• Journal of Veterinary Clinics
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• v.35 no.5
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• pp.195-199
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• 2018

Zinc is necessary for normal functions in the immune system. The objective of the study is to examine the effect of zinc on the chemotactic activity of porcine peripheral blood polymorphonuclear cells (PMNs). A modified Boyden chamber was used to determine the directional migration distance of PMNs. Various concentrations of zinc showed no chemotactic activity to PMNs. However, culture supernatant from peripheral blood mononuclear cells (PBMCs) treated with zinc remarkably increased the chemotactic activity of PMNs when compared with culture supernatant from PBMCs treated without zinc. Culture supernatant from PBMCs treated without zinc also increased the migration distance of PMNs relative to vehicle control (medium alone). Increasing effect in chemotactic activity of PMNs by culture supernatant from PBMCs treated with zinc was inhibited by treatment of porcine anti-interleukin (IL)-8 polyclonal antibody (pAb). This effect was not affected by heat treatment ($4-85^{\circ}C$). This corresponded with heat stable physical characteristics of IL-8. These results suggest that zinc can upregulate the chemotaxis of PMNs, which is primary mediated by IL-8 chemotactic factor released from PBMCs treated with zinc.

• Journal of Veterinary Clinics
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• v.37 no.5
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• pp.249-254
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• 2020

Neutrophil extracellular trap (NET) formation is an immune response for the invasion of microbes. The purpose of this study is to examine the effect of zinc on NET formation of porcine peripheral blood polymorphonuclear cells (PMNs). The NET formation of PMNs was measured by fluorescence microplate reader. The production of tumor necrosis factor (TNF)-α in the culture supernatants from zinc-treated peripheral blood mononuclear cells (PBMCs) was measured by enzyme-linked immunosorbent assay (ELISA). Zinc itself did not have no effect on NET formation. However, the NET formation of PMNs was increased by culture supernatants from PBMCs treated with zinc. Also, the NET formation of PMNs was increased by recombinant porcine (rp) TNF-α. The production of TNF-α in PBMCs culture supernatants was shown to increase upon zinc treatments. These NET formations of PMNs increased by either culture supernatant from PBMCs treated with zinc or rpTNF-α were inhibited by treatment of anti-rpTNF-α polyclonal antibody (pAb). These results suggested that zinc has an immunostimulating effect on the NET formation of PMNs, which is mediated by TNF-α released from zinc-treated PBMCs. Therefore, zinc may play an important role for NET formation in the defense of porcine inflammatory diseases.

• Journal of Veterinary Clinics
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• v.37 no.2
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• pp.61-66
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• 2020

Nickel is a nutritionally essential trace element that plays an important role in the immune system of several animal species. The aim of this study was to examine the effect of nickel chloride on chemotactic activity of peripheral blood polymorphonuclear cells (PMNs) and whether this effect is associated with interleukin (IL)-8 and a nuclear factor-kappa B (NF-κB)-dependent pathway. Peripheral blood mononuclear cells (PBMCs) and PMNs were isolated by Percoll solution (Specific gravity; 1.080) and 1.5% dextran treatment, respectively. A modified Boyden chamber assay was used to measure the chemotactic activity of PMNs. The level of IL-8 in culture supernatant from PBMCs was measured by enzyme-linked immunosorbent assay (ELISA). Both of PBMCs and PMNs exhibited a low viability when cultured with concentration of greater than 1,000 μM of nickel chloride for 24 h. Thus, nickel chloride was used at concentration of 500 μM, which preserved cell viability. Treatment with nickel did not directly affect the chemotactic activity of PMNs. However, the chemotactic activity of PMNs was remarkably increased by culture supernatant from PBMCs treated with nickel chloride (500 μM) for 24 h. Recombinant porcine IL-8 polyclonal antibody (pAb) neutralized the enhancing effect on the chemotactic activity of PMNs by culture supernatant from PBMCs treated with nickel and this culture supernatant had higher IL-8 levels than the culture supernatant from untreated PBMCs. In addition, n-tosyll-phenylalanine chloromethyl ketone (TPCK), a NF-κB inhibitor, antagonized the enhancing effect on the chemotactic activity of PMNs by the culture supernatant from PBMCs treated with nickel. These results suggested that nickel stimulates porcine PBMCs to produce IL-8, which increases the chemotaxis of PMNs via NF-κB-dependent pathway.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.4
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• pp.248-258
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• 2022

Objective: This research investigated the effects of human chorionic gonadotropin (HCG)-producing peripheral blood mononuclear cells (PBMCs) on the implantation rate and embryo attachment in mice. Methods: In this experimental study, a DNA fragment of the HCG gene was cloned into an expression vector, which was transfected into PBMCs. The concentration of the produced HCG was measured using enzyme-linked immunosorbent assay. Embryo attachment was investigated on the co-cultured endometrial cells and PBMCs in vitro. As an in vivo experiment, intrauterine administration of PBMCs was done in plaque-positive female mice. Studied mice were distributed into five groups: control, embryo implantation dysfunction (EID), EID with produced HCG, EID with PBMCs, and EID with HCG-producing PBMCs. Uterine horns were excised to characterize the number of implantation sites and pregnancy rate on day 7.5 post-coitum. During an implantation window, the mRNA expression of genes was evaluated using real-time polymerase chain reaction. Results: DNA fragments were cloned between the BamHI and EcoRI sites in the vector. About 465 pg/mL of HCG was produced in the transfected PBMCs. The attachment rate, pregnancy rate, and the number of implantation sites were substantially higher in the HCG-producing PBMCs group than in the other groups. Significantly elevated expression of the target genes was observed in the EID with HCG-producing PBMCs group. Conclusion: Alterations in gene expression following the intrauterine injection of HCG-producing PBMCs, could be considered a possible cause of increased embryo attachment rate, pregnancy rate, and the number of implantation sites.

• Journal of Veterinary Clinics
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• v.40 no.3
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• pp.175-181
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• 2023

Fucoidan extracted from brown seaweed has a variety of biological activities. Neutrophil extracellular traps (NETs) formation is an immune response for the invasion of pathogens. Neutrophils release granule protein and chromatin that form extracellular fibers that bind microbes. These NETs degrade virulence factors and kill bacteria. The aim of this study was to investigate the effect of fucoidan on NET formation of porcine peripheral blood polymorphonuclear cells (PMNs). The NET formation was determined by fluorescence emission of propidium iodide (PI) in PMNs by a fluorescence microplate reader. The production of tumor necrosis factor (TNF)-α from peripheral blood mononuclear cells (PBMCs) was measured by ELISA method. Fucoidan itself did not show any direct effect on NET formation. However, NET formation of PMNs was increased by the culture supernatant from PBMCs treated with fucoidan. The NET formation of PMNs were also enhanced by treatment with recombinant porcine (rp) TNF-α. The ability of culture supernatant from PBMCs treated with fucoidan to increase the NET formation of PMNs was inhibited by addition of goat anti-rp TNF-α polyclonal antibody (pAb) (IgG) prior to the culture. The increase of NET formation by rp TNF-α was also inhibited by goat anti-rp TNF-α pAb (IgG). The level of TNF-α in culture supernatant from PBMCs was increased by treatment with fucoidan. These results suggest that fucoidan increases porcine NET formation, which is mediated by TNF-α produced from PBMCs.

• Clinical and Experimental Vaccine Research
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• v.12 no.4
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• pp.328-336
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• 2023

Purpose: Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Materials and Methods: Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. Results: With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Conclusion: Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

• Molecular & Cellular Toxicology
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• v.4 no.4
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• pp.323-330
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• 2008

Surrogate tissue analysis incorporating -omics technologies has emerged as a potential alternative method for evaluating toxic effect of the tissues which are not accessible for sampling. Among the recent applications, blood including whole blood, peripheral blood lymphocytes and peripheral blood mononuclear cells (PBMCs) was suggested as a suitable surrogate tissue in determining toxicant exposure and effect at the pre- or early clinical stage. In this application, we investigated transcriptomic profiles in astemizole treated Cynomolgus monkey's PBMCs. PBMCs were isolated from 4-6 years old male monkeys at 24 hr after administration45 Helvetica Light (10 mg/kg, 30 mg/kg). Gene expression profiles of astemizole treated monkey's PBMCs were determined using Affymetrix $GeneChip^{(R)}$ Human Genome U133 plus 2.0 arrays. The expression levels of 724 probe sets were significantly altered in PBMCs at 10 or 30 mg/kg after astemizole administration following determination of paired t-test using statistical criteria of ${\geq}$$1.5-fold changes at P<0.05. Gene expression patterns in PBMCs showed a considerable difference between astemizole 10 and 30 mg/kg administration groups in spite of an administration of the same chemical. However, close examination using Ingenuity Pathway Analysis (IPA) software revealed that several gene sets related to cardiotoxicity were deregulated at astemizole 10 and 30 mg/kg administration groups. The deregulation of cardiac hypertrophy related genes such as TXN, GNAQ, and MAP3K5 was observed at 10 mg/kg group. In astemizole 30 mg/kg group, genes involved in cardiotoxicity including cardiac necrosis/cell death, dilation, fibrosis, and hypertrophy were also identified. These results suggest that toxicogenomic approach using PBMCs as surrogate tissues will contribute to assess toxicant exposures and identify biomarkers at the pre-clinical stage.

• Clinical and Experimental Vaccine Research
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• v.12 no.1
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• pp.47-59
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• 2023

Purpose: The development and study of hepatitis C virus (HCV) vaccine candidates' individualized responses are of great importance. Here we report on an HCV DNA vaccine candidate based on selected envelope (E1/E2) epitopes. Besides, we assessed its expression and processing in human peripheral blood mononuclear cells (PBMCs) and in vivo cellular response in mice. Materials and Methods: HCV E1/E2 DNA construct (EC) was designed. The antigen expression of EC was assayed in PBMCs of five HCV-uninfected donors via a real-time quantitative polymerase chain reaction. Serum samples from 20 HCV antibody-positive patients were used to detect each individual PBMCs expressed antigens via enzyme-linked immunosorbent assay. Two groups, five Swiss albino mice each, were immunized with the EC or a control construct. The absolute count of lymph nodes' CD4⁺ and CD8⁺ T-lymphocytes was assessed. Results: Donors' PBMCs showed different levels of EC expression, ranging between 0.83-2.61-fold in four donors, while donor-3 showed 34.53-fold expression. The antigens expressed in PBMCs were significantly reactive to the 20 HCV antibody repertoire (all p=0.0001). All showed comparable reactivity except for donor-3 showing the lowest reactivity level. The absolute count % of the CD4⁺ T-cell significantly increased in four of the five EC-immunized mice compared to the control group (p=0.03). No significant difference in CD8⁺ T-cells % was observed (p=0.89). Conclusion: The inter-individual variation in antigen expression and processing dominance was evident, showing independence in individuals' antigen expression and reactivity levels to antibodies. The described vaccine candidate might result in a promising natural immune response with a possibility of CD4⁺ T-cell early priming.

• Journal of Periodontal and Implant Science
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• v.40 no.6
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• pp.265-270
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• 2010

Purpose: The aim of this study was to investigate the immunomodulatory effects of canine periodontal ligament stem cells on allogenic and xenogenic immune cells in vitro. Methods: Mixed cell cultures consisting of canine stem cells (periodontal ligament stem cells and bone marrow stem cells) and allogenic canine/xenogenic human peripheral blood mononuclear cells (PBMCs) were established following the addition of phytohemagglutinin. The proliferation of PBMCs was evaluated using the MTS assay. The cell division of PBMCs was analyzed using the CFSE assay. The apoptosis of PBMCs was assessed using the trypan blue uptake method. Results: Periodontal ligament stem cells and bone marrow stem cells inhibited the proliferation of allogenic and xenogenic PBMCs. Both periodontal ligament stem cells and bone marrow stem cells suppressed the cell division of PBMCs despite the existence of a mitogen. No significant differences in the percentages of apoptotic PBMCs were found among the groups. Conclusions: Canine periodontal ligament stem cells have an immunomodulatory effect on allogenic and xenogenic PBMCs. This effect is not a product of apoptosis of PBMCs but is caused by the inhibition of cell division of PBMCs.

• 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.