Purpose : Patients with chronic granulomatous disease (CGD) have genetic mutations in a component of the NADPH oxidase enzyme that is necessary for the generation of the superoxide anion. The profound defect in innate immunity is reflected by the patients susceptibility to catalase-positive bacteria and fungi. In addition, CGD patients display signs of persistent inflammation, which is not associated only with deficient superoxide anion production. The aim of this study was to elucidate the cytokine responses in CGD patients after $TNF-{\alpha}$ stimulation. Methods : Heparinized blood samples were collected from 8 CGD patients and 10 healthy volunteers. Monocytes ($1{\times}10^6cell/well$) isolated by the magnet cell isolation system were incubated with a constant amount of $TNF-{\alpha}$ (10 ng/mL) at $37^{\circ}C$ for 6 h. Incubated cells were harvested at 60-min intervals for IL-8 and IL-10 mRNA analysis, and the supernatant was collected at the same intervals to determine IL-8 and IL-10 expression. Monocytes from healthy volunteers were also incubated with antioxidants followed by $TNF-{\alpha}$ stimulation for IL-8 and IL-10 expression. Results : In CGD patients, a high expression of IL-8 together with a significantly higher IL-10 expression than in the healthy controls was seen after $TNF-{\alpha}$ stimulation. Moreover, normal monocytes treated with antioxidants exhibited increased IL-8 responses. Conclusion : The absence of phagocyte-derived reactive oxidants in CGD might be associated with a dysregulated production of pro- and antiinflammatory cytokines. Additional research related to reactive oxidants is needed to clarify the role of cytokines in CGD patients.
N-methyl-D-aspartate (NMDA) receptors have received considerable attention regarding their involvement in glutamate-induced neuronal excitotoxicity. Resveratrol has been shown to exhibit neuroprotective effects against this kind of overactivation, but the underlying cellular mechanisms are not yet clearly understood. In this study, HT-22 neuronal cells were treated with NMDA in Mg2+-free buffer and subsequently used as an experimental model of glutamate excitotoxicity to elucidate the mechanisms of resveratrol-induced neuroprotection. We found that NMDA treatment causes a drop in MTT reduction ability, disrupts inside-negative transmembrane potential of mitochondria, depletes cellular ATP levels, and stimulates intracellular ROS production. Double fluorescence imaging studies demonstrated an increased formation of mitochondrial permeability transition (MPT) pores accompanied by apoptotic cell death, while cobalt protoporphyrin and bilirubin showed protective effects against NMDA-induced mitochondrial injury. On the other hand, zinc protoporphyrin IX significantly attenuated the protective effects of resveratrol which was itself shown to enhance heme oxygenase-1 (HO-1) mRNA and protein expression levels. In cells transfected with HO-1 small interfering RNA, resveratrol failed to suppress the NMDA-induced effects on MTT reduction ability and MPT pore formation. The present study suggests that resveratrol may prevent mitochondrial injury in NMDA- treated HT-22 cells and that enhanced expression of HO-1 is involved in the underlying cellular mechanism.
Lee, Joon Ha;Baek, Minhee;Lee, Hwa Jeong;Kim, In-Woo;Kim, Sun Young;Seo, Minchul;Kim, Mi-Ae;Kim, Seong Hyun;Hwang, Jae Sam
Journal of Life Science
/
v.29
no.11
/
pp.1218-1226
/
2019
The white-spotted flower chafer Protaetia brevitarsis seulensis is a medicinally beneficial and important edible insect species. We previously performed an in silico analysis of the Protaetia brevitarsis seulensis transcriptome to identify putative antimicrobial peptides and then tested their antimicrobial and hemolytic activities. These peptides had potent antimicrobial activities against bacteria and yeast without inducing hemolysis. In the present study, the cationic antimicrobial peptide, protaetiamycine 2, was selected for further assessment of its anti-inflammatory properties in mouse macrophage Raw264.7 cells. Protaetiamycine 2 treatment of Raw264.7 cells suppressed LPS-induced nitric oxide production and reduced the expression of inducible nitric oxide synthase and cyclooxygenase-2, as determined by real-time PCR and western blotting. The expression of proinflammatory cytokines ($TNF-{\alpha}$, IL-6, and $IL-1{\beta}$) was also attenuated through the MAPKs and $NF-{\kappa}B$ signaling. We also confirmed that protaetiamycine 2 bound to bacterial cell membranes by a specific interaction with LPS. Collectively, these data obtained from LPS-induced Raw264.7 cells indicated that protaetiamycine 2 could have both antimicrobial and anti-inflammatory properties.
Purpose: Current studies have demonstrated the neuroprotective effects of 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) in many animal models of brain injury, including hypoxic-ischemic (HI) encephlopathy, trauma and excitotoxicity, but limited data are available for those during the neonatal periods. Here we investigated whether CNQX can protect the developing rat brain from HI injury via mediation of nitric oxide synthase. Methods: In an in vivo model, left carotid artery ligation was done in 7-day-old Sprague-Dawley (SD) rat pups. The animals were divided into six groups; normoxia (N), hypoxia (H), hypoxia with sham-operation (HS), hypoxia with operation (HO), HO treated with vehicle (HV), and HO treated with CNQX at a dose of 10 mg/kg (HC). Hypoxia was made by exposure to a 2 hr period in the hypoxic chamber (92% $N_2$, 8% $O_2$). In an in vitro model, embryonic cortical neuronal cell culture of SD rats at 18-day gestation was done. The cultured cells were divided into three groups: normoxia (N), hypoxia (H), and hypoxia treated with CNQX (HC). The N group was prepared in 5% $CO_2$ incubators and the other groups were placed in 1% $O_2$) incubators (94% $N_2$, 5% $CO_2$) for 16 hr. Results: In the in vitvo and in vivo models, the expressions of iNOS and eNOS were reduced in the hypoxia group when compared to the normoxia group, whereas they were increased in the CNQX-treated group compared to the hypoxia group. In contrast, the expression of nNOS was showed reversely. Conclusion: CNQX has neuroprotective property over perinatal HI brain injury via mediation of nitric oxide synthase.
The giant mealworm beetle, Zophobas atratus (Coleoptera: Tenebrionidae) has been used as a protein source for small pets and mammals. Recently, it was temporarily registered in the list of the Food Code. We previously performed an in silico analysis of the Zophobas atratus transcriptome to identify putative antimicrobial peptides and identified several antimicrobial peptide candidates. Among them, we assessed the antimicrobial and anti-inflammatory activities of zophobacin 1 that was selected bio-informatically based on its physicochemical properties against microorganisms and mouse macrophage Raw264.7 cells. Zophobacin 1 showed antimicrobial activities against microorganisms without inducing hemolysis and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Moreover, ELISA and Western blot analysis revealed that zophobacin 1 reduced expression levels of pro-inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We also investigated expression of pro-inflammatory cytokines (interleukin-6 and interleukin-1β) production through quantitative real time-PCR and ELISA. Zophobacin 1 markedly reduced the expression level of cytokines through the regulation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling. We confirmed that zophobacin 1 bound to bacterial cell membranes via a specific interaction with lipopolysaccharides. These data suggest that zophobacin 1 could be promising molecules for development as antimicrobial and anti-inflammatory therapeutic agents.
The development of novel peptide antibiotics with potent antimicrobial activity and anti-inflammatory activity is urgently needed. In a previous work, we performed an in-silico analysis of the Papilio xuthus transcriptome to identify putative antimicrobial peptides and identified several candidates. In this study, we investigated the antibacterial and anti-inflammatory activities of papiliocin 3, which was selected bioinformatically based on its physicochemical properties against bacteria and mouse macrophage Raw264.7 cells. Papiliocin 3 showed antibacterial activities against E. coli and S. aureus without inducing hemolysis and decreased the nitric oxide production of the lipopolysaccharide-induced Raw264.7 cells. Moreover, ELISA and Western blot analysis revealed that papiliocin 3 reduced the expression levels of pro-inflammatory enzymes, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2). In addition, we examined whether papiliocin 3 could inhibit the expression of pro-inflammatory cytokines (interleukin-6 and interleukin-1β) in LPS-induced Raw264.7 cells. We found that papiliocin 3 markedly reduced the expression level of cytokines through the regulation of mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) signaling. We also confirmed that papiliocin 3 binds to bacterial cell membranes via a specific interaction with lipopolysaccharides. Collectively, these findings suggest that papiliocin 3 could be a promising molecule for development as a novel peptide antibiotic.
Lee, Chul-Ho;Lee, Kye Young;Hong, Yun-Chul;Choe, Kang-Hyeon;Kim, Yong-Dae;Kang, Jong-Won;Kim, Heon;Hong, Jang Soo
Tuberculosis and Respiratory Diseases
/
v.58
no.5
/
pp.490-497
/
2005
Background : The paraoxonase enzyme plays a significant role in the detoxification of various organophosphorous compounds in mammals, and paraoxonase (PON) 1 is one of the endogenous free-radical scavenging systems in the human body. In this study, we investigated the interaction between cigarette smoking and the genetic polymorphism of PON1 with lung cancer in Korean males. Methods : Three hundred thirty five patients with lung cancer and an equal number of age-matched controls were enrolled in this study. Every subject was asked to complete a questionnaire concerning their smoking habits and alcohol drinking habits. A 5' exonuclease assay (TaqMan) was used to genotype the PON1 Q192R polymorphism. The effects of smoking habits and drinking habits, the PON1 Q192R polymorphism and their interactions were statistically analyzed. Results : Cigarette smoking and the Q/Q genotype of PON1 were significant risk factors for lung cancer. Individuals carrying the Q/Q genotype of PON1 were at a higher risk for lung cancer as compared with those individuals carrying the Q/R or R/R genotype (odds ratio, 2.84; 95% confidence interval, 1.69 - 4.79). When the groups were further stratified by the smoking status, the Q/Q PON1 was associated with lung cancer among the current or ex-smokers (odds ratio, 2.56; 95% confidence interval, 1.52 - 4.31). Current smokers or ex-smokers who had the Q/Q genotype showed an elevated risk for lung cancer (odds ratio: 15.50, 95% confidence interval: 6.76 - 35.54) as compared with the group of subjects who never smoked, and had the Q/R or R/R genotype. The odds ratios (95% confidence interval) of smokers with the PON1 Q/Q type compared to the nonsmokers with the PON1 Q/R or R/R type were 53.77 (6.55 - 441.14) for squamous cell carcinoma, 6.25 (1.38 - 28.32) for adenocarcinoma, and 59.94 (4.66 - 770.39) for small cell carcinoma, and these results were statistically significant. Conclusion : These results suggest that cigarette smoking and the PON1 Q/Q genotype are risk factors for lung cancer. The combination of cigarette smoking and the PON1 Q/Q genotype significantly increased the lung cancer risk irrespective of the histologic type of cancer.
Song, Gi Seon;Lee, You-Rim;Kim, Sungmin;Kim, Wontae;Choi, Jungwon;Yoo, Dahyeon;Yoo, Jungyoung;Jang, Kyung-Tae;Lee, Jaewang;Jun, Jin Hyun
Korean Journal of Clinical Laboratory Science
/
v.52
no.3
/
pp.284-295
/
2020
In December 2019, the first coronavirus disease- 2019 (COVID-19) patient was reported in Wuhan, Hubei Province, China. Since then, the number of patients who suffered severe acute respiratory syndrome caused by the novel Coronavirus (SARS-CoV-2 or 2019-nCoV) has increased dramatically in Korea. This new variant virus induces pulmonary diseases, including cough, sore throat, rhinorrhea, dyspnea, and pneumonia. Because SARS-CoV-2 is an RNA virus, real-time reverse-transcriptase PCR has been used widely to diagnose COVID-19. As the Korea Centers for Disease Prevention and Control (KCDC) and Ministry of Food & Drug Safety (MFDS) approved emergency use authorization, clinical specimens collected from COVID-19 patients and even healthy people have been clinically diagnosed by laboratory medicine. Based on a literature search, this paper reviews the epidemiology, symptoms, molecular diagnostics approved by KCDC, a current diagnosis of COVID-19 in the laboratories, the difference between molecular and serological diagnosis, and guidelines for clinical specimens. In addition, the Korean guidelines of biosafety for clinical laboratory scientists are evaluated to prevent healthcare-associated infection. The author's experience and lessons as clinical laboratory scientists will provide valuable insights to protect the domestic and international health community in this COVID-19 pandemic around the world.
Objective: This study was performed to compare the expression of osteopontin (OPN) mRNA and protein in the eutopic and ectopic endometrial tissues in women with endometriosis and endometrial tissues in women without endometriosis. Methods: A total of 32 women with histologically confirmed endometriosis were recruited for study group. For controls, 34 women undergoing operative treatment for cervical intraepithelial neoplasia (CIN) or benign gynecologic condition other than endometriosis were recruited. At the time of laparoscopy or laparotomy, a biopsy specimen was taken from the endometrial cavity and peritoneal endometrial implant or endometrioma whenever appropriate. We employed real time quantitative RT-PCR to quantify OPN mRNA expression of these tissues and performed western blot analysis to measure the quantity of OPN. Results: The expression of OPN mRNA was significantly higher in both eutopic and ectopic endometrial tissues of women with endometriosis than in endometrial tissues of controls during both proliferative and secretory phase. In the eutopic endometrial tissue of women with endometriosis, OPN mRNA expression significantly increased during the secretory phase compared to the proliferative phase in women with endometriosis as well as controls. However, in the ectopic endometrial tissue, OPN mRNA expression significantly decreased during the secretory phase compared to the proliferative phase. The expression of OPN protein was significantly higher in women with endometriosis than in controls. Conclusion: This study shows the marked expression of OPN mRNA and protein in eutopic and ectopic endometrial tissues in women with endometriosis may be associated with the adhesion and invasion of endometrial explants.
Jo, Heui-Seung;Chang, Yoon-Hwan;Kim, Han-Suk;Kim, Byeong-Il;Choi, Jung-Hwan
Neonatal Medicine
/
v.18
no.1
/
pp.42-48
/
2011
Purpose: Several factors including prolonged inflammatory response are thought to contribute to the pathogenesis of bronchopulmonary dysplasia (BPD). The clinical findings can be explained by an increased production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-$\alpha$ ). We investigated the relationship between susceptibility to BPD and TNF-$\alpha$ promoter polymorphisms to identify genetic factors of the disease. Methods: Thirty-eight preterm infants who had developed BPD and 55 controlled infants with a birth weight <1,500 g were analyzed for TNF-$\alpha$ genotypes. The alleles of five promoter sites (-1031/-863/-857/-308/-238) of TNF-$\alpha$ gene were determined using $Taqman^{(R)}$-based allelic discrimination assays. Results: Gestational age ($27^{+5}{\pm}2^{+0}$ wk vs. $29^{+2}{\pm}1^{+4}$ wk, P<0.0001) and birth weight (990${\pm}$270 g vs. 1,220${\pm}$230 g, P<0.0001) were lower in the BPD group compared to the control group. The incidence of respiratory distress syndrome (71.1% vs. 49.1%, P=0.035) and patent ductus arteriosus (71.1% vs. 50.9%, P=0.052) was higher in the BPD group compared to the control group. The frequencies of the alleles and genotypes of five promoter sites (-1031/-863/-857/-308/-238) of TNF-$\alpha$ gene did not show differences between the BPD group and the control group. Conclusion: TNF-$\alpha$ promoter polymorphisms are not associated with susceptibility to BPD in Korean preterm infants.
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