• IMMUNE NETWORK
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• v.9 no.5
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• pp.192-202
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• 2009

Background: Little information is available the role of Nitric Oxide (NO) in host defenses during human tuberculosis (TB) infection. We investigated the modulating factor(s) affecting NO synthase (iNOS) induction in human macrophages. Methods: Both iNOS mRNA and protein that regulate the growth of mycobacteria were determined using reverase transcriptase-polymerase chain reaction and western blot analysis. The upstream signaling pathways were further investigated using iNOS specific inhibitors. Results: Here we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-${\gamma}$ synergistically enhanced NO synthesis and iNOS expression induced by Mycobacterium tuberculosis (MTB) or by its purified protein derivatives in human monocyte-derived macrophages. Both the nuclear factor-${\kappa}B$ and MEK1-ERK1/2 pathways were indispensable in the induction of iNOS expression, as shown in toll like receptor 2 stimulation. Further, the combined treatment with 1,25-D3 and IFN-${\gamma}$ was more potent than either agent alone in the inhibition of intracellular MTB growth. Notably, this enhanced effect was not explained by increased expression of cathelicidin, a known antimycobacterial effector of 1,25-D3. Conclusion: These data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.

• The Journal of Pediatrics of Korean Medicine
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• v.23 no.2
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• pp.29-50
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• 2009

Objectives : The purpose of this study is to investigate effectiveness of KamiCheongsimyeonjatang(KCSYJT) medicines to suppress atopic dermatitis in mouse model experimentally. Methods : First, in vitro, we isolated B cells from 18 weeks of atopicdermatitis-like skin NC/Nga mouse. Then we analyzed FACS(Fluorescence Activated Cell Sorter) by intracellular staining of IFN-$\gamma$, GATA-3+ analyzed cytokines by using real-time PCR. Secondly, in vivo, after administration of KCSYJT to atopic dermatitis NC/Nga mouse at 12 weeks of age, we analyzed serum IgE and the change of activated cell in PBMCs(Peripheral Blood Mononuclear Cells). Results : In vitro, KCSYJT medicines supressed IL-1$\beta$, IL-6, TNF-$\alpha$, and TGF-$\beta$ mRNA and increased IL-10 mRNA in B cells. Also, KCSYJT medicines decreased the levels of GATA-3$^+$CD4$^+$ and increased the levels of IFN-$\gamma^+$CD4$^+$T Cell. In vivo, serum IgE levels dreased in KCSYJT group than control group and In PBMCs, the activated cell percentage of granulocytes, CD3+, CD3+/CD4+, B220+/CD23+, and CCR3+ decreased and CD19+, CD3+/CD8+ increased in KCSYJT group than control group. Conclusions : This study demonstrates immunological activity of KCSYJT on atopic dermatitis-like model mice.

• Biomedical Science Letters
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• v.13 no.4
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• pp.325-332
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• 2007

Cell-mediated immune response (CMI) is a major immune protective mechanism against tuberculosis (TB) infection. Among several components involved in CMI, recent studies suggest that CD8+ T cells are important in controlling TB infection. In our previous report, we defined four Mycobacterium tuberculosis (MTB) derived epiotpe peptides specific for HLA-A*0201-restricted CD8+ T cells. These four peptides are $PstAl_{75-83}$, $ThyA_{30-38}$, $RpoB_{127-135}$ and $85B_{15-23}$. In this study, these epitope peptides specific CD8+ T cell responses in tuberculous pleurisy were investigated using ex vivo $IFN-\gamma$ elispot assay and intracellular $IFN-\gamma$ staining method. As a result, we observed these epitope peptide specific CD8+ T cell responses are induced in all three patients with tuberculous pleurisy suggesting that CD8+ T cells are involved in protective immune mechanism against MTB infection in tuberculous pleurisy. However, the CMI to mitogens and MTB antigens from pleural fluids of patients with tuberculous pleurisy does not seem to correlate with that from peripheral blood, although the sample size is too small to make any conclusion. In sum, the MHC I restricted CD8+ T cell responses seem to be induced efficiently in the pleural fluids, at the site of TB infection, in which the CMI is actively induced. In addition, these experiments suggest that MHC I restricted CD8+ T cell mediated immune responses are also involved in protective mechanism against MTB infection in extra-pulmonary TB.

• Korean Journal of Veterinary Research
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• v.49 no.2
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• pp.167-172
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• 2009

Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma (A.) phagocytophilum. Natural killer T (NKT) cells are key players in host defense against various microbial infections. We investigated the role of NKT cells in immune response to A. phagocytophilum infection using NKT-knockout ($J\alpha$18-/-) mice. $J\alpha$18-/- and wild-type (WT) mice were infected with low-passage A. phagocytophilum and assayed for hepatic histopathology and cytokine production during 7 days post-infection. Compared to WT controls, the infected $J\alpha$18 -/- mice had much less histopathologic lesions and less apoptosis through day 7, and lower concentrations of ${IFN\gamma}$ and IL- 12, but not of IL-10. This result suggests that NKT cells are major components in the pathogenesis of HGA.

• Tuberculosis and Respiratory Diseases
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• v.53 no.1
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• pp.5-16
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• 2002

Background : Though mononuclear phagocytes serve as the final effectors in killing intracellular Mycobacterium tuberculosis, the bacilli readily survive in the intracellular environment of resting cells. The mechanisms through which cellular activation results in the intracellular killing is unclear. In this study, we sought to explore an in vitro model of a low-level infection of human mononuclear phagocytes with MAC and $H_{37}Ra$ and determine the extent of the lymphocyte dependent cytotoxicity of human monocytes and alveolar macrophages. Materials and Methods : The peripheral monocytes were prepared using the Ficoll gradient method from PPD positive healthy people and tuberculosis patients. The alveolar macrophages were prepared from PPD positive healthy people via a bronchoalveolar lavage. The human mononuclear phagocytes were infected at a low infection rate (bacilli:phagocyte 1:10) with MAC(Mycobacterium avium) and Mycobacterium tuberculosis $H_{37}Ra$. Non-adherent cells(lymphocyte) were added at a 10:1 ratio. After 1,4, and 7 days culture in $37^{\circ}C$, 5% CO2 incubator, the cells were harvested and inoculated in a 7H10/OADC agar plate for the CFU assay. The bacilli were calculated with the CFU/$1{\times}10^6$ of the cells and the cytotoxicity was expressed as the log killing ratio. Results : The intracellular killing of MAC and $H_{37}Ra$ within the monocyte was greater in patients with tuberculosis compared to the PPD positive controls (p<0.05). Intracellular killing of MAC and $H_{37}Ra$ within the alveolar macrophage appeared to be greater than that within the monocytes of the PPD positive controls. There was significant lymphocyte dependent inhibition of intracellular growth of the mycobacteria within the monocytes in both the controls and tuberculosis patients and within the macrophages in the controls(p<0.05). There was no specific difference in the virulence between the MAC and the $H_{37}Ra$. Conclusion : This study is an in vitro model of a low-level infection with MAC and $H_{37}Ra$ of human mononuclear phagocytes. The intracellular cytotoxicity of the mycobacteria within the phagocytic cells was significantly lymphocyte dependent. During the 7 days culture after the intracellular phagocytosis, the actual confinement of the mycobacteria was observed within the monocytes of tuberculosis patients and the alveolar macrophages of the controls as in the case of adding lymphocytes.

• Biomolecules & Therapeutics
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• v.12 no.1
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• pp.25-33
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• 2004

In this study we investigated whether ATP loss was involved in the potentiated death of immunostimulated rat primary astrocytes in glucose-deprived condition. Rat primary astrocytes immunostimulated with LPS plus IFN-${\gamma}$ for 48 h underwent death upon glucose deprivation, which dependent on the production of peroxynitrite. Intracellular ATP level synergistically decreased by glucose deprivation in immunostimulated astrocytes but not in control cells, and the loss of ATP occurred well ahead of the LDH release. The synergistic cell death and ATP loss by immunostimulation and glucose deprivation were inhibited by iNOS inhibitor (L-NAME and L-NNA) or peroxynitrite decomposition catalyst (also a superoxide anion scavenger), Mn(III)tetrakis(N-methyl-4'-pyridyl)porphyrin (MnTMPyP). Exogenous addition of peroxynitrite generator, SIN-l timedependently induced ATP loss and cell death in the glucose-deprived astrocytes. Depletion of intracellular glutathione (GSH) and dis겨ption of mitochondrial transmembrane potential (MTP) were also observed under same conditions. Supply cellular ATP by the addition of exogenous adenosine or ATP during glucose deprivation inhibited ATP depletion, GSH depletion, MTP disruption and cell death in SIN-l treated or immunostimulated astrocytes. This study showed that perturbation in the regulation of intracellular ATP level in immunostimulated astrocytes might make them more vulnerable to energy challenging stimuli.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.45 no.1
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• pp.57-67
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• 2019

Particulate matters with a diameter of < $10{\mu}m$ (PM10) exert oxidative stress and inflammatory events in various organs. The purpose of this study was to examine the molecular mechanism of reactive oxygen species (ROS) production induced by PM10 in the human epidermal keratinocytes (HEKs). When cultured HEKs were exposed to PM10, ROS production was induced and it was inhibited by apocynin, an antioxidant. The mRNA expression of NADPH oxidase (NOX) family was analyzed in order to examine their role in PM10-induced ROS production. PM10 increased the mRNA expression of NOX1, NOX2, dual oxidase (DUOX) 1 and DUOX2. HEKs expressed DUOX1 and DUOX2 at higher levels compared to other NOXs. The mRNA expression of dual oxidase maturation factors, DUOXA1 and DUOXA2, was also increased by PM10. We examined whether these calcium-dependent enzymes, DUOX1 and DUOX2, mediate the PM10-induced ROS production. A selective intracellular calcium chelator, BAPTA-AM, attenuated ROS production induced by PM10 or calcium ionophore A23187. The small intereference RNA (siRNA)-mediated down-regulation of DUOX2, but not DUOX1, attenuated the ROS production induced by PM10. PM10 increased the expression of inflammatory cytokines such as interleukin $(IL)-1{\beta}$, IL-6, IL-8 and interferon $(IFN)-{\gamma}$. SiRNA-mediated down-regulation of DUOX2 suppressed the PM10-induced expression of $IFN-{\gamma}$ but not other cytokines. This study suggests that DUOX2 plays a crucial role in ROS production and inflammatory response in PM10-exposed keratinocytes.

• IMMUNE NETWORK
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• v.11 no.6
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• pp.416-419
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• 2011

Dextran sodium sulfate (DSS) is a widely used chemical model for inflammatory bowel disease (IBD). It is thought that imbalances in the T helper (Th) cell subsets contribute to IBD. Recent studies suggest that the acute DSS-colitis model is polarized toward a Th1/Th17 profile based on RT-PCR analysis of colonic tissues. In the current study we determined whether colonic Th cells from DSS-colitis mice were skewed toward the Th17 profile. Mice were treated with 5% DSS for 7 days and colonic T cells isolated and examined for production of IFN-${\gamma}$ (Th1 cell), IL-4 (Th2 cell) and IL-17 (Th17 cell) by intracellular flow cytometry. We found that the percentage of colonic Th17 cells were similar to non-treated controls but the percentage of Th1 cells were elevated in DSS-colitis mice. These results suggest that in the acute DSS-colitis model the colonic Th cells exhibit a Th1 profile and not a Th17 profile.

• Parasites, Hosts and Diseases
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• v.54 no.1
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• pp.31-38
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• 2016

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked $IFN-{\gamma}$. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, $5{\mu}M$) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine ($5{\mu}M$) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, $20{\mu}M$) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, $10{\mu}M$) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.

• IMMUNE NETWORK
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• v.11 no.5
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• pp.268-280
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• 2011

Background: Dengue virus, which belongs to the Flavivirus genus of the Flaviviridae family, causes fatal dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with infection risk of 2.5 billion people worldwide. However, approved vaccines are still not available. Here, we explored the immune responses induced by alternating prime-boost vaccination using DNA vaccine, adenovirus, and vaccinia virus expressing E protein of dengue virus type 2 (DenV2). Methods: Following immunization with DNA vaccine (pDE), adenovirus (rAd-E), and/or vaccinia virus (VV-E) expressing E protein, E protein-specific IgG and its isotypes were determined by conventional ELISA. Intracellular CD154 and cytokine staining was used for enumerating CD4+ T cells specific for E protein. E protein-specific CD8+ T cell responses were evaluated by in vivo CTL killing activity and intracellular IFN-${\gamma}$ staining. Results: Among three constructs, VV-E induced the most potent IgG responses, Th1-type cytokine production by stimulated CD4+ T cells, and the CD8+ T cell response. Furthermore, when the three constructs were used for alternating prime-boost vaccination, the results revealed a different pattern of CD4+ and CD8+ T cell responses. i) Priming with VV-E induced higher E-specific IgG level but it was decreased rapidly. ii) Strong CD8+ T cell responses specific for E protein were induced when VV-E was used for the priming step, and such CD8+ T cell responses were significantly boosted with pDE. iii) Priming with rAd-E induced stronger CD4+ T cell responses which subsequently boosted with pDE to a greater extent than VV-E and rAd-E. Conclusion: These results indicate that priming with live viral vector vaccines could induce different patterns of E protein-specific CD4+ and CD8+ T cell responses which were significantly enhanced by booster vaccination with the DNA vaccine. Therefore, our observation will provide valuable information for the establishment of optimal prime-boost vaccination against DenV.