• Tuberculosis and Respiratory Diseases
• /
• v.52 no.4
• /
• pp.395-404
• /
• 2002

Background : Surfactant protein A (SP-A) is important for regulating surfactant secretion, synthesis and recycling. However, It's regulation in vivo is unclear. SP-A has important roles in regulating surfactant metabolism as well as determining its physical properties. Glucocorticoid accelerates the morphologic differentiation of epithelial cells into type II cells and increase the rate of phosphatidylcholine synthesis. Methods : The authors investigated the effects of glucocorticoid on the accumulation of mRNA encoding SP-A and SP-A protein content. Adult rats were given various doses of subcutaneous dexamethasone and sacrificed after 24 hours and one week. SP-A mRNA was measured using a filter hybridization method. The lung SP-A protein content was determined using a double sandwich ELISA assay with polyclonal antiserum raised in rabbits against purified rat SP-A. Results : 1) The accumulation of SP-A mRNA in the dexamethasone treated group 24 hours after 0.2 mg/kg dexamethasone treatment was increased 38.8% compared to the control group. 2) The accumulation of SP-A mRNA in the dexamethasone treated group 1 week after 2 mg/kg dexamethasone treatment was 49.7% higher than the control group(P<0.01). 3) The total lung SP-A level was not altered after 24 hours by the 0.2mg/kg treatment. The total lung SP-A content one week after 2mg/kg dexamethasone administration was 373.7% higher than the control group(P<0.005). Conclusion : Dexamethasone treatment results in an increase in the SP-A mRNA and SP-A protein levels, suggesting that the pretranslational events in vivo may in part contribute to this process.

• Tuberculosis and Respiratory Diseases
• /
• v.62 no.4
• /
• pp.299-307
• /
• 2007

Background: High mobility group box 1 (HMGB1) is a novel, late mediator of inflammation. This study compared the pro-inflammatory effects of LPS and HMGB1. The transcriptional factors that play an important role in mediating the HMGB1-induced stimulation of IL-8 were also evaluated. Methods: RAW264.7 cells were stimulated with either LPS (100 ng/ml) or HMGB1 (500 ng/ml). The $TNF-{\alpha}$, MIP-2 and $IL-1{\beta}$ levels in the supernatant were evaluated by ELISA at 0, 2, 4, 8, 12 and 24h after stimulation. An acute lung injury was induced by an injection of LPS (5 mg/kg) or HMGB1 (2.5 mg/kg) into the peritoneum of the Balb/c mice. The lung cytokines and MPO activity were measured at 4h (for LPS) or 24h (for HMGB1) after the injection. The transcriptional factor binding sites for NF-IL6, $NF-{\kappa}B$ and AP-1 in the IL-8 promoter region were artificially mutated. Each mutant was ligated with pIL-6luc and transfected into the RAW264.7 cells. One hour after stimulation with HMGB1 (500 ng/ml), the cell lysate was analyzed for the luciferase activity. Results: The expression of MIP-2, which peaked at 8h with LPS stimulation, increased sequentially until 24h after HMGB1 stimulation. An intraperitoneal injection of HMGB1, which induced a minimal increased in $IL-1{\beta}$ expression, provoked the accumulation of neutrophils the lung. A mutation of AP-1 as well as $NF-{\kappa}B$ in the IL-8 promoter region resulted in a lower luciferase activity after HMGB1 stimulation. Conclusion: The proinflammatory effects of HMGB1, particularly on IL-8, are mediated by both $NF-{\kappa}B$ and AP-1.

• Clinical and Experimental Pediatrics
• /
• v.50 no.3
• /
• pp.248-254
• /
• 2007

Purpose : Inflammation plays a major role in the pathogenesis of RDS and BPD in the immature lung. We investigated the possible role of IL-10 and IL-12 in the cord blood of preterm newborns with RDS or BPD. Methods : Forty preterm newborns whose mothers received antenatal care at Ewha Womans University Mokdong Hospital between January 2003 to June 2005, and agreed to testing their cord blood samples were enrolled. The gestational ages were below 34 weeks. Cord blood level of IL-10 and IL-12 were determined by ELISA. We separated the patients into 2 groups (RDS group and non-RDS group, BPD group and non-BPD group) and compared the cytokine levels and clinical records of the groups. Results : Cord blood IL-10 level showed a significant inverse correlation with gestational age and birth weight (P=0.001, P=0.005). Preterm infants with RDS showed higher IL-10 level (1.0 vs 0.1 pg/mL; P=0.001) in the cord blood than those without RDS. The differences remained statistically significant after correction for the effect of gestational age between both preterm groups. Despite similar cord blood IL-10 levels, preterm infants with BPD showed no significant difference with those without BPD. Conclusion : Cord blood IL-10 levels are increased in preterm infants which may be due to the immuno-suppression occurring during pregnancy and to fetal immaturity because these levels are inversely correlated with the gestational age. So, Cord blood IL-10 level can be used as the predictor of RDS.

• Reproductive and Developmental Biology
• /
• v.32 no.1
• /
• pp.27-31
• /
• 2008

This study was performed to investigate the correlations between steroids and TGF-${\beta}_1$ levels and delayed parturition in SCNT clone calving. The recipients pregnant by AI were used as control (AI-R). All AI-R were labored by natural delivery (n=5, day $284{\pm}0.71$ of pregnancy). The recipients pregnant by SCNT embryo (SCNT-R) showing no signs of delivery about 10 days after expected date were operated by Caesarean section (n=5, day 292). The blood and placentome samples were obtained and weighed at parturition. The concentrations of plasma progesterone (P4) and Estradiol-$17{\beta}$ (E2) were measured by radioimmunoassay (RIA). The levels of plasma and placental TGF-${\beta}_1$ levels were examined by ELISA. The placentomes from SCNT-R were overweight (p<0.05) compared to those of AI-R. The plasma P4 (p<0.01) level in SCNT-R at parturition was significantly higher compared to that of AI-R. In contrast, the plasma E2 level in the SCNT-R was significantly lower compared to that of AI-R (p<0.05). The plasma and placental TGF-${\beta}_1$ protein levels in the SCNT-R were significantly higher than those of AI-R at parturition, respectively (p<0.01). Based on these results, aberrant expressions of steroid hormones and high levels of plasma and placental TGF-${\beta}_1$ protein at parturition may be one of the key indicators on delayed parturition of SCNT clone calving.

• Reproductive and Developmental Biology
• /
• v.31 no.3
• /
• pp.161-167
• /
• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• The Journal of the Korean Society for Microbiology
• /
• v.21 no.4
• /
• pp.487-501
• /
• 1986

Previous studies from our laboratory suggested that Korean LJP patients might habor A. actinomycetemcomitans of different serotype from Caucasian LJP patients in whom serotype b was predominant. In order to observe the prevalence and serotype distribution of A. actinomycetemcomitans in localized juvenile periodontitis patients and to evaluate leukotoxic activity of oral isolates, this study was performed. A. actinomycetemcomitans was isolated by using a selective medium(tryptic soy agar supplemented with 10% serum, $75{\mu}g$ of bacitracin and $5{\mu}g$ of vancomycin per ml). Using immunoabsorbed, ammonium sulfate-fractionated serotype-specific antisera, a total of 69 strains were serologically categorized by ELISA. Leukotoxicity was monitored biochemically by measuring lactate dehydrogenase indicator of cell viability in culture supernatant of PMNL plus viable A. actinomycetemcomitans mixture. The results were as follows: 1. A. actinomycetemcomitans was detected in 75% of 16 LJP patients, and 71% in the LJP lesions and 6% in the control sites. 2. Presence or absence of A. actinomycetemcomitans in the sampled disease sites has no in fluence on clinical measurements. 3. Three serotypes were approximately equally distributed in overall 9 patients. Three patients harbored 2 different serotypes of A. actinomycetemcomitans in the same disease site or different disease sites. 4. The proportion of leukotoxic oral isolates was 22% of a total of 46 strains and the prevalence was 69% in 13 sampled sites. The same disease site could harbor both leukotoxic and nonleukotoxic strains. 5. Distribution of leukotoxic strains in 3 serotypes were not different.

• Tuberculosis and Respiratory Diseases
• /
• v.46 no.1
• /
• pp.44-52
• /
• 1999

Background : Airway infiltration by inflammatory cells, particularly of eosinophils, is one of the characteristic features of asthma. Several mechanisms for the recruitment of eosinophil is focused on the CD4+ T lymphocyte for the preferential production of Th2-c1erived cytokines. Interleukin-10(IL-10) is identified cytokine with potent antiinflammatory activity. This molecule has been shown to inhibit the release of cytokine from inflammatory cells including Th2 cell, and also to inhibit eosinophil survival. We therefore attempted to determine whether decreased synthesis of IL-10 in the lung of bronchial asthma may contribute to inflammation that is characteristics of this dease. Method: Subjects were patients with bronchial asthma(n=23) and normal controls(n=11). IL-10 produced from peripheral mononuclear cell(PBMC) and in bronchoalveolar lavage(BAL) fluid was measured by ELISA method. Degree of bronchial inflammation was assessed by total cell counts and eosinophil percents in BAL fluid, eosinophil infiltration on bronchial biopsy tissue and $PC_{20}$ for methacholine. Results: The IL-10 level produced by PBMC and in BAL fluid from patient with bronchial asthma were not different with normal controls(respectively, $901.6\pm220.4$ pg/ml, $810.9\pm290.8$ pg/ml for PBMC, $24.5\pm9.5$ pg/mL $30.5\pm13.5$ pg/ml for BAL fluid p>0.05). There were significant negative correlation between IL-10 in BAL fluid and eosinophil percents in BAL fluid or degree of eosinophil infiltration in bronchial biopsy (respectively r=-0.522, r=-0.4486 p<0.05). However there was no difference of IL-10 level according to $PC_{20}$ for methacholine. There were no correlation between IL-10 production by PBMC and peripheral blood eosinophil counts or serum eosinophilic cationic protein levels(respectively r=0.1146, r=0.0769 p>0.05). Conclusion: These observation suggest that IL-10 may participate but not acts the crucial role in regulation of the airway inflammation in bronchial asthma.

• Tuberculosis and Respiratory Diseases
• /
• v.49 no.2
• /
• pp.217-224
• /
• 2000

Background : It is well known that when macrophages are stimulated with endotoxin, they produce a wide variety of cytokine mediators, including TNF-$\alpha$ and IL-1$\beta$. However, there is an alteration in the macrophages' responsiveness when they are challenged with repeated bouts of endotoxin, termed "endotoxin tolerance" which is regarded as a self-protective phenomenon from continuous stimulation. In this study, endotoxin tolerance in the peripheral blood monocytes of sepsis patients was evaluated. Methods : Fourteen patients with organism-documented sepsis were included. The severity of illness was evaluated by APACHE II score. Peripheral blood monocytes were isolated from the patients and diluted to $1{\times}10^5$ well. After stimulation with endotoxin (LPS of E. coli O114 : B4, 100 ng/ml), they were incubated at $37^{\circ}C$ in 5% $CO_2$ incubator for 24 hours. Supernatant was collected for the measurement of TNF-$\alpha$ and IL-1$\beta$ with ELISA method. Peripheral blood monocytes of seven healthy volunteers were used as control. Results : The APACHE II score (mean$\pm$SD) of the patients at the time of blood sampling was 12.2$\pm$5.7. The primary infection foci were urinary tract infection, pneumonia, subacute bacterial endocarditis, and catheter related infection, etc. The causative organisms were gram negative rods (10 cases), gram positive cocci (6 cases) with two cases of mixed infection. Serum TNF-$\alpha$ could be measured in 4 cases with 29.9$\pm$27.7 pg/ml. Serum IL-1$\beta$was measurable in only one patient. The TNF-$\alpha$ level of supernatant of cultured peripheral blood monocytes was 2,703$\pm$2,066 pg/ml in patients and 2,102$\pm$1914 pg/ml in controls. The IL-1$\beta$level of supernatant was 884$\pm$1,050 pg/ml in patients and 575$\pm$558 pg/ml in controls. There was no difference of TNF-$\alpha$ and IL-1$\beta$ level between patients and controls. Conclusion : We cannot prove the phenomenon of endotoxin tolerance in this study. Future study needs to be focused on the more severe sepsis patients who were taken for sampling earlier. Addition of serum to the culture medium could be an another valuable option for the success of this study.

• 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.

• Korean Journal of Food Science and Technology
• /
• v.44 no.1
• /
• pp.55-60
• /
• 2012

The aim of this study was to determine the optimal physical treatment to reduce the antigenicity of gliadin in wheat dough. Medium wheat dough was treated with an autoclave (5, 10, 30, and 50 min at $121^{\circ}C$, 1 atm), a microwave (1, 5, and 10 min) or both (10, 30, and 50 min/5, 10 min). The proteins in the dough extracts were analyzed by SDSPAGE and the binding ability of anti-gliadin IgG to gliadin was examined by ci-ELISA and immunoblotting. Results showed that the ability of anti-gliadin IgG to bind to gliadin in wheat dough treated with an autoclave alone or in combination with a microwave was decreased. Especially, it declined to ~77% after autoclaving for 30 min and 35% after both autoclaving for 50 min and microwaving for 5 min. In addition, the intensity of gliadin bands in SDS-PAGE were weakened and anti-gliadin IgG did not recognize gliadin in immunoblotting. However, microwaving alone did not affect the antigenicity of gliadin in wheat dough. These results indicate that autoclaving may affect the reduction of the antigenicity of gliadin in medium wheat dough. Moreover, autoclaving in combination with microwaving is more effective for reducing the antigenicity of wheat dough.