• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.509-516
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• 2003

A model system for the immnunochemical detection of Escherichia coli O157:H7 using a combined immunomagnetic separation (IMS) and test-strip liposome immunoassay (LIA) procedure was developed. Immunomagnetic beads coated with anti-E. coli O157 IgG antibodies were used to separate the E. coli O157 (including the H7 serotype) from culture. Immunoliposomes, whose surface was conjugated to goat anti-E. coli O157:H7 IgG and which encapsulated the marker dye, sulforhodamine B, were used as a detection label. The test strip, onto which antibodies to goat IgG were immobilized, was the immunosensor capturing immunoliposomes that did not bind to E. coli O157:H7 on the immunomagnetic bead-E. coli O157:H7 complexes. In experiments, pure cell culture suspensions of $10^5 E.$ coli O157:H7 organisms per ml produced a measurable signal inhibition, whereas a weak yet detectable signal inhibition occurred with $10^3CFU/ml$. The inhibition signals increased, when the incubation time for IMS was extended to 90 min and higher IgG-tag density (0.4mol%) was used on the liposomes. With 0.2 and 0.4mol% IgG-tagged liposomes, the IMS-LIA procedure showed more improved signal inhibitions than those of a direct (no IMS) LIA. The combined assay, which measures the instantaneous signal from immunoliposomes, can be completed within 90 min, making it significantly faster than conventional plating methods and enzyme-linked immunosorbent assay (ELISA). Accordingly, it is quite feasible to use the combined immunoassay format of IMS and dye-loaded immunoliposomes for the detection of E. coli O157:H7.

• Molecular & Cellular Toxicology
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• 제2권1호
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• pp.67-73
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• 2006

In previous studies, it has been discovered that cancer cells not only overexpress regulatory subunit I (Rl)/protein kinase type I (PKA-I) but also secrete outside the cell an extracellular form of PKA (ECPKA) and that the ECPKA secretion detected in patients' serum is obviously greater than that found in non-cancer patients or healthy subjects. We now found that ECPKA elicits the formation of serum autoantibodies that can serve as a cancer diagnostic and prognostic marker. To measure the presence of anti-ECPKA autoantibody in the human sera, basic methodology for ECPKA assay was established an enzyme-linked immunosorbent assay (ELISA). We obtained serum samples from 199 patients with different types of cancer, and also obtained 31 serum samples to compare with ECPKA concentrations from non-cancer patients and 119 normal volunteers. Compared with normal or non-cancer patient sera, we found that the frequency of anti-ECPKA autoantibody was significantly higher in cancer patients (88%) than in those without cancer (17%). Furthermore the presence of anti-ECPKA autoantibodies in the serum of cancer patients was highly correlated with the site of metastasis. The immunoassay developed for anti-ECPKA antibodies is highly sensitive and specific. Therefore, this discovery of an autoantibody-based cancer diagnostic may have serious clinical application and may become an important advance over current technology.

• Journal of Microbiology and Biotechnology
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• 제31권8호
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• pp.1115-1122
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• 2021

Rotavirus (RV), as the main cause of diarrhea in children under 5 years, contributes to various childhood diseases. Valeriana jatamansi Jones is a traditional Chinese herb and possesses antiviral effects. In this study we investigated the potential mechanisms of V. jatamansi Jones in RV-induced diarrhea. MTT assay was performed to evaluate cell proliferation and the diarrhea mice model was constructed using SA11 infection. Mice were administered V. jatamansi Jones and ribavirin. Diarrhea score was used to evaluate the treatment effect. The enzyme-linked immunosorbent assay was performed to detect the level of cytokines. Western blot and quantitative reverse transcription-PCR were used to determine protein and mRNA levels, respectively. Hematoxylin-eosin staining was applied to detect the pathological change of the small intestine. TdT-mediated dUTP nick-end labeling was conducted to determine the apoptosis rate. The results showed V. jatamansi Jones promoted MA104 proliferation. V. jatamansi Jones downregulated phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in protein level, which was consistent with the immunohistochemistry results. Moreover, V. jatamansi Jones combined with ribavirin regulated interleukin-1β (IL-1β), interferon γ, IL-6, tumor necrosis factor α, and IL-10, and suppressed secretory immunoglobulin A secretion to remove viruses and inhibit dehydration. V. jatamansi Jones + ribavirin facilitated the apoptosis of small intestine cells. In conclusion, V. jatamansi Jones may inhibit RV-induced diarrhea through PI3K/AKT signaling pathway, and could therefore be a potential therapy for diarrhea.

• International Journal of Oral Biology
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• 제47권1호
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• pp.1-8
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• 2022

Inflammation is a protective mechanism against pathogens, but if maintained continuously, it destroys tissue structures. Aggregatibacter actinomycetemcomitans is a gram-negative, facultative anaerobic bacterium often found in severe periodontitis. A. actinomycetemcomitans invades epithelial cells and triggers inflammatory response in the immune cells. In this study, we investigated the effect of water-soluble rosehip extract on A. actinomycetemcomitans-induced inflammatory responses. A human monocytic cell line (THP-1) was differentiated to macrophages by phorbol 12-mystristate 13-acetate treatment. The cytotoxic effect of extract was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effects of extract on bacterial growth were examined by measuring the optical densities using a spectrophotometer. THP-1-derived macrophages were infected A. actinomycetemcomitans after extract treatment, and culture supernatants were analyzed for cytokine production using enzyme-linked immunosorbent assay. Protein expression was measured by western blotting. Extract was not toxic to THP-1-derived macrophages. A. actinomycetemcomitans growth was inhibited by 1% extract. The extract suppressed A. actinomycetemcomitans-induced tumor necrosis factor-α, interleukin (IL)-1β, and IL-8 production. It also decreased mitogen-activated protein kinase (MAP kinase) and nuclear factor-κB (NF-κB) phosphorylation. Moreover, the extract inhibited the expression of inflammasome components, including nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3, Absent in Melanoma 2, and apoptosis associated speck-like protein containing a CARD. And cysteine-aspartic proteases-1 and IL-1β expression were decreased by the extract. In summary, extract suppressed A. actinomycetemcomitans growth and decreased inflammatory cytokine production by inhibiting activation of MAP kinase, NF-κB, and inflammasome signaling. Rosehip extract could be effective in the treatment of periodontal inflammation induced by A. actinomycetemcomitans infection.

• 대한본초학회지
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• 제29권1호
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• pp.53-60
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• 2014

Objectives : Bojungikgitang (BJT), the Oriental medical prescription has been traditionally used about improvement of immune response and infective disease at Asian nation. In this study, we has compared about the anti-inflammatory and antioxidative effects on BJT of three countries including Korea (Korean Traditional Medicine, KTM), China (Traditional Chinese Medicine, CTM) and Japan (Japanese Traditional Medicine, JTM). Methods : We has basically using LPS-stimulated RAW 264.7 cells. The expression of these inflammatory mediators has measured using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Also, free radical scavenging assay has tested for anti-oxidative activity as well as the contents of total flavonoid and polyphenol. Results : As a result, we were founded the inhibitory effects of BJT (KTM, CTM, JTM) on LPS-induced production of NO, TNF-${\alpha}$ and IL-6 as well as the anti-oxidative activities. Especially the KTM was most effective in anti-inflammatory and anti-oxidative activities. Conclusions : These results indicate that BJT (KTM, CTM, JTM) has a good anti- inflammatory and anti-oxidative effects. But, there were degree of effects on between pharmacopoeia of the countries. Thus, further study is required that find appropriate methods for extracting as well as establish of standardized processes in order to improve the quality of BJT (KTM, CTM, JTM) as an anti-inflammatory and anti-oxidative agent for treatment of inflammatory diseases.

• 대한본초학회지
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• 제34권6호
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• pp.117-124
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• 2019

Objective : Broccoli is edible green plant that has a wide variety of health benefits including cancer prevention and cholesterol reduction. However, leaves of broccoli are not eaten and are mostly left as waste. This study was conducted to evaluate the effects of the broccoli leaf extract (BLE) on prostaglandin E₂ (PGE₂) production related to nuclear factor kappa B (NF-κB) signaling in lipopolysaccharide (LPS)-activated macrophages. Methods : BLE was prepared by extracting dried leaf with ethanol. Cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PGE₂ and inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). Expression level of each protein was monitored by Western blot analysis. Results : In LPS-activated Raw264.7 cells, PGE₂ release into culture medium was dramatically enhanced compared to control cells. However, increased PGE₂ was attenuated dose-dependently by treatment with BLE. Inhibition of PGE₂ production by BLE was due to the suppression of cyclooxygenase-2 (COX-2) expression determined by Western blot analysis. BLE also inhibited the production of inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). Inhibition at PGE₂ and cytokine was mediated from inhibition of nuclear translocation of NF-κB due to the repression of inhibitory kappa B alpha (IκBα) phosphorylation and degradation. Conclusion : This study showed that BLE exerted inhibitory activities against PGE₂, which is critical for the initiation and resolution of inflammatory responses, and that inhibition of PGE₂ was mediated by suppression of NF-κB signaling. These results suggest that the waste broccoli leaves could be used for controlling inflammation.

• Anatomy and Cell Biology
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• 제56권2호
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• pp.219-227
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• 2023

Adult neurogenesis has been reported in the hypothalamus, subventricular zone and subgranular zone in the hippocamp. Recent studies indicated that new cells in the hypothalamus are affected by diet. We previously showed beneficial effects of safflower seed oil (SSO), a rich source of linoleic acid (LA; 74%), on proliferation and differentiation of neural stem cells (NSCs) in vitro. In this study, the effect of SSO on hypothalamic neurogenesis was investigated in vivo, in comparison to synthetic LA. Adult mice were treated with SSO (400 mg/kg) and pure synthetic LA (300 mg/kg), at similar concentrations of LA, for 8 weeks and then hypothalamic NSCs were cultured and subsequently used for Neurosphere-forming assay. In addition, serum levels of brain-derived neurotrophic factor (BNDF) were measured using enzyme-linked immunosorbent assay. Administration of SSO for 8 weeks in adult mice promoted the proliferation of NSCs isolated from SSO-treated mice. Immunofluorescence staining of the hypothalamus showed that the frequency of astrocytes (glial fibrillary acidic protein⁺ cells) are not affected by LA or SSO. However, the frequency of immature (doublecortin⁺ cells) and mature (neuronal nuclei⁺ cells) neurons significantly increased in LA- and SSO-treated mice, compared to vehicle. Furthermore, both LA and SSO caused a significant increase in the serum levels of BDNF. Importantly, SSO acted more potently than LA in all experiments. The presence of other fatty acids in SSO, such as oleic acid and palmitic acid, suggests that they could be responsible for SSO positive effect on hypothalamic proliferation and neurogenesis, compared to synthetic LA at similar concentrations.

• Korean Circulation Journal
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• 제53권6호
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• pp.387-403
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• 2023

Background and Objectives: Myocardial ischemia and reperfusion injury (MIRI) has high morbidity and mortality worldwide. We aimed to explore the role of long noncoding RNA lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) in cardiomyocyte pyroptosis. Methods: Hypoxia/reoxygenation (H/R) injury was constructed in human cardiomyocyte (HCM). The level of LOXL1-AS1, miR-761, phosphatase and tensin homolog (PTEN) and pyroptosis-related proteins was monitored by quantitative real-time polymerase chain reaction or western blot. Flow cytometry examined the pyroptosis level. Lactate dehydrogenase (LDH), creatine kinase-MB and cardiac troponin I levels were detected by test kits. Enzyme-linked immunosorbent assay measured the release of inflammatory cytokines. Dual-luciferase assay validated the binding relationship among LOXL1-AS1, miR-761, and PTEN. Finally, ischemia/reperfusion (I/R) animal model was constructed. Hematoxylin and eosin staining assessed morphological changes of myocardial tissue. NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and casepase-1 expression was determined by immunohistochemistry. Results: After H/R treatment, LOXL1-AS1 and PTEN were highly expressed but miR-761 level was suppressed. LOXL1-AS1 inhibition or miR-761 overexpression increased cell viability, blocked the release of LDH and inflammatory cytokines (interleukin [IL]-1β, IL-18), inhibited pyroptosis level, and downregulated pyroptosis-related proteins (ASC, cleaved caspase-1, gasdermin D-N, NLRP3, IL-1β, and IL-18) levels in HCMs. LOXL1-AS1 sponged miR-761 to up-regulate PTEN. Knockdown of miR-761 reversed the effect of LOXL1-AS1 down regulation on H/R induced HCM pyroptosis. LOXL1-AS1 aggravated the MIRI by regulating miR-761/PTEN axis in vivo. Conclusions: LOXL1-AS1 targeted miR-761 to regulate PTEN expression, then enhance cardiomyocyte pyroptosis, providing a new alternative target for the treatment of MIRI.

• 치위생과학회지
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• 제19권2호
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• pp.133-140
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• 2019

Background: The light-emitting diode (LED) curing light used is presumed to be safe. However, the scientific basis for this is unclear, and the safety of LED curing light is still controversial. The purpose of this study was to investigate the effect of LED curing light irradiation according to the conditions applied for the polymerization of composite resins in dental clinic on the cell viability and inflammatory response in Raw264.7 macrophages and to confirm the stability of LED curing light. Methods: Cell viability and cell morphology of Raw264.7 macrophages treated with 100 ng/ml of lipopolysaccharide (LPS) or/and LED curing light with a wavelength of 440~490 nm for 20 seconds were confirmed by methylthiazolydiphenyl-tetrazolium bromide assay and microscopic observation. The production of nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) was confirmed by NO assay and $PGE_2$ enzyme-linked immunosorbent assay kit. Expression of interleukin $(IL)-1{\beta}$ and tumor necrosis factor $(TNF)-{\alpha}$ in total RNA and protein was confirmed by reverse transcription polymerase chain reaction and Western blot analysis. Results: The LED curing light did not affect the viability and morphology of normal Raw264.7 cells but affected the cell viability and induced cytotoxicity in the inflammation-induced Raw264.7 cells by LPS. The irradiation of the LED curing light did not progress to the inflammatory state in the inflammation-induced Raw264.7 macrophage. However, LED curing light irradiation in normal Raw264.7 cells induced an increase in NO and $PGE_2$ production and mRNA and protein expression of $(IL)-1{\beta}$ and $(TNF)-{\alpha}$, indicating that it is possible to induce the inflammatory state. Conclusion: The irradiation of LED curing light in RAW264.7 macrophage may induce an excessive inflammatory reaction and damage oral tissues. Therefore, it is necessary to limit the long-term irradiation which is inappropriate when applying LED curing light in a dental clinic.

• Journal of Acupuncture Research
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• 제22권1호
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• pp.155-164
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• 2005

사람 기관지 상피세포에 알러지 환경을 유발 하고자 사이토카인을 처리하여 TARC의 분비를 유도하고, 이 케모카인 분비에 반하(半夏) 약쇄액(藥鎖液)이 미치는 효과를 실험한 결과 다음과 같은 결론을 얻었다. 1. 사람의 기관지 상피 세포주에 각각 IL-4, TNF-${\alpha}$, IFN-${\gamma}$ 및 IL-$1{\beta}$를 처리하는 경우와 IL-4와 TNF-${\alpha}$, INF-${\alpha}$와 IFN-${\gamma}$, IFN-${\gamma}$와 IL-$1{\beta}$를 병용 처리할 경우 TARC의 분비량를 측정한 결과 IL-4와 TNF-${\alpha}$와 TNF-${\alpha}$와 IFN-${\gamma}$를 병용 처리하였을 경우 TARC의 분비량이 유의하게 증가하였다. 2. 반하(半夏) 약쇄액(藥鎖液) 처리군의 24시간 및 48시간에서 통계적으로 유의한 감소를 관찰할 수 있었다. 3. 반하(半夏) 약쇄액(藥鎖液)에 의한 TARC 분비억제를 관찰 한 결과 농도의존적인 분비 감소 효과를 관찰 할 수 있었다. 4. MTT assay법을 이용한 세포 독성 측정에선 대조군과 반하(半夏) 약침액(藥鍼液) 처리군간에 유의성이 없었으므로 50, 100 및 200${\mu}g/m{\ell}$의 농도에선 세포독성이 없었음을 관찰할 수 있었다. 이에 반하(半夏) 약광액(藥鑛液)은 TARC 케모카인 억제를 통해 천식에 대한 치료효과를 나타낼 수 있을 것으로 사려된다.