• Journal of Pharmaceutical Investigation
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• v.32 no.4
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• pp.285-290
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• 2002

In order to assay the efficacy of newly synthesized antiviral compounds, in vitro studies of their active intracellular phosphorylated metabolites were established as compared with Zidovudine (ZDV). Antiviral base analogs require intracellular phosphorylation prior to the inhibition of HIV replication. Therefore, antiviral drugs concentrations in plasma have not reflected any direct relationship with activity or toxicity. A method has been developed to measure the concentration of total phosphorylated metabolites inside peripheral blood mononuclear cells using modified commercial radioimmunoassay (RIA). ZDV 5'-monophosphate was synthesized and used as a procedural control for RIA modification. PBMCs were isolated from whole blood and incubated with ZDV for 20 h to allow metabolic phosphorylation. Viable cells were extracted overnight with 60% methanol. After evaporation, the extract was reconstituted in Tris buffer. Samples were split into two fractions, one of which was treated with alkaline phosphatase (AP) to liberate phosphate groups. Concentrations of phosphorylated metabolites were determined by subtracting thε concentration of non-AP-treated fraction from that of the treated fraction. Recovery of phosphorylated ZDV from cell extracts was approximately 90%, and reproducibility was acceptable (coefficients of variation <15% for concentrations${\geq}$0.25 ng/mL). Intracellular concentrations $(0.135{\sim}5.019\;nmole/10^6\;cells)$ followed a nonlinear dose-response relationship over the range $0.015{\sim}2.996mM$ extracellular ZDV, with concentration-dependant saturation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.29 no.2
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• pp.108-115
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• 2003

Suppression of cellular immunity is the host responses to surgical stress. When the body is exposed to surgical stress, decreased immunocyte function is one of the surgical stress-induced biologic responses. In all patients exposed to the surgical stress, peripheral blood lymphocyte numbers and function were suppressed until at least 2 weeks postoperatively. This immunosuppression was mainly due to a decrease of helper-inducer T cells, cytotoxic T cells, natural killer cells, and an increase of suppressor T cells. The blood levels of interleukin-6(IL-6) cytokine increase in response to surgical stress and cause an increase of so-called acute phase reactants, including C-reactive protein(CRP). In the previously damaged patients group, expected to early stress expose, immunosuppression was more developed than other normal groups. Cellular immunosuppression by surgical stress was mainly due to an increase of lymphocyte subsets that depress cellular immunity coupled with a decrease of the subsets that promote it. Overproduction of CRP in response to surgical stress may play an important role in the development of immunosuppression.

• Korean Journal of Veterinary Research
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• v.37 no.1
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• pp.129-135
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• 1997

Peripheral blood mononuclear cells(PBMNC) of Korean native cattle rosetted with Korean goat erythrocytes(KGRBC) and blood monocytes were evaluated for four cytochemical reactions such as acid phosphatase(ACP), alkaline phosphatase-anti-boby(ALP-Ab), ${\alpha}$-naphthyl butyrate esterase(${\alpha}$-NBE) and peroxidase. The results obtained were as follows; In rosetted cells, the positivities of ACP in E AET-DeX, EA and EAC were 70.3%, 22.4% and 25.2%, those of ${\alpha}$-NB were 27.4%, 44.2% and 79.8%, and those of ALP-Ab were 9.5%, 88.3% and 91.5%, respectively. Whereas, the positivity for Peroxidase in monocytes was 100%. In non-rosetted (remained) cells, the positivities of ACP in E AET+DeX. EA and EAC were 41.4%, 57.2% and 61.9%, those of ${\alpha}$-NB were 38.6%, 16.5% and 18.9% and those of ALP-Ab were 98.2%, 5.3% and 6.3%, in order.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.4
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• pp.471-478
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• 2014

Investigating gene expression of immune cells of whole blood or peripheral blood mononuclear cells (PBMC) under polyinosinic:polycytidylic acid (poly I:C) stimulation is valuable for understanding the immune response of organism to RNA viruses. Quantitative real-time PCR (qRT-PCR) is a standard method for quantification of gene expression studies. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. In the study, using two different analysis programs, geNorm and NormFinder, we systematically evaluated the gene expression stability of six candidate reference genes (GAPDH, ACTB, B2M, RPL4, TBP, and PPIA) in samples of whole blood and PBMC with or without poly I:C stimulation. Generally, the six candidate genes performed a similar trend of expression stability in the samples of whole blood and PBMC, but more stably expressed in whole blood than in PBMC. geNorm ranked B2M and PPIA as the best combination for gene expression normalization, while according to NormFinder, TBP was ranked as the most stable reference gene, followed by B2M and PPIA. Comprehensively considering the results from the two programs, we recommended using the geometric mean of the three genes, TBP, PPIA and B2M, to normalize the gene expression of whole blood and PBMC with poly I:C stimulation. Our study is the first detailed survey of the gene expression stability in whole blood and PBMC with or without poly I:C stimulation and should be helpful for investigating the molecular mechanism involved in porcine whole blood and PBMC in response to poly I:C stimulation.

• Journal of Korean Society of Occupational and Environmental Hygiene
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• v.31 no.3
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• pp.286-293
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• 2021

Objectives: Inorganic dust is known to be a risk factor for chronic obstructive pulmonary disease (COPD) regardless of smoking and pneumoconiosis. Adaptive and innate immunity, including lymphocyte infiltrate, are involved in the pathogenesis of COPD. The purpose of this study was to analyze the lymphocyte subsets in the blood of workers exposed to inorganic dust and confirm the influencing factors. Methods: The general characteristics of the subjects (n=107) were analyzed through a personal questionnaire. Diagnosis of COPD was established according to pulmonary function tests with FEV₁/FVC post bronchodilator lower than 70%, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines. For lymphocyte analysis, blood was stained with a fluorescent CD marker and analyzed by flow cytometry. Results: The increase in CD4⁺ T lymphocytes was associated with a decrease in age (𝛽=-0.273, p=0.008) and an increase in the cumulative smoking amount (𝛽=0.205, p=0.034). The increase in NK cells was associated with an increase in age (𝛽=0.325, p=0.001) and a decrease in cumulative smoking (𝛽=-0.220, p=0.019). The period of exposure to dust, %FVC predicted and %FEV₁/FVC, and the relative population of peripheral blood lymphocytes did not show a statistically significant relationship. Conclusions: CD4⁺ T lymphocytes and CD56⁺CD16⁺ NK cells in peripheral blood were more related to age and cumulative smoking than the duration of dust exposure. Age and smoking are major risk factors for the development of COPD, so it can be predicted that peripheral blood CD4⁺ T lymphocytes and CD56⁺CD16⁺ NK cells are related to the development of COPD in workers exposed to inorganic dust.

• Tuberculosis and Respiratory Diseases
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• v.74 no.6
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• pp.264-268
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• 2013

Background: Idiopathic pulmonary fibrosis (IPF) is a lethal pulmonary fibrotic disease. In general, the exaggerated activation of the coagulation cascade has been observed during initiation or maintenance of the fibrotic disease. In our recent study, immunohistochemical expression of protease-activated receptor-2 (PAR-2), which plays a key role in coagulation cascade, was observed in surgical specimen of IPF patients, and associated with poor clinical outcome. The aim of this study was to evaluate the overexpression of PAR-2 in inflammatory cells from peripheral blood and bronchoalveolar lavage fluid in IPF patients. Methods: From May 2011 to March 2012, IPF patients and controls were enrolled in Seoul National University Hospital. Peripheral blood and bronchoalveolar lavage fluid were collected for analysis of PAR-2 expression. Flow cytometry and reverse transcription polymerase chain reaction were used for PAR-2 receptor and mRNA assessment. Results: Twelve IPF patients and 14 controls were included in this study. Among them, flow cytometry analysis was conducted from 26 peripheral blood (patient group, 11; control group, 13) and 7 bronchoalveolar lavage fluid (patient group, 5; control group, 2). The expression of PAR-2 receptor was not different between patient and control groups (p=0.074). Among all 24 population, PAR-2 mRNA assessment was performed in 19 persons (patient group, 10; control group, 9). The mRNA expression of PAR-2 was not significant different (p=0.633). Conclusion: In IPF patients, PAR-2 receptor and mRNA expression were not different from control group.

• Korean Journal of Agricultural Science
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• v.46 no.3
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• pp.579-589
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• 2019

Due to the ban on the use of antibiotics, interest has been increasing for the development of therapeutic agents to treat various diseases using natural resources. Osthole, a natural coumarin compound used in traditional Chinese medicines, exerts an anti-inflammatory effect, but its effects in cows remain unknown. In this study, the effect of osthole on lipopolysaccharide (LPS)- or concanavalin-A (Con-A)- stimulated peripheral blood mononuclear cells (PBMCs) was assessed. Jugular venous blood was collected from Korean calves, and PBMCs were isolated. They were then used to study the immune response of PBMCs to treatment with osthole and LPS or Con-A for 72 h by measuring inflammatory cytokines including tumor necrosis factor-${\alpha}$ ($TNF-{\alpha}$) and interferon-${\gamma}$ ($IFN-{\gamma}$). Osthole significantly inhibited the mRNA secretion of $TNF-{\alpha}$ and $IFN-{\gamma}$ in a dose-dependent manner. Therefore, osthole inhibited LPS- or Con-A- induced $TNF-{\alpha}$ and Con-A-induced $IFN-{\gamma}$ production significantly in dose-dependent manner. These results clearly suggest that osthole inhibited the LPS- or Con-A- stimulated upregulation of pro-inflammatory cytokines in a dose-dependent manner, without causing obvious cytotoxic effects. Osthole could also protect cows from LPS- or Con-A- induced endotoxin shock, possibly by inhibiting the production of pro-inflammatory cytokines, which suggests that osthole might be a novel therapeutic agent for the prevention of inflammatory diseases.

• BMB Reports
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• v.52 no.4
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• pp.259-264
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• 2019

Social requirements are needed for living in an aging society and individual longevity. Among them, improved health and medical cares, appropriate for an aging society are strongly demanded. Human cord blood-derived plasma (hUCP) has recently emerged for its unique anti-aging effects. In this study, we investigated brain rejuvenation, particularly olfactory function, that could be achieved by a systemic administration of young blood and its underlying mechanisms. Older than 24-month-old mice were used as an aged group and administered with intravenous injection of hUCP repetitively, eight times. Anti-aging effect of hUCP on olfactory function was evaluated by buried food finding test. To investigate the mode of action of hUCP, brain, serum and spleen of mice were collected for further ex vivo analyses. Systemic injection of hUCP improved aging-associated olfactory deficits, reducing time for finding food. In the brain, although an infiltration of activated microglia and its expression of cathepsin S remarkably decreased, significant changes of proinflammatory factors were not detected. Conversely, peripheral immune balance distinctly switched from predominance of Type 1 helper T (Th1) cells to alternative regulatory T cells (Tregs). These findings indicate that systemic administration of hUCP attenuates age-related neuroinflammation and subsequent olfactory dysfunction by modulating peripheral immune balance toward Treg cells, suggesting another therapeutic function and mechanism of hUCP administration.

• Journal of Chest Surgery
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• v.55 no.3
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• pp.214-224
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• 2022

Background: Studies of the prognostic role of circulating tumor cells (CTCs) in early-stage non-small cell lung cancer (NSCLC) are still limited. This study investigated the prognostic power of CTCs from the pulmonary vein (PV), peripheral blood (PB), and bone marrow (BM) for postoperative recurrence in patients who underwent curative resection for NSCLC. Methods: Forty patients who underwent curative resection for NSCLC were enrolled. Before resection, 10-mL samples were obtained of PB from the radial artery, blood from the PV of the lobe containing the tumor, and BM aspirates from the rib. A microfabricated filter was used for CTC enrichment, and immunofluorescence staining was used to identify CTCs. Results: The pathologic stage was stage I in 8 patients (20%), II in 15 (38%), III in 14 (35%), and IV in 3 (8%). The median number of PB-, PV-, and BM-CTCs was 4, 4, and 5, respectively. A time-dependent receiver operating characteristic curve analysis showed that PB-CTCs had excellent predictive value for recurrence-free survival (RFS), with the highest area under the curve at each time point (first, second, and third quartiles of RFS). In a multivariate Cox proportional hazard regression model, PB-CTCs were an independent risk factor for recurrence (hazard ratio, 10.580; 95% confidence interval, 1.637-68.388; p<0.013). Conclusion: The presence of ≥4 PB-CTCs was an independent poor prognostic factor for RFS, and PV-CTCs and PB-CTCs had a positive linear correlation in patients with recurrence.

• Korean Journal of Veterinary Research
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• v.29 no.2
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• pp.33-39
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• 1989

Pure separation of various leukocytes is required for the assessment of their roles in immunological and phisiological function. In this study, pure separation of monocytes from canine peripheral blood was attempted. At first, mononuclear cells (PBMC) were separated by ficoll-hypaque gradient method and then monocytes were recovered from PBMC suspensions in sucrose gradient Sol. (PBMC-Sucrose), autologous plasma (PBMC-Plasma) and autologous serum (PBMC-Serum) incubated at $37^{\circ}C$ for 2 hours. 1. In the separation of PBMC by ficoll-hypaque gradient method in canine blood, higher relative centrifugal force (RCF) was required, as high as more than 1,300xg RCF for 40 minutes, for clear formation of PBMC layer than that in human blood as usually used 400xg RCF for 40 minutes. 2. In monocytes-separation from three PBMC suspensions following PBMC separation, recovery-, purity- and viability-rate of monocytes showed better results in PBMC-Plasma and PBMC-Serum than in PBMC-Sucrose suspension, particulary showing better results from PBMC suspensions performed by centrifugation at 1,500xg RCF for 40 minutes.