• Title/Summary/Keyword: PBMC

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Molecular Detection of Korean-type Bovine Immunodeficiency Virus by Polymerase Chain Reaction (DNA 중합효소 연쇄반응을 이용한 한국형 젖소 면역 결핍 바이러스의 검출)

  • 권오식
    • Biomedical Science Letters
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    • v.5 no.1
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    • pp.101-107
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    • 1999
  • Bovine immunodeficiency virus (BIV) which was grouped into the Lentivirinae of family Retroviridae, was known to be causing many immunodeficiency syndromes among cows. The BIV was studied worldwide during last several years for its importance in cattle industries but nothing was reported in Korea until now Thus we initially tried to study the existence of BIV in cattle around the Daegu·Kyungpook area by PCR related molecular techniques. As a prerequisite investigation for detecting Korean-type BIV, we had focused our aim into BLV infected cows because the BLV infected cows tend to show BIV infection with 5% ranges. Hence we randomly sampled fresh bloods from 248 cows and bulls near the Daegu·Kyungpook area and collected peripheral blood monocytes (PBMC) from the sample bloods. After extracting genomic DNA from the PBMC, we subjected it to PCR and Soluthern blot analysis for BIV/BLV detection. Overall, 66.9% (81/121) of the cow PBMC samples turned out to be BLV positive by PCR and the result was reconfirmed by Southern blot analysis. The value was two times higher than the previously reported results of BLV infection in Korea. The significant difference was mainly due to 1) applying highly specific methods for BLV detection such as PCR 2) that BLV was continuously spreaded in the Daegu Kyungpook area without any notice during last ten years. We also tested the BLV positive samples with the same techniques for BIV detection. And we found some BIV positives among the lot 3C samples by PCR, which had showed 100% BLV positive.

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Effect of Dietary Krill Meal Levels on Performance and Immune Response of Broiler Chicks Injected with Salmonella typhimurium Lipopolysaccharide (살모넬라 LPS를 주입한 육계병아리에 있어서 사료 중 크릴 밀 수준이 생산성과 면역반응에 미치는 영향)

  • Im, J.T.;Park, I.K.;Koh, T.S.
    • Journal of Animal Science and Technology
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    • v.49 no.2
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    • pp.225-238
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    • 2007
  • In this study, the effects of dietary krill meal levels on cellular immunity in LPS-injected broiler chicks was evaluated. One day-old male broiler chicks(Ross) were fed on the experimental basal meal(0.0% krill meal), or diets containing 0.5%, 1.0% and 2.0% krill for 3 weeks, and the acute phase response was activated by intraperitoneally injection of Salmonella typhimurium lipopolysaccharide(LPS) 3 times at 9, 11, and 13 days of age. 1. Acute phase response induced a significant reduction in(p<0.05) daily weight gain and feed intake, and increases in liver and spleen weight. However, it was not affected by dietary krill meal levels. 2. The krill meal diets reduced TNF-α activity as compared to the basal diet after 24 hours (acute phase response) and 1 week(recovery from the acute phase response) following LPS injection (p<0.05). The acute phase response induced a significant increase(p<0.05) in TNF-α activity relative to the control in chicks fed on a basal diet, but this was also unaffected by dietary krill meal levels. 3. Acute phase response-mediated ovotransferrin levels(relative to what was measured in the control bird) were increased in birds fed on the basal, 1.0% and 2.0% krill diets, and were reduced in birds fed on the 0.5 % krill diet. 4. In LPS-injected chicks, 1.0% and 2.0% krill meal diets induced a significant reduction in(p<0.05) the Con A-induced proliferation of PBMC and splenocytes relative to what was observed in the chicks fed on a 0.5% krill diet, whereas the splenocytes proliferated in a linear fashion with the krill levels in the diets of the control birds. The results showed that the dietary levels of krill meal reduced TNF-α activity in the blood and also influenced blood ovotransferrin levels and the proliferation of PBMC and splenocytes, and krill meal is considered to be associated with both innate and cellular immunity in broiler chicks.

Trans-10, cis-12 Conjugated Linoleic Acid Modulates Nuclear Factor-${\kappa}B$ p65 Activity on the Production of Tumor Necrosis Factor-${\alpha}$ in Porcine Peripheral Blood Mononuclear Cells (돼지 말초혈액 단핵구세포에서 trans-10, cis-12 conjugated linoleic acid의 TNF-${\alpha}$ 생산에 대한 nuclear factor-${\kappa}B$ p65 활성 조절 효과)

  • Kim, Young-Beum;Lee, Ill-Woo;Kang, Ji-Houn;Yang, Mban-Pyo
    • Journal of Veterinary Clinics
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    • v.28 no.2
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    • pp.190-195
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    • 2011
  • Nuclear factor ${\kappa}B$ (NF-${\kappa}B$) is a nuclear transcription factor that modulates the expression of inflammatory cytokines such as tumor necrosis factor (TNF)-${\alpha}$. trans-10, cis-12 (t10c12)-conjugated linoleic acid (CLA) participates in the inhibition of TNF-${\alpha}$ production upon lipopolysaccharide (LPS)-stimulation. However, in our previous study, t10c12-CLA enhanced the production of TNF-${\alpha}$ by LPS-unstimulated porcine peripheral blood mononuclear cells (PBMCs) and RAW 264.7 macrophages in vitro. To resolve this apparent contradiction, we hypothesized that the effect of t10c12-CLA on TNF-${\alpha}$ production depends on NF-${\kappa}B$ activation induced by LPS stimulation. To test this hypothesis, we assessed the in vitro effect of t10c12-CLA on TNF-${\alpha}$ production and NF-${\kappa}B$ p65 activity in LPS-stimulated and LPS-unstimulated porcine PBMCs. t10c12-CLA treatment resulted in increased TNF-${\alpha}$ production by LPS-unstimulated PBMCs but decreased TNF-${\alpha}$ production by LPS-stimulated PBMCs. t10c12-CLA increased the degradation of inhibitory ${\kappa}B$ ($I{\kappa}B$)-${\alpha}$ protein and activated NF-${\kappa}B$ p65 in LPS-unstimulated PBMCs, but had the opposite effect in LPS-stimulated PBMCs. Notably, t10c12-CLA enhanced NF-${\kappa}B$ p65 binding activity in LPS-unstimulated PBMCs exposed to caffeic acid phenethyl ester (CAPE), a NF-${\kappa}B$ inhibitor. Conversely, it inhibited NF-${\kappa}B$ p65 binding activity in LPS-stimulated PBMCs exposed to CAPE. These results suggest that t10c12-CLA may have different actions under different physiological conditions, and that its effect may be associated with a change in NF-${\kappa}B$ p65 activity.

In Vitro Selection of Cancer-Specific RNA Aptamers

  • Lee Young-Ju;Lee Seong-Wook
    • Journal of Microbiology and Biotechnology
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    • v.16 no.7
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    • pp.1149-1153
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    • 2006
  • In this study, nuclease-resistant RNA aptamers that are specific for Jurkat T leukemia cells were selected by a subtractive systemic evolution of ligands by exponential enrichment (SELEX) method. A randomized nuclease-resistant RNA library was incubated with normal peripheral blood mononuclear cells (PBMC) in each round to preclude RNAs that recognize the common cellular components on the surface of normal and cancer cells. The precluded RNAs were used for the selection of Jurkat T cell-specific aptamers, and the specific RNAs were then gradually enriched from start to the following selections. After 16 rounds of the subtractive SELEX, the selected aptamers were found to preferentially bind to Jurkat T cells, but not to the normal PBMC, evidenced by fluorescence-activated cell sorting analysis. Thus, the subtractive SELEX can be used to identify ligands to cancer-specific biological markers without prior knowledge of the nature of markers. The aptamers could be applied to specific cell sorting, tumor therapy, and diagnosis, and moreover, to find cancer cell-specific markers.

Suppressive Effect of GJB-7 on Collagen-Induced Arthritis in Mice (관절7호방이 Type II collagen으로 유발된 흰쥐의 관절염에 미치는 영향)

  • Min, Boo-Ki;Oh, Min-Seok
    • The Journal of Korean Medicine
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    • v.31 no.4
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    • pp.63-78
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    • 2010
  • Objectives: This study was carried out to know the effects of Gwan-Jul-Bang-7 (hereafter referred to GJB-7) on the inhibition of arthritis induced by collagen on the mouse. Methods: To assess the effects of GJB-7 on mouse with arthritis induced by collagen II, we conducted several experiments such as analysis of cytotoxicity, hepatotoxicity, arthritis index, total cell number of draining lymph nodes and paw joints, value of immunocyte in PBMC (peripheral blood mononuclear cell), DLN (draining lymph node) and paw joint, measurement of cytokine and anti-collagen II, observation of the histological changes of joint. Results: 1. Cytotoxicity against HFC (human fibroblast cells) was not observed in any concentration and hepatotoxicity was not observed in the GJB-7 treated group. 2. The incidence of arthritis significantly decreased. 3. Total cell number of draining lymph nodes significantly increased and total cell number of paw joints significantly decreased. 4. The percentage of $CD8^+$ cells in PBMC (peripheral blood mononuclear cell) significantly increased. The percentage of $CD3^+/CD69^+$, and $CD3^+/CD49b^+$ cells in PBMC significantly decreased. 5. The percentage of $CD19^+,\;CD3^+$, and $CD4^+/CD25^+$ cells in DLN (draining lymph nodes) significantly increased. The percentage of $B220^+/CD23^+$ cells in DLN significantly decreased. 6. The percentage of $CD3^+,\;CD4^+$, and $CD11b^+/Gr-1^+$ cells in paw joints significantly decreased. 7. The production of TNF-$\alpha$, IL-6, and MCP-1 significantly decreased. 8. Anti-collagen II in serum significantly decreased. 9. With the hematoxylin and eosin stain, inflammation of joint decreased. Under Masson's trichrome stain, the cartilage destruction and synovial cell proliferation and the expression of collagen fibers decreased. Conclusions: Comparison of the results for this study showed that GJB-7 had immunomodulatory effects. So we expect that GJB-7 could be used as an effective drug for not only rheumatoid arthritis but also another auto-immune diseases.

Individual LPS Responsiveness Depends on the Variation of Toll-like Receptor (TLR) Expression Level

  • JaeKal, Jun;Abraham, Edward;Azam, Tania;Netea, Mihai G.;Dinarello, Charles A.;Lim, Jong-Seok;Yang, Young;Yoon, Do-Young;Kim, Soo-Hyun
    • Journal of Microbiology and Biotechnology
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    • v.17 no.11
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    • pp.1862-1867
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    • 2007
  • An individual's immune response is critical for host protection from many different pathogens, and the responsiveness can be assessed by the amount of cytokine production upon stimulating bacterial components such as lipopolysaccharide (LPS). The difference between individuals in their peripheral blood mononuclear cells (PBMC) responsiveness to LPS, a Gram-negative endotoxin, was investigated from 27 healthy individuals. We observed a large variation in $IFN{\gamma}$ production among different individuals. The PBMC of the consistently three highest and three lowest $IFN{\gamma}$ producers were investigated. Since previous studies described that a single point mutation in the coding region of TLR2 and TLR4 is linked to the individual responsiveness to pathogenic bacterial infections, we first examined the known point mutations in the coding region of $TLR2^{Pro681His}$, $TLR4^{Pro714His}$ located in the cytoplasmic regions of the Toll-like domain as well as $TLR4^{Asp299Gly}$ located in the extracellular region. None of these mutations were associated with an individual's responsiveness to LPS, despite the presence of $TLR4^{Asp299Gly}$ mutation. Further investigation revealed that the variation of PBMC responsiveness to LPS among healthy individuals was due to constitutive expression levels of TLR4 and TLR2. This result is consistent with an aging-related low expression of Toll-like receptors in the mouse model of LPS responsiveness. The present study therefore suggests that the constitutive expression levels of TLR2 and TLR4 may contribute to the individual response to LPS.

Dietary Glutamine Supplementation Enhances Weaned Pigs Mitogen-Induced Lymphocyte Proliferation

  • Lee, D.N.;Weng, C.F.;Cheng, Y.H.;Kuo, T.Y.;Wu, J.F.;Yen, H.T
    • Asian-Australasian Journal of Animal Sciences
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    • v.16 no.8
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    • pp.1182-1187
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    • 2003
  • Two experiments involving 92 crossbred, 21 day old weaned pigs were used to evaluate the effect of glutamine supplement in a dietary or culture medium on lymphocyte proliferation. In Exp. 1, 88 pigs were fed diets supplemented with 0, 0.5, 1.0, or 1.5% glutamine for 28 days. Lymphocytes were prepared from peripheral blood mononuclear cells (PBMC), ileal Peyer's patches (PP), the mesenteric lymph node (MLN), and the spleen in each dietary supplement group on days 7, 14, or 28 postweaning. Lymphocytes were cultured at $37^{\circ}C$ for 72 h in a RPMI-1640 medium with or without mitogen-stimulated, and pulsed with 3Hthymidine for an additional 18 h. The stimulation index of PBMC proliferation in 1.0% dietary glutamine supplement group and both of the MLN and splenocytes proliferation in 1.5% dietary glutamine supplement group was significantly (p<0.05) increased at 14 days postweaning. In Exp. 2, four weaned pigs were fed a basal diet for 14 days. The 3H-thymidine incorporation of PBMC, PP, and MLN cells, incubated with 0.125 to 0.25 mM glutamine in culture medium were markedly enhanced with Con A-stimulated, however, the splenocyte proliferation was not affected in the addition of glutamine medium. These observations suggest that dietary glutamine supplement might enhance the lymphocyte proliferation of weaned pigs.

Increased Oxidative Stress and RUNX3 Hypermethylation in Patients with Hepatitis B Virus-Associated Hepatocellular Carcinoma (HCC) and Induction of RUNX3 Hypermethylation by Reactive Oxygen Species in HCC Cells

  • Poungpairoj, Poonsin;Whongsiri, Patcharawalai;Suwannasin, Surasit;Khlaiphuengsin, Apichaya;Tangkijvanich, Pisit;Boonla, Chanchai
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.13
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    • pp.5343-5348
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    • 2015
  • Promoter hypermethylation of the runt-related transcription factor 3 (RUNX3) gene is associated with increased risk of hepatocellular carcinoma (HCC). Oxidative stress plays a vital role in both carcinogenesis and progression of HCC. However, whether oxidative stress and RUNX3 hypermethylation in HCC have a cause-and-effect relationship is not known. In this study, plasma protein carbonyl and total antioxidant capacity (TAC) in patients with hepatitis B virus (HBV)-associated HCC (n=60) and age-matched healthy subjects (n=80) was determined. RUNX3 methylation in peripheral blood mononuclear cells (PBMC) of subjects was measured by methylation-specific PCR. Effect of reactive oxygen species (ROS) on induction of RUNX3 hypermethylation in HCC cells was investigated. Plasma protein carbonyl content was significantly higher, whereas plasma TAC was significantly lower, in HCC patients than healthy controls. Based on logistic regression, increased plasma protein carbonyl and decreased plasma TAC were independently associated with increased risk for HCC. PBMC RUNX3 methylation in the patient group was significantly greater than in the healthy group. RUNX3 methylation in hydrogen peroxide ($H_2O_2$)-treated HepG2 cells was significantly higher than in untreated control cells. In conclusion, increase in oxidative stress in Thai patients with HBV-associated HCC was demonstrated. This oxidative increment was independently associated with an increased risk for HCC development. RUNX3 in PBMC was found to be hypermethylated in the HCC patients. In vitro, RUNX3 hypermethylation was experimentally induced by $H_2O_2$. Our findings suggest that oxidative stress is a cause of RUNX3 promoter hypermethylation in HCC cells.

Preliminary assessment of correlation between T-lymphocyte responses and control of porcine reproductive and respiratory syndrome virus (PRRSV) in piglets born after in-utero infection of a type 2 PRRSV

  • Cha, Sang-Ho;Bandaranayaka-Mudiyanselage, Carey;Bandaranayaka-Mudiyanselage, Chandima B.;Ajiththos, Dharani;Yoon, Kyoung-Jin;Gibson, Kathleen A.;Yu, Ji-Eun;Cho, In-Soo;Lee, Stephen S.;Chung, Chungwon J.
    • Korean Journal of Veterinary Research
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    • v.58 no.1
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    • pp.9-16
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    • 2018
  • A preliminary study into the protective mechanisms of adaptive immunity against porcine reproductive and respiratory syndrome virus (PRRSV) in piglets (n = 9) born to a gilt challenged intranasally with a type-2 PRRSV. Immune parameters (neutralizing antibodies, $CD3^+CD4^+$, $CD3^+CD8^+$, $CD3^+CD4^+CD8^+$ T-lymphocytes, and PRRSV-specific interferon $(IFN)-{\gamma}$ secreting T-lymphocytes) were compared with infection parameters (macro- and microscopic lung lesion, and PRRSV-infected porcine alveolar macrophages ($CD172{\alpha}^+PRRSV-N^+\;PAM$) as well as with plasma and lymphoid tissue viral loads. Percentages of three T-lymphocyte phenotypes in 14-days post-birth (dpb) peripheral blood mononuclear cell (PBMC) had significant negative correlations with percentages of $CD172{\alpha}^+PRRSV-N^+\;PAM$ (p < 0.05) as well as with macroscopic lung lesion (p < 0.01). Plasma and tissue viral loads had significant (p < 0.05) negative correlations with $CD3^+CD4^+CD8^+$ T-lymphocyte percentage in PBMC. Frequencies of $CD3^+CD8^+$ and $CD3^+CD4^+$ T-lymphocytes in 14-dpb PBMC had significant negative correlations with of lymph node (p = 0.04) and lung (p = 0.002) viral loads. $IFN-{\gamma}$-secreting T-lymphocytes frequency had a significant negative correlation with gross lung lesion severity (p = 0.002). However, neutralizing antibody titers had no significant negative correlation (p > 0.1) with infection parameters. The results indicate that T-lymphocytes contribute to controlling PRRSV replication in young piglets born after in-utero infection.

Perfluorocarbon Does Not Inhibit Chemokine Expression in Airway Epithelial Cells (Perfluorocarbon이 기도 상피세포 Chemokine 발현에 미치는 영향에 관한 연구)

  • Suh, Gee-Young;Kang, Kyeong-Woo;Park, Sang-Joon;Chung, Man-Pyo;Kim, Ho-Joong;Choi, Dong-Chull;Rhee, Chong-H;Kwon, O-Jung
    • Tuberculosis and Respiratory Diseases
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    • v.48 no.2
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    • pp.223-235
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    • 2000
  • Background: Liquid ventilation is associated with decreased inflammatory response in an injured lung. This study was performed to investigate if whether perfluorocarbon(PFC) can decrease chemokine expression in airway epithelial cells. Methods: A549 cells were used for airway epithelial cells and perfluorodecalin for PFC. To expose cells to PFC, lower chamber of Transwell$^{(R)}$plate was used. This study was performed in two parts. In the first part, we examined whether PFC could decrease chemokine expression in airway epithelial cells through inhibition of other inflammatory cells. Peripheral blood mononuclear cells(PBMC's) were isolated and stimulated with lipopolysaccharide(LPS, 10 ${\mu}g/mL$) for 24 hours with or without exposure to PFC. Then A549 cells were stimulated with conditioned media(CM) containing the culture supernatants of PBMC. After 24 hours, the expressions of interleukin-8(IL-8) and RANTES were measured. In the second part of the study, we studied whether PFC could directly suppress chemokine expression in airway epithelial cells. A549 cells were stimulated for 24 hours with interleukin-l$\beta$ and/or tumor necrosis factor-$\alpha$ with or without exposure to PFC, and then the chemokine expression was measured. Northern analysis was used to measure the mRNA expression, and ELISA was used for immunoreactive protein measurements in culture supernatant. Results: 1. IL-8 and RANTES mRNA expression and immunoreactive protein production were increased significantly by CM from LPS-stimulated PBMC in A459 cells compared to with CM from unstimulated PBCM (p<0.05), but exposure of PFC had no significant effect on either mRNA expression or immunoreactive protein expression. 2. IL-8 and RANTES mRNA expression and immunoreactive protein production were increased significantly by IL-1$\beta$ and TNF-$\alpha$ in A549 cells(p<0.05), but exposure of PFC had no significant effect on neither either mRNA expression nor immunoreactive protein production. Conclusion : Decreased chemokine expression of airway epithelial cells may not be involved in decreased inflammatory response observed in liquid ventilation. Further studies on possible mechanisms of decreased inflammatory response are warranted.

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