-63
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• 2006

Purpose : This study was designed to document the etiologies and the characteristics of parapneumonic effusion in children. Methods : During a 17-year period from 1987 to 2004, parapneumonic effusion was confirmed in 86 children at Gyeongsang National University Hospital. The clinical records of these children were reviewed and radiological findings and laboratory data, especially results of thoracentesis, were analyzed retrospectively. Results : M. pneumoniae(34 subjects) was the most common pathogen at all over age, especially above 1-years-old. There were diagnosed with clinical characteristics and serologic tests. The $2^{nd}$ most common pathogen revealed non tuberculous bacteria(14 subjects). A species of bacteria at no tuberculous bacteria revealed S. aureus(5), S. pneumoniae(3), P. aeroginosa(3), other staphylococcus (2), and K. pneumoniae(1). There were confirmed with sputum culture or pleural fluid culture or blood culture. S. aureus was most common pathogen in infants. The $3^{rd}$ common pathogen was M. tuberculosis(7). There were confirmed with skin tuberculin tests and AFB stains. Another that was classified as a non bacteria was adenovirus(2). Complications of parapneumonic effusion such as pleural thickness occurred on M. tuberculosis(1). Non tuberculous bacteria, especially S. aureus revealed a serious predominance of polymorphocyte at pleural fluid, and lowest pleural pH and glucose, and highest pleural protein and LDH. Tuberculosis revealed high pleural protein and LDH. Conclusion : Age and chemistries of pleural fluid might be helpful in differentiating various etiologies of parapneumonic effusion. If there were suspicious of tuberculosis and non-tuberculous bacteria, more aggressive approaches were needed to prevent complication.

• Clinical and Experimental Pediatrics
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• v.49 no.4
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• pp.431-438
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• 2006

Purpose : Insulin-like growth factor binding protein(IGFBP)-3 has been known as a tumor suppressor gene, and its anti-tumor function was divided into insulin-like growth factor(IGF)-dependent and IGF-independent mechanism. In IGF-independent mechanism, IGFBP-3 directly interacts with a cell without binding of IGFs, becoming an interesting object in oncology. Several studies demonstrate that one of the well-known tumor suppressor genes, p53, induces directly IGFBP-3 transcription, and the increment of IGFBP-3 expression induces apoptosis of many cancer cells. Recently, the anti-tumor mechanisms of IGFBP-3 have been reported, but post-translational modification of IGFBP-3 and its anti-tumor mechanism are not well known. In this study, we examined whether p53 regulated the glycosylation of IGFBP-3, and analysed the meaning of IGFBP-3 glycosylation related to the apoptosis of cancer cell. Methods : The p53-mutated status of MDA-MB-231 human breast cancer cells was used in this experiment. The expression and glycosylation of IGFBP-3 were tested by Western blot analysis after infection of adenovirus mediated Ad/p53 and/or Ad/IGFBP-3. Results : Ad/p53 infected cells resulted in growth retardation and the induced apoptosis. p53 induced direct expression and glycosylation of IGFBP-3. The increase of glcosylated IGFBP-3 was able to promote cellular apoptosis, and the glycosylation of IGFBP-3 was more activated by the double treatment of Ad/p53 and Ad/IGFBP-3. Conclusion : From this study, the anti-tumor activity of IGFBP-3 was shown to improve the stabilization of IGFBP-3 through the increment of glycosylation of IGFBP-3 by p53. This result suggests that the combined gene therapy of p53 and IGFBP-3 may appropriate treatment of cancer.

• Tuberculosis and Respiratory Diseases
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• v.46 no.2
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• pp.229-240
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• 1999

Background: The impact of the immune response on cancer gene therapy using viral vectors to deliver a "suicide gene" is currently unclear. A vigrous immune response targeted at viral proteins or transgene may enhance the efficacy of tumor destruction and even augment responses to tumor antigens. These responses may involve the release of cytokines and stimulation of tumor specific cytotoxic T-lymphocytes that enhance therapeutic efficacy. On the other hand, a vigorous rapid cellular immune response may destroy cells expressing the therapeutic gene and attenuate the response to therapy. Furthermore, development of neutralizing antibody responses may prevent readministration of virus, a potentially significant limitation. Evaluating the significance of these limitations in animal models and developing solutions are therefore of obvious importance. Methods: After retroviral transduction of mouse mesothelioma cell line(AB12) with Herpes Simplex Virus thymidine kinase (HSVtk) gene in vitro, subcutaneous flank tumors were established. To study the effect of intact immune system on efficacy of tumor erradication, the ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Balb/c mice, immunodeficient Balb/c-nude and SCID mice, and Balb/c mice immunosuppressed with cyclosporin. Results: Ganciclovir treatment resulted in greater inhibition of tumor growth in Balb/c mice compared with immunodeficient Balb/c-nude mice and SCID mice(in immunodeficient mice, there were no growth inhibition by ganciclovir treatment). Ganciclovir treatment resulted in greater inhibition of tumor growth in noncyclosporin (CSA) treated Balb/c mice compared with CSA treated Balb/c mice. On day 8, mean ganciclovir-treated tumor volume were 65% of control tumor volume in Balb/c mice versus 77% control tumor volume in CSA-treated Balb/c mice. This effect was still evident during therapy (day 11 and 13). On day 13, non-CSA treated tumor volume was 35% of control tumor volume versus 60% of control tumor volume in CSA treated Balb/c mice. Duration of expression of HSVtk was not affected by the immunosuppression with CSA. Conclusion: These results indicate that the immune responses against retrovirally transduced cells enhance the efficacy of the HSVtk/ganciclovir system. These findings have important implications for clinical trials using currently available retrovirus vectors as well as for future vector design.

• Tuberculosis and Respiratory Diseases
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• v.51 no.2
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• pp.122-134
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• 2001

Background : Some chemotherapeutic drugs induce NF-${\kappa}B$ activation by degrading the $I{\kappa}B{\alpha}$ protein in cancer cells which contributes to anticancer drug resistance. We hypothesized that inhibiting $I{\kappa}B{\alpha}$ degradation would block NF-${\kappa}B$ activation and result in increased tumor cell mortality in response to chemotherapy. Methods : The "superrepressor" form of the NF-${\kappa}B$ inhibitor was transferred by an adenoviral vector (Ad-$I{\kappa}B{\alpha}$-SR) to the human lung cancer cell lines (NCI H157 and NCI H460). With a MIT assay, the level of sensitization to cisplatin and paclitaxel were measured. To confirm the mechanism, an EMSA and Annexin V assay were performed. Results : EMSA showed that $I{\kappa}B{\alpha}$-SR effectively blocked the NF-${\kappa}B$ activation induced by cisplatin. Transduction with Ad-$I{\kappa}B{\alpha}$-SR resulted in an increased sensitivity of the lung cancer cell lines to cisplatin and paclitaxel by a factor of 2~3 in terms of $IC_{50}$. Annexin-V analysis suggests that this increment in chemosensitivity to cisplatin probably occurs through the induction of apoptosis. Conclusion : The blockade of chemotherapeutics induced NF-${\kappa}B$ activation by inducing Ad-$I{\kappa}B{\alpha}$-SR, increased apoptosis and increasing the chemosensitivity of the lung cancer cell lines tested, subsequently. Gene transfer of $I{\kappa}B{\alpha}$-SR appears to be a new therapeutic strategy of chemosensitization in lung cancer.

• Clinical and Experimental Pediatrics
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• v.52 no.3
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• pp.330-338
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• 2009

Purpose : The causes of acute lower respiratory tract infection (ALRTI) are mostly attributable to viral infection, including respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus A/B (IFV A/B), or adenovirus (ADV). Several Korean studies reported human metapneumovirus (hMPV) as a common pathogen of ALRTI. However, studies on seasonal distribution and clinical differences relative to other viruses are insufficient, prompting us to perform this study. Methods : From November 2006 to October 2007, we tested nasopharyngeal aspiration specimens in children hospitalized with ALRTI with the multiplex reverse transcriptase-polymerase chain reaction to identify 6 kinds of common pathogen (hMPV, RSV, PIV, IFV A/B, and ADV). We analyzed positive rates and clinical features by respiratory chart review. Results : We detected 38 (8.4%) hMPV-positive cases out of 193 (41.8%) virus-positive specimens among 462 patients. HMPV infection prevailed from March to June with incidence peaking in April. HMPV-positive patients were aged 15 years (76.3%), and the ratio of boys to girls was 1.2:1. The median age was 27 months. HMPV primarily caused pneumonia (76.3 %) (P=0.018). Average hospitalization of HMPV-associated ALRTI patients was 5.8 days. In addition, they showed parahilar peribronchial infiltration (100%) on chest X-ray, normal white blood cell count (73.7%), and negative C-reactive protein (86.8 %) (P>0.05). All hMPV-positive patients recovered without complication. Conclusion : HMPV is a common pathogen of ALRTI in Korean children, especially in 1-5 year olds, from March to May. Immunocompetent children diagnosed with hMPV-associated ALRTI may have a good prognosis.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5767-5772
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• 2014

Background and Aim: B7-H1, a co-inhibitory molecule of the B7 family, is found aberrantly expressed in ovarian cancer cells and infiltrating macrophage/dendritic-like cells, and plays a critical role in immune evasion by ovarian cancer. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects on ovarian cancer. However, whether IL-12 regulates B7-H1 expression in human ovarian cancer associated-macrophages has not been clarified. Therefore, we investigated the effects of IL-12 on the expression of B7-H1 in ovarian cancer-associated macrophages and possible mechanisms. Methods: PMA induced THP-1-derived macrophages or human monocyte-derived macrophages were treated with recombinant IL-12 (rIL-12) or infected with adenovirus carrying human IL-12 gene (Ad-IL-12-GFP) for 24 h, then cocultured with the SKOV3 ovarian cancer cell line for another 24 h. Macrophages were collected for real-time PCR and Western blot to detect the expression of B7-H1, and activation of the NF-${\kappa}B$ signaling pathway. Moreover, supernatants were collected to assay for IL-12, IFN-${\gamma}$ and IL-10 by ELISA. In addition, monocyte-derived macrophages treated with IFN-${\gamma}$ were cocultured with SKOV3 and determined for the expression of B7-H1. Furthermore, the expression of B7-H1 in monocyte-derived macrophages was also evaluated after blocking NF-${\kappa}B$ signaling. Results: The expression of B7-H1 was significantly upregulated in monocyte-derived macrophages treated with rIL-12 or Ad-IL-12-GFP compared with the control groups (p<0.05), accompanied by a remarkable upregulation of IFN-${\gamma}$ (p<0.05), a marked downregulation of IL-10 (p<0.05) and activation of NF-${\kappa}B$ signaling. However, the upregulation of B7-H1 was inhibited by blocking the NF-${\kappa}B$ signaling pathway (p<0.05). Expression of B7-H1 was also increased (p<0.05) in monocyte-derived macrophages treated with IFN-${\gamma}$ and cocultured with SKOV3. By contrast, the expression of B7-H1 in THP-1-derived macrophages was significantly decreased when treated in the same way as monocyte-derived macrophages (p<0.05), and IL-10 was also significantly decreased but IFN-${\gamma}$ was almost absent. Conclusions: IL-12 upregulates the expression of B7-H1 in monocyte-derived macrophages, which is possible though inducing the secretion of IFN-${\gamma}$ and further activating the NF-${\kappa}B$ signal pathway. However, IL-12 downregulates the expression of B7-H1 in THP-1-derived macrophages, associated with a lack of IFN-${\gamma}$ and inhibition of expression of IL-10.

• KSBB Journal
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• v.21 no.4
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• pp.248-254
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• 2006

Animal cell culture industry has a large market and an exponential growth rate among biological industry field. Chines hamster ovary(CHO) cells are the most widely used cell lines for recombinant protein production. They can avoid infection from polio, herpes, hepatitis B, HIV, measles, adenovirus and etc. Moreover it is easy to transfection recombinant genes and possible to suspension culture. Serum free media is one of the most important factor of protein production. Because serum has problems. Serum is not defined the contents until now, it has a number of proteins, lipids, carbohydrates and unknown molecules that cause of risk involve in infection and high cost of product purification. CHO cell line cultured using serum free media were the basis of a very successful method to produce(glyco-)protein in mammalian cells, which are then used as pharmaceutical products. Also, the low protein content of the developed medium facilitates downstream processing and product purification. But non-adapted CHO cells have a limit of proliferation cultured using serum free media and it takes very long time to adapt non-adapted cells to serum free media. There are a number of causes of a limit of proliferation using serum free media. Absence of growth factors and growth stimulating molecules is a major factor of the reasons. It makes growth signals and moves cell cycle. And increase of cellular stress is another reason. It induces increase of intraceullar ROS concentration. The purpose of this study is about improvement of proliferation capacity of non-adapted CHO cells cultured using serum free media without adaptation process.