• Tuberculosis and Respiratory Diseases
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• v.39 no.2
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• pp.120-130
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• 1992

Background: Although there have been many studies on the pathogenetic mechanism of acute lung injury, it is still elusive. Recently interests have been focused on the role of oxygen free radicals. But the effect of hydroxyl radical on the neutrophil mobilization and the alveolar-capillary permeability is not clear especially in the endotoxin-induced acute lung injury model of rats. This investigation was performed to evaluate the pathogenetic role of hydroxyl radical on the neutrophil accumulation into the lung and the increased alveolar-capillary permeability in the endotoxin-induced acute lung injury in rats. Method: Fifty rats were divided into four groups: vehicle control group (n=5, 6hrs; n=5, 24hrs), endotoxin-treated group (n=10, 6hrs; n=10, 24hrs), Dimethylthiourea (DMTU)-pretreated group (n=10, 6hrs), and deferoxamine (DFX)-pretreated group (n=10, 6hrs). Thirty minutes before sacrifice, $^{125}I$-tagged bovine serum albumin was injected. Six and twenty four hours after endotoxin injection, the rats were sacrificed, and the radioactivity of lung tissue and peripheral blood was counted. Permeability index was defined as the ratio of radioactivity between lung tissue and peripheral blood. Another set of rats (n=52) were divided into the same four groups as before [vehicle control group (n=5, 6hrs; n=5, 24hrs), endotoxin·treated group (n=7, 6hrs; n=8, 24hrs), DMTU-pretreated group (n=6, 6hrs; n=9, 24hrs), and DFX-pretreated group (n=5, 6hrs; n=7, 24hrs)], and were sacrificed 6 and 24 hours after endotoxin injection. In these rats, cell profile of peripheral blood and bronchoalveolar lavage fluid was evaluated, and the pathologic examination of lung tissue was performed. Results: 1) Increased alveolar-capillary permeability was observed 6 hours after endotoxin injection, which was normalized after 24 hours, and this increase was attenuated by pretreatment with DMTU and DFX. 2) Neutrophil sequestration into the lung was observed 24 hours after endotoxin administration, but this was not influenced by DMTU and DFX pretreatment. Conclusion: These results suggest that hydroxyl radical would not be involved in the sequestration of neutrophils into the lung, but plays an important role in the increase of alveolar-capillary permeability in the endotoxin-induced acute lung injury in rats.

• Tuberculosis and Respiratory Diseases
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• v.63 no.3
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• pp.242-250
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• 2007

Background: Abnormal angiogenesis can induce hypoxia within a highly proliferating tumor mass, and these hypoxic conditions can in turn create clinical problems, such as resistance to chemotherapy. However, the mechanism by which hypoxia induces these changes has not yet been determined. Therefore, this study was conducted to determine how hypoxia induces changes in cell viability and extracellular microenvironments in an in vitro culture system using non-small cell lung cancer cells. Methods: The non-small cell lung cancer cell line, A549 was cultured in DMEM or RPMI-1640 media that contained fetal bovine serum. A decrease in the oxygen tension of the media that contained the culture was then induced in a hypoxia microchamber using a $CO_2-N_2$ gas mixture. A gas analysis and an MTT assay were then conducted. Results: (1) The decrease in oxygen tension was checked the anaerobic gas mixture for 30 min and then reoxygenation was induced by adding a 5% $CO_2-room$ air gas mixture to the chamber. (2) Purging with the anaerobic gas mixture was found to decrease the further oxygen tension of cell culture media. (3) The low oxygen tension resulted in a low pH, lactic acidosis and a decreased glucose concentration in the media. (4) The decrease in glucose concentration that was observed as a result of hypoxia was markedly different when different types of media were evaluated. (5) The decrease in oxygen tension inhibited proliferation of A549 cells. Conclusion: These data suggests that tumor hypoxia is associated with acidosis and hypoglycemia, which have been implicated in the development of resistance to chemotherapy and radiotherapy.

• Tuberculosis and Respiratory Diseases
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• v.43 no.4
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• pp.579-589
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• 1996

Background : Activation of neutrophil is critical for the clearance of microorganisms and toxic host mediators during sepsis. Unfortunately the activated neutrophil and its toxic byproducts can produce tissue injury and organ dysfunction. The leucocyte CD11/18 adhesion complex regulates neutrophil-endothelial cell adhesion, the first step in neutrophil migration to sites of injection and inflammation. To investigate the potential of neutrophil inhibition as a treatment strategy for sepsis, we evaluated the effects of monoclonal antibody against CD11b (MAb 1B6) in rats intrabronchial challenged with Escherichia coli. Methods : Animals were randomly assigned to receive monoclonal antibody against CD11b (1 mg/kg, sc) and bovine serum albumin(BSA, 1 mg/kg, sc) 6 hr before, at 0 and 6 hr after intrabronchial challenge of $20x10^9$ CFU/kg E. coli 0111. Animals were randomized to treat either 24, 60 or 90% oxygen after bacterial challenge and begining 4 hr after inoculation, all animals were received 100 mg/kg ceftriaxone qd for 3 days. Peripheral and alveolar neutrophil(by bronchoalveolar lavage) counts and lung injury parameters such as alveolar-arte rial $PO_2$ difference, wet to dry lung weight ratio and protein concentration of alveolar fluid were measured in survived rats at 12 hr and 96 hr. Results : Monoclonal antibody against CD11b decreased circulating and alveolar neutrophil especially more in 12 hr than in 96 hr The lung injury parameters of antibody-treated animals were not different from those of BSA-treated animals. but It was meaningless due to small number of survived animals. The early(6 hr) mortality rate was significantly increased in antibody-treated group(51%) compared to BSA-treated group(31%) (P=0.02) but late(from 12 hr to 72 hr) mortality rate was not different in antibody-treated group(44%) from BSA-treated group(36%) (P =0.089). Conclusion : Leucocyte CD11b/18 adhesion molecule is known to regulate neutrophil migration to the site of infection and inflammation. The monoclonal antibody against CD11b decreased alveolar neutrophil in rats with pulmonary sepsis and increased early mortality rate. Therefore, we can speculate that monoclonal antibody against CD11b blocks of alveolar recruitment of neutrophils, impairs host defense mechanism and increases early mortality rate of pulmonary sepsis in rat.

• Tuberculosis and Respiratory Diseases
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• v.66 no.3
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• pp.178-185
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• 2009

Background: Epigallocatechin-3-gallate (EGCG) is the major catechin in green tea, and has shown antiproliferative, antiangiogenic, antimetastatic and cell cycle pertubation activity in various tumor models. Hypoxia can be induced because angiogenesis is insufficient for highly proliferating cancer. Hypoxia-inducible factor-1$\alpha$ (HIF-1$\alpha$) and its downstream target, vascular endothelial growth factor (VEGF), are important for angiogenesis, tumor growth and metastasis. The aim of this study was to determine how hypoxia could cause changes in the cellular phenomena and microenvironment in a non-small cell culture system and to examine the effects of EGCG on a HIF-1$\alpha$ and VEGF in A549 cell line. Methods: A549 cells, a non-small cell lung cancer cell line, were cultured with DMEM and 10% fetal bovine serum. A decrease in oxygen tension was induced using a hypoxia microchamber and a $CO_2-N_2$ gas mixture. Gas analysis and a MTT assay were performed. The A549 cells were treated with EGCG (0, 12.5, 25, 50 ${\mu}mol/L$), and then examined by real-time-PCR analysis of HIF-1$\alpha$, VEGF, and $\beta$-actin mRNA. Results: Hypoxia reduced the proliferation of A549 cells from normoxic conditions. EGCG inhibited HIF-1$\alpha$ transcription in A549 cells in a dose-dependent manner. Compared to HIF-1$\alpha$, VEGF was not inhibited by EGCG. Conclusion: HIF-1$\alpha$ can be inhibited by EGCG. This suggests that targeting HIF-1$\alpha$ with a EGCG treatment may have therapeutic potential in non-small cell lung cancers.

• Tuberculosis and Respiratory Diseases
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• v.48 no.4
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• pp.487-499
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• 2000

Background : Acute lung injury is an hypoxic respiratory failure resulting from damage to the alveolar-capillary membrane, which can be developed by a variety of systemic inflammatory diseases. In this study the therapeutic effects of intra-tracheal pulmonary surfactant instillation was evaluated in the intratracheal endotoxin induced acute lung injury model of a rat. Methods : Twenty Sprague-Dawley rats were divided into 4 groups, and normal saline (2 ml/kg, for group 1) or LPS (5 mg/kg, for group 2, 3, and 4) was instilled into the trachea respectively. Either normal saline (2 ml/kg, for group 1 & 2, 30 min later) or bovine surfactant (15 mg/kg, 30 min later for group 3, 5 hr later for group 5) was instilled into the trachea. The therapeutic effect of intratracheal surfactant therapy was evaluated with one chamber body plethysmography (respiratory frequency, tidal volume and enhanced pause), ABGA, BAL fluid analysis (cell count with differential, protein concentration) and pathologic examination of the lung. Results : Intratracheal endotoxin instillation increased the respiration rate decreased the tidal volume and int creased the Penh in all group of rats. Intratracheal instillation of surfactant decreased Penh, increased arterial oxygen tension, decreased protein concentration of BAL fluid and decreased lung inflammation at both times of administration (30 minute and 5 hour after endotoxin instillation). Conclusion : Intratracheal instillation of surfactant can be a beneficial therapeutic modality as discovered in the acute lung injury model of rats induced by intratracheal LPS intillation. It deserves to be evaluated for treatment of human acute lung injury.

• Tuberculosis and Respiratory Diseases
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• v.63 no.6
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• pp.497-506
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• 2007

Background: The present investigation was performed in rats and isolated human neutrophils in order to confirm the presumptive role of the positive feedback loop of cytosolic phospholipase $A_2$ ($cPLA_2$) activation by plateletactivating factor (PAF). Methods: The possible formation of the positive feedback loop of the $cPLA_2$ activation and neutrophilic respiratory burst was investigated in vivo and in vitro by measurement of the parameters denoting acute lung injury. In addition, morphological examinations and electron microscopic cytochemistry were performed for the detection of free radicals in the lung. Results: Five hours after intratracheal instillation of PAF ($5{\mu}g/rat$), the lung leak index, lung myeloperoxidase (MPO) activity, the number of neutrophils and the concentration of cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid were increased by PAF as compared with those of control rats. The NBT assay and cytochrome-c reduction assay revealed an increased neutrophilic respiratory burst in isolated human neutrophils following exposure to PAF. Lung and neutrophilic $cPLA_2$ activity were increased following PAF exposure and exposure to hydrogen peroxide increased $cPLA_2$ activity in the lung. Histologically, inflammatory findings of the lung were observed after PAF treatment. Remarkably, as determined by $CeCl_3$ cytochemical electron microscopy, increased production of hydrogen peroxide was identified in the lung after PAF treatment. Conclusion: PAF mediates acute oxidative lung injury by the activation of $cPLA_2$, which may provoke the generation of free radicals in neutrophils.

• Tuberculosis and Respiratory Diseases
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• v.48 no.6
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• pp.887-897
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• 2000

Background : Since the exact pathogenesis of sepsis-induced ARDS has not been elucidated, the mechanisms of enhanced neutrophilic respiratory burst were probed in endotoxin primed neutrophils associated with the roles of phospholipase A2(PLA2), platelet activating factor(PAF) and apoptosis. Methods : In isolated fresh human neutrophils, effects of the inhibition of PLA2 and PAF on the apoptosis were examined by the method of Annexin-FITC/dual PIflow cytometry. The roles of PLA2 and PAF on the neutrophilic respiratory burst were also examined by measuring oxidant generation in cytochrome-c reduction assay. Activities of the PLA2 and lysoPAF acetyltransferase (lysoPAF AT) of the neutrophils were determined to understand the effect of endotoxin on these enzymatic activities which may be related to the neutrophilic respiratory burst and apoptosis. In addition, the role roles of PLA2 and PAF in neutrophilic adhesion to bovine endothelial cells were examined in vitro by neutrophil adhesion assay. To investigate the effect of oxidants on pulmonary surfactant, cytochemical ultrastructural microscopy was performed. To inhibit PLA2 and PAF, non-specific PLA2 inhibitor mepacrine (100 nM) and WEB 2086 (100 nM) or ketotifen fumarate (10 ${\mu}g$/ml) were used respectively in all in vitro experimental sets. WEB 2086 is PAF receptor antagonist, and ketotifen fumarate is a lyso PAF AT inhibitor. Results: The mapacrine treatment, provided and the endotoxin (ETX) treatment, resulted in increased apoptosis of neutrophils (p<0.001) while treatments of WEB 2086 and ketotifen did not. The inhibition of PLA2 and PAF decreased (p<0.001) production of oxidants from PMA-stimulated neutrophils. While endotoxin increased the PLA2 activity of neutrophils (p<0.01), mepacrine supressed (p<0.001) the activity, provided after treatment of ETX. The lyso PAF actyltransferase activity (lyso PAF AT) increased (p<0.01) after treatment of ETX. In contrast, mepacrine, WEB 2086 and ketotifen showed a tendency of decreasing the activity after treatment of ETX. The treatment of ETX incresed (p<0.001) neutrophil adhesion to endothelial cells, which was reversed by inhibition of PLA2 and PAF (p<0.01). The binding of oxidants to pu1monary surfactant was identified histologically. Conclusions : The enhanced neutrophilic respiratory burst by ETX plays a pivotal role in the pathogenesis of ARDS in terms of oxidayive oxidative stress. Increased production of oxidants from neutrophils is mediated by the activations of PLA2 and lyso PAF AT.

• Tuberculosis and Respiratory Diseases
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• v.49 no.3
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• pp.310-322
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• 2000

Background : Phospholipase C(PLC) plays an important role in cellular signal transduction and is thought to be critical in cellular growth, differentiation and transformation of certain malignancies. Two second messengers produced from the enzymatic action of PLC are diacylglycerol (DAG) and inositol 1, 4, 5-trisphosphate (IP3). These two second messengers are important in down stream signal activation of protein kinase C and intracellular calcium elevation. In addition, functional domains of the PLC isozymes, such as Src homology 2 (SH2) domain, Src homology 3 (SH3) domain, and pleckstrin homology (PH) domain play crucial roles in protein translocation, lipid membrane modificailon and intracellular memrane trafficking which occur during various mitogenic processes. We have previously reported the presence of PLC-${\gamma}1$, ${\gamma}2$, ${\beta}1$, ${\beta}3$, and ${\delta}1$ isozymes in normal human lung tissue and tyrosine-kinase-independent activation of phospholipase C-${\gamma}$ isozymes by tau protein and AHNAK. We had also found that the expression of AHNAK protein was markedly increased in various mstologic types of lung can∞r tissues as compared to the normallungs. However, the report concerning expression of various PLC isozymes in lung canærs and other lung diseases is lacking. Therefore, in this study we examined the expression of PLC isozymes in the paired surgical specimens taken from lung cancer patients. Methods : Surgically resected lung cancer tissue samples taken from thirty seven patients and their paired normal control lungs from the same patients, The expression of various PLC isozymes were studied. Western blot analysis of the tissue extracts for the PLC isozymes and immunohistochemistry was performed on typical samples for localization of the isozyme. Results : In 16 of 18 squamous cell carcinomas, the expression of PLC-${\gamma}1$ was increased. PLC-${\gamma}1$ was also found to be increased in all of 15 adenocarcinoma patients. In most of the non-small cell lung cancer tissues we had examined, expression of PLC-${\delta}1$ was decreased. However, the expression of PLC-${\delta}1$ was markedly increased in 3 adenocarcinomas and 3 squamous carcinomas. Although the numbers were small, in all 4 cases of small cell lung cancer tissues, the expression of PLC-${\delta}1$ was nearly absent. Conclusion : We found increased expression of PLC-${\gamma}1$ isozyme in lung cancer tissues. Results of this study, taken together with our earlier findings of AHNAK protein-a putative PLD-${\gamma}$, activator-over-expression, and the changes observed in PLC-${\delta}1$ in primary human lung cancers may provide a possible insight into the derranged calcium-inositol signaling pathways leading to the lung malignancies.