• Tuberculosis and Respiratory Diseases
• /
• v.49 no.2
• /
• pp.189-197
• /
• 2000

Background : Tissue inhibitor of metalloproteinase is a natural inhibitor that counteracts pro teolytic enzymes essential to the invasion of cancer cell. Whether or not TIMP-2 gene transfer via adenovirus could inhibit the invasion of lung cancer cell iη vitro was evaluated for the future purpose of gene therapy against lung cancer. Methods : Recombinant adenovirus-TIMP-2(Ad-TIMP-2) was generated by homologous recombination after pACCMV-TIMP-2 and pJM17 were cotransfected into 293 cell by standard calcium phosphate coprecipitate method. Calu-6, one of the most invasive lung cancer cells, was transduced with Ad-TIMP-2 or Ad-$\beta$gal. Anchorage-independent growth and invasiveness were assessed by soft agar clonogenicity assay and invasion assay using two-chamber, well divided by matrigel. Results : Ad-TIMP-2 transduced calu-6 cells produced biologically active TIMP-2 more than 50 times more than parental calu-6. TIMP-2 gene transfer did not suppress the in vitro tumorigenicity. However, two chamber well assay revealed that Ad-TIMP-2 transduction reduced the invasiveness of calu-6 efficiently (12% compared with parental cell) even at low 10moi. Conclusion : Even though TIMP-2 gene transfer did not inhibit in vitro tumorigenicity, it did inhibit invasion of lung cancer cell in vitro. The inhibition of invasion by Ad-TIMP-2 may be a useful strategy for the treatment of lung cancer.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.28 no.5
• /
• pp.375-395
• /
• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Tuberculosis and Respiratory Diseases
• /
• v.42 no.4
• /
• pp.455-464
• /
• 1995

Background: Considering that both humoral and cell mediated immunities play an important role for human tuberculosis infection, enzyme-linked immunosorbent assay(ELISA) measurement of immunoglobulin G (IgG) antibody to mycobacterial antigens can be used for the serologic diagnosis of tuberculous pleural effusion. Method: We measured absorbance values of IgG antibodies to purified-protein-derivative (PPD) and lipoarabinomannan-B (LAM-B) in the pleural fluid (PF) and the serum in 40 tuberculous (TPE) and 19 nontuberculous pleural effusions (NTPE). Results: 1) The IgG antibodies to PPD and LAM-B were significantly (P<0.0005) higher in the PF and the serum of TPE compared to NTPE. 2) The IgG antibodies to PPD and LAM-B in the serum were higher than that in PF. 3) Significant correlations were found between pleural and serum IgG antibodies to PPD and LAM-B. 4) With a cutoff value for IgG antibody to PPD in the PF of 0.091, sensitivity was 55.0% and specificity 94.7% in the diagnosis of TPE. 5) With a cutoff value for IgG antibody to LAM-B in the PF of 0.337, sensitivity was 50.0% and specificity 94.7% in the diagnosis of TPE. 6) The seropositive rates in TPE were not related to PPD skin test status, the amount of PF and coexisting active pulmonary tuberculosis. Conclusion: The assay of IgG antibodies to PPD and LAM-B might be useful for the diagnosis of TPE. Our study suggests the mechanism of passive transfer of IgG antibodies to PPD and LAM-B from the serum to the PF through pleural tissue.

• Tuberculosis and Respiratory Diseases
• /
• v.49 no.6
• /
• pp.703-714
• /
• 2000

Background : Surfactant protein A (SP-A) is important in the regulation of surfactant secretion, synthesis and recycling. SP-A has important roles in regulating surfactant metabolism as well as in determining surfactant's physical properties. Since systemic sepsis is one of the common causes of acute respiratory distress syndrome (ARDS) and abnormalities in surfactant function have been described in ARDS, the authors investigated the effects of endotoxemia on the accumulation of mRNA encoding SP-A and SP-A protein content. Methods : Adult rats were given various doses of intraperitoneal endotoxin from Salmonella enteritidis and sacrificed at different times. SP-A mRNA was measured by filter hybridization method. Lung SP-A protein content was determined by double sandwich ELISA assay using a polyclonal antiserum raised in rabbits against purified rat SP-A. Results : 1) The accumulation of SP-A mRNA in the endotoxin treated group 24 hours after 2mg/kg and 5mg/kg endotoxin treatments was significantly increased 50.9% and 27.3%, respectively, compared to the control group (P<0.001, P<0.025). 2) The accumulation of SP-A mRNA 24 hours in the 5mg/kg endotoxin treated group was significantly increased by 26.5% compared to the control group (P<0.01). 3) Total amount of lung SP-A was not altered at 24 hours by various doses of treatment. Total lung SP-A content 144 hours after endotoxin administration was significantly decreased by 51.4% compared to the control group (P<0.01). Conclusions : The specific regulation of SP-A by various time course in vivo is evident. The late decline in SP-A protein content was unexpected and suggests that SP-A may be differentially regulated during lung inflammation. The functional significance of these alterations remains to be clarified.

• Journal of Life Science
• /
• v.33 no.4
• /
• pp.305-314
• /
• 2023

Ultraviolet B (UVB) irradiation causes skin diseases by inducing cellular oxidative stress, photoaging, and inflammation. This study aimed to investigate the protective effects of morin against UVB-induced oxidative stress in normal human dermal fibroblasts (NHDFs). Morin has been reported to be a potential therapeutic candidate for oxidative stress-mediated diseases, neurodegenerative diseases, and inflammation. Since morin has been identified as a potential antioxidant, we speculated that morin could alleviate UVB-induced apoptosis in NHDFs. Cell viability and intracellular reactive oxygen species (ROS) levels were measured using the MTT assay, H2DCFDA, and the DHE staining method, respectively. Lipid peroxidation and protein carbonyl formation were tested using ELISA kits. DNA fragmentation and comet assay were used to assess DNA damage. Apoptotic bodies were analyzed using Hoechst 33342 staining and TUNEL assay. The expression of apoptosis-related proteins was examined using Western blot analysis. Morin showed a cyto-protective effect by scavenging UVB-induced ROS, increasing the expression of antioxidant-related proteins and inhibiting UVB-induced oxidative alterations such as lipid peroxidation, protein carbonylation, and DNA damage. Morin protects against UVB-induced cell apoptosis by inhibiting Bcl-2-associated X protein, caspase-9, and caspase-3 expression, while increasing the expression of the anti-apoptotic protein Bcl-2. These effects of morin were conferred through decreased phosphorylation of p38 and c-Jun N-terminal kinase 1/2. The results demonstrated that morin may be developed as a preventive/therapeutic drug to be used to prevent UVB-induced skin damage.

• Journal of Animal Science and Technology
• /
• v.48 no.6
• /
• pp.933-942
• /
• 2006

This experiment was carried out to investigate effects of honeybee venom treatment on the humoral immune response in pigs. Corresponding author : S. K. Cho, Dept. of Animal Sci. Chung-Buk National University, Kaesin-dong, Cheongju, 361-763, Korea. phone : 043-261-2551. E-mail : deercho@chungbuk.ac.kr To investigate effects of natural honeybee venom on the concentration of immunoglobulin G, A, and M, 20 piglets(LY×D) from 3 sows were allocated into two groups bee venom-treated group(10 piglets) and non-treated control(10 piglets). Natural honeybee venom was treated at 0, 3, 6 days after birth and the acupoints were Hai-men(ST-25), Du-kou(CV-8) and Jiao-chao(GV-1) points at 0, 3 days after birth and the regions of castration and tail amputation point at 6 days. Control group was injected 1㎖ of saline to the same site. Concentrations of IgG, A, and M were measured with immunoturbidimetric method at 0, 3, 7, 14, and 21 days after treatment. To investigate the effect of bee venom on the production of antibodies against hog cholera and atrophic rhinitis vaccines that were used as indicator antigens, 40 piglets(LYxD) from 5 sows were grouped as bee venom-treated group (20 piglets) and control group(20 piglets). Natural honeybee venom was treated at 0, 3days(castration, tail amputation) and 21days after birth. The acupoints were Hai-men(ST-25), Du-kou(CV-8) and Jiao-chao (GV-1) points at 0 day, the regions of castration and tail ampution at 3 days and Jiao-chao(GV-1) and Bai-hui(GV-20) points at 21days after birth(weaning). Control group was injected 1ml of saline to the same site. Atrophic rhinitis vaccine was injected twice at 24 and 44 days after birth and hog cholera vaccine was also injected twice at 44 and 64 days after birth. Antibody titers against Bordetella bronchiseptica and hog cholera virus were measured by using tube agglutination and ELISA tests at 24, 34, 44, 54 and 74 days after birth. Concentrations of IgG of treated group were 339.52, 366.48, 296.52, 242.06 and 219.06mg/dl at 0, 3, 7, 14 and 21 days after birth, respectively. In contrast, concentrations of IgG in control group were respectively 347.10, 334.14, 243.28, 205.18 and 191.58mg/dl during same periods with treated group. Concentrations of IgG at 0 day was not significantly different between the treated group and control group but treated group were significantly increased by 10.28% at 3 days after birth (P<0.02), 21.88% at 7 days after birth(P<0.01), 18.0% at 14 days after birth(P<0.07) and 14.3% at 21 days after birth(P<0.01). Concentrations of IgA and Ig M were not significantly different. Antibody titers against hog cholera virus were significantly increased by 57.0% at 24 days after birth(P<0.03), 74.6% at 34 days after birth (P<0.006), 48.6% at 44 days after birth(P<0.017), 45.0% at 54 days after birth(P<0.16) and 44.4% at 74 days after birth (P<0.006) in bee venom treated group in comparison with control group. Antibody titers against the Bordetella bronchiseptica was significantly increased in Beevenom treated group as 9.1% (P<0.32) at 24days, 39.7% (P<0.002) at 34days, 31.9% (P<0.02) at 44days, 33.4% (P<0.01) at 54days and 57.3% (P<0.007) at 74 days after birth when compared with those of control group pigs. Collecting together, the results in this study showed that immune responses were increased by treatment of natural honeybee venom to pigs. These results suggested that the treatment of bee venom could be used effectively for the increase of productivity in livestock industry.

• Biomedical Science Letters
• /
• v.19 no.2
• /
• pp.90-97
• /
• 2013

The tuberculin skin test (TST) and interferon gamma (IFN-${\gamma}$) release assay (IGRA) have been widely used for diagnosis of latent tuberculosis infection (LTBI). In order to overcome limitations of current LTBI diagnostic methods, the development of a novel molecular assay which is able to measure the IFN-${\gamma}$ messenger RNA (mRNA) expression level after stimulation with Mycobacterium tuberculosis (MTB) specific antigen was recently developed. The ability of a molecular assay to detect MTB infection was similar to commercial IGRA however, the optimal incubation time for stimulating IFN-${\gamma}$ was not yet established. Therefore, in this study the direct comparisons of MTB Ag stimulation times (4 and 24 hrs) were performed for diagnosis of MTB infection. Data showed that the coincident rate between QFT-GIT IFN-${\gamma}$ ELISA and IFN-${\gamma}$ RT-PCR (4 hrs) was 88.35% and that of QFT-GIT and IFN-${\gamma}$ RT-PCR (24 hrs) was 70.85%. Based on a receiver operating characteristic (ROC) curve, the 4 hrs-MTB specific Ag stimulation time for IFN-${\gamma}$ RT-PCR had the significant P value, 95% CI value, and AUC (P < 0.0001, 95% CI=0.82 to 1.02, and AUC=0.9214) in comparison with 24 hrs-MTB specific Ag stimulation time (P = 0.009, 95% CI=0.06 to 0.94, and AUC=0.7711). These results show that 4-hr was the most optimal MTB Ag stimulation time for performing IFN-${\gamma}$ RT-PCR. Although semi-quantitative RT-PCR had a few analytical limitations, it might be useful as an alternative molecular diagnostic method for detecting MTB infection.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.6
• /
• pp.3877-3880
• /
• 2013

Objective: To explore the expression and significance of tumor specific growth factor (TSGF), carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP) in cancer tissue and serum of patients with colon cancer. Materials and Methods: Radical surgery for colon cancer was performed on 43 patients with laparoscopu under conditions of general anesthesia. The Elisa method was used to detect the levels of serum TSGF, CEA and AFP before and after radical operation, and cancer tissue underwent TSGF, CEA and AFP immunohistochemistry staining after laparoscopic surgery. The decreased conditions of serum TSGF, CEA and AFP in patients with colon cancer at different levels of differentiation and clinical stagings were analyzed, and the relationships of expression rates between histological types, colon cancer morphology, lymph node metastasis and TSGF, CEA as well as AFP in cancer tissue were assessed. Results: Compared with before radical surgery, the levels of serum TSGF, CEA and AFP decreased notably in patients after operations (p<0.01). The decreased degree of TSGF and CEA was the largest in patients with poorly differentiated cancer tissue (p<0.01), while that of AFP was noted in patients with moderately differentiated cancer tissue (p<0.01). The decreased degree of TSGF and AFP was the largest in patients at phase Dukes A (p<0.01), while that of CEA in patients at phase Dukes C (p<0.01). There were no significant differences among the positive expression rates of TSGF, CEA and AFP with different histological types and colon cancer morphologies (p>0.05). The positive expression rates of TSGF and CEA in patients with lymph node metastasis were significantly higher than those without lymph node metastasis (p<0.01). Conclusions: TSGF, CEA and AFP can be used to evaluate the effect of radical operation for colon cancer, and the changed levels of different markers are associated with tumor differentiation, clinical stating and presence or absence of lymph node metastasis.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.5
• /
• pp.2081-2086
• /
• 2015

Gecko is a kind of traditional Chinese medicine with remarkable antineoplastic activity. However, undefined mechanisms and ambiguity regarding active ingredients limit new drug development from gecko. This study was conducted to assess anti-angiogenic properties of the aqueous extracts of fresh gecko (AG) or macromolecular components separated from AG (M-AG). An enzyme-linked immunosorbent assay (ELISA) approach was applied to detect the vascular endothelial growth factor (VEGF) secretion of the tumor cells treated with AG or M-AG. The effect of AG or M-AG on vascular endothelial cell proliferation and migratory ability was analyzed by tetrazolium dye colorimetric method, transwell and wound-healing assays. Chick embryo chorioallantoic membrane (CAM) assays were used to ensure the anti-angiogenic activity of M-AG in vivo. The results showed that AG or M-AG inhibited the VEGF secretion of tumor cells, the relative inhibition rates of AG and M-AG being 27.2% and 53.2% respectively at a concentration of $20{\mu}L/mL$. AG and M-AG inhibited the vascular endothelial (VE) cell proliferation with IC50 values of $11.5{\pm}0.5{\mu}L/mL$ and $12.9{\pm}0.4{\mu}L/mL$ respectively. The VE cell migration potential was inhibited significantly (p<0.01) by the AG (${\geq}24{\mu}L/mL$) or M-AG (${\geq}12\mu}L/mL$) treatment. In vivo, neovascularization of CAM treated with M-AG was inhibited significantly (p<0.05) at a concentration of ${\geq}0.4{\mu}L/mL$. This study provided evidence that anti-angiogenesis is one of the anti-tumor mechanisms of AG and M-AG, with the latter as a promising active component.

• Tuberculosis and Respiratory Diseases
• /
• v.58 no.2
• /
• pp.142-152
• /
• 2005

Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and $IFN-{\gamma}$ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and $IFN-{\gamma}$ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. Conclusion : Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.