• Korean Journal of Microbiology
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• v.50 no.2
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• pp.169-172
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• 2014

Recently, Hepatitis C virus (HCV) replication system has been established using human hepatoma cells (huh cell) and a variety of HCV clones. In this study, we established an infectious HCV replication system using huh7.5 cells and J6/JFH1 clone (genotype 2a). In addition, we investigated the antigen presentation capability of HCV-infected huh7.5 cells to HCV-specific T cells. Interestingly, HCV-infected huh7.5 cells were not capable of activating HCV-specific T cells. However, huh7.5 cells stimulated by exogenous HCV peptide were able to activate HCV-specific T cells, which was shown to produce TNF-${\alpha}$ and IFN-${\gamma}$. We further examined if HCV infection has an inhibitory effect on the expression of MHC class I molecule of huh7.5 cells. We found that HCV infection did not change the expression level of MHC class I molecule on huh7.5 cells.

• Korean Journal of Clinical Laboratory Science
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• v.54 no.1
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• pp.38-48
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• 2022

Carcinoembryonic antigen (CEA) is an oncofetal antigen primarily detected in the peripheral blood of cancer patients, particularly in those with colorectal cancer. CEA is considered a valuable target for antigen-specific immunotherapy. In this study, we induced the anti-tumor immunity for CEA through the administration of a dendritic cell (DC) vaccine. However, there was a limitation in inducing tumor regression in the DC vaccinated mice. To enhance the efficacy of anti-tumor immunity in MC38/CEA2 tumor-bearing mice, we evaluated the effects of DC vaccine in combination with cyclophosphamide (CYP). Administration of CYP 100 mg/kg in mice resulted in significant inhibition of tumor growth in the 2-day tumor model, whereas a lower inhibition of tumor growth was seen in the 10-day tumor model. Therefore, the 10-day tumor model was selected for testing chemo-immunotherapy. The combined CYP and DC vaccine not only increased tumor antigen-specific immune responses but also induced synergistic anti-tumor immunity. Furthermore, the adverse effects of CYP such as weight loss and immunosuppression by regulatory T cells and myeloid-derived suppressor cells showed a significant reduction in the combined chemo-immunotherapy treatment compared with CYP alone. Our data suggest that chemoimmunotherapy with the DC vaccine may offer a new therapeutic strategy to induce a potent anti-tumor effect and reduce the adverse effects of chemotherapy.

• IMMUNE NETWORK
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• v.2 no.1
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• pp.35-40
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• 2002

Background: CTLA-4 (Cytotoxic T Lymphocyte associated Antigen 4, CD152) has been known as a homologue of CD28, an accessory molecule providing a key costimulatory signal for successful antigen-driven activations of T lymphocyte. Most of biochemical and cell biological characteristics of the CD152 protein remain unknown while those of CD28 have been characterized in detail. Methods: In this study CD152 expression in both $CD4^+$ and $CD8^+$ PBLs was studied by using flow cytometry. And intracellular CD152 multiprotein complex was purified and used for generating antibodies recognizing proteins composing of intracellular CTLA-4 multi protein complex. Results: Level of surface expression of this molecule was peaked at 2 days of PHA stimulation in flow cytometric analysis. 40~45% of PHA blast cells were $CD152^+$ in both of two subsets at this stage and the level of expression were equivalent in both two subsets. Contrary to this surface expression, intracellular expression was peaked at day 3 and it was preferentially induced in $CD8^+$ cells and about 60% of $CD8^+$ cells were $CD152^+$ at this stage. High molecular weight (>350 kD) intacellular CD152 protein complex purified by using preparative electrophoresis were immunized into rabbits and then 3 different anti-P34PC4, anti-P34PC7 and anti-P34PC8 antibodies were obtained. Using these 3 antibodies two unknown antigens associated with intracellular CD152 multiprotein complex were found and their molecular weights were 54 kD and 75 kD, respectively. Among these, the former was present as 110 kD homodimer in non-reducing condition. Conclusion: It seemed that 34 kD intracellular CD152 molecule forms high molecular weight multiprotein complex at least with 2 proteins of 75 kD monomer and 110 kD homodimer.

• Parasites, Hosts and Diseases
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• v.26 no.2
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• pp.73-86
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• 1988

The sensitivity and specificity of crude and affinity-purified antigens of Clcnorchis sinensis obtained from the infected rabbits were studied. Stage-specific antigenic proteins from the eggs, metacercariae and adult worms were characterized by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent astray (ELISA). The results were as follows: 1. The antibody.binding antigen (ABA) purified from whole worm crude antigen (IVWA) by CNBr-activated Sepharose 4B affinity chromatography made :l specific bands against rabbit antisera on Ouchterlony gel diffusion plate, while WWA made 7 bands. Major WWA protein bands by SDS-PAGE were found at 16, 300~18, 500 and 28, 000~29, 000 daltons, while major ABA protein bands were at 18, 000~21, 000 and 29, 000~31, 000 daltons. The reactivity of ABA with rabbit anti-sera in ELISA was remarkably less sensitive than that of WWA. 2. Molecular weights of egg antigen (EGA), metacercarial antigen (MEA) and adult worm antigen (WWA) of C. sinensis ranged from 15, 000-200, 000 daltons, 15, 000-100, 000 daltons and 11, 000~80, 000 daltons, respectively. Major WWA proteins consisted mainly of polypeptide bands of low molecular weight, less than 31, 000 daltons, while those of EGA and MEA consisted of higher molecular T.eights than 30, 000 daltons. 3. The ELISA reactivities of WWA to rabbit anti.sera were remarkably greater than those of MEA. EGA showed negative reaction throughout the experiments. WWA showed higher optical density (O.D.) than 1.0, when reacted with rabbit anti-sera obtained at 4~6 weeks after the infection. In the rabbit anti-sera later than 12 weeks after the infection, the O.D. reacting witll WWA showed a plateau without variation. MEA shoT.ed relatively low O.D. values (<0.6), when reacted with anti-sera from lightly in(ected groups throughout the experiments, althougll there were some wealth positive cases (O.D.>0.6) ill heavily infected groups. MEA reacted with rabbit anti-sera showed negative results on Ouchterlony gel diffusion plates. Summarizing the above results, it is suggested that the whole worm antigen prepared from the adult worms of C. sinensis is most highly antigenic. However, this antigen might reveal cross reactions with other trematodes such as Paragonimus westermani, therefore, purification of antigenic proteins from the crude antigen is essential 18 increase the sensitivity and specificity for the immuncdiagnosis of clonorchiasis.

• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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• pp.62-64
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• 2008

A major hurdle to the development of RNA interference as therapy for HIV infection is the delivery of siRNA to T lymphocytes which are difficult cells to transfect even in vitro. We have employed a single chain antibody to the pan T cell surface antigen CD7 was conjugated to an oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2${\gamma}$-/- mice reconstituted with human peripheral blood lymphocytes (Hu-PBL). Using a novel delivery, we first show that scFvCD7-9R efficiently delivered CD4 siRNA into human T cells in vitro. In vivo administration to Hu-PBL mice resulted in reduced levels of surface CD4 expression on T cells. Mice infected with HIV-1 and treated on a weekly basis with scFvCD7-9R-siRNA complexes targeting a combination of viral genes and the host coreceptor molecule CCR5 successfully maintained CD4/CD3 T cell ratios up to 4 weeks after infection in contrast to control mice that displayed a marked reduction in CD4 T cell numbers. p24 antigen levels were undetectable in 3 of the 4 protected mice. scFvCD7-9R/antiviral siRNA treatment also helped maintain CD4 T cell numbers with reduced plasma viral loads in Hu-PBL mice reconstituted with PBMC from donors seropositive for HIV, indicating that this method can contain viral replication even in established HIV infections. Our results show that scFvCD7-9R could be further developed as a potential therapeutic for HIV-1 infection.

• Parasites, Hosts and Diseases
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• v.55 no.2
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• pp.143-148
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• 2017

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.511-518
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• 2011

To avoid hyperacute rejection of xenografts, ${\alpha}1,3$-galactosyltransferase knock-out (GalT KO) pigs have been produced. In this study, we examined whether Sia-containing glycoconjugates are important as an immunogenic non-Gal epitope in the pig liver with disruption of ${\alpha}1,3$-galactosyltransferase gene. The target cells were then used as donor cells for somatic cell nuclear transfer (scNT). A total of 1,800 scNT embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. Real-time RT-PCR and glycosyltransferase activity showed that ${\alpha}2,3$-sialyltransferase (${\alpha}2,3ST$) and ${\alpha}2,6$-sialyltransferase (${\alpha}2,6ST$) in the heterozygote GalT KO liver have higher expression levels and activities compared to controls, respectively. According to lectin blotting, sialic acidcontaining glycoconjugate epitopes were also increased due to the decreasing of ${\alpha}$-Gal in heterozygote GalT KO liver, whereas GalNAc-containing glycoconjugate epitopes were decreased in heterozygote GalT KO liver compare to the control. Furthermore, the heterozygote GalT KO liver showed a higher Neu5Gc content than control. Taken together, these finding suggested that the deficiency of GalT gene in pigs resulted in increased production of Neu5Gc-bounded epitopes (H-D antigen) due to increase of ${\alpha}2,6$-sialyltransferase. Thus, this finding suggested that the deletion of CMAH gene to the GalT KO background is expected to further prolong xenograft survival.

• IMMUNE NETWORK
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• v.13 no.2
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• pp.63-69
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• 2013

IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membranebound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-${\alpha}$. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the $CD8^+$ T cell-depleted mice than in $CD4^+$ T cell-depleted or normal mice. These results suggest that $CD8^+$ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and $CD4^+$ helper T cells.

• YAKHAK HOEJI
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• v.51 no.6
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• pp.482-489
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• 2007

Kaempferitrin, isolated from Kenaf (Hibiscus cannabinus), was examined to evaluate its modulatory effects on antigen-presenting cell functions of macrophages/monocytes such as phagocytosis of foreign materials, up-regulation of costimulatory molecules (CD40, CD80 and CD86), adhesion molecule activation, and antigen processing and presentation. Kaempferitrin strongly blocked up-regulation of CD40, CD80 and CD86, but not pattern recognition receptor (PRR) (e.g., TLR2). It also suppressed functional activation of CD29 (${\beta}1$-integrins), as assessed by cell-cell adhesion assay, required for T cell-antigen-presenting cell (APC) interaction. Furthermore, this compound did not block a simple activation of CD29, as assessed by cell-fibronectin adhesion assay. However, the compound did not diminish phagocytic uptake, an initial step for antigen processing, and ROS generation in RAW264.7 cells. In particular, to understand molecular mechanism of kaempferitrin-mediated inhibition, the regulatory role of LPS-induced signaling events was examined using immunoblotting analysis. Interestingly, this compound dose dependently suppressed the phosphorylation of $I{\kappa}B{\alpha}$, Src, Akt and Syk, demonstrating that it can negatively modulate the activation of these signaling enzymes. Therefore, our data suggested that kaempferitrin may be involved in regulating APC function-relevant immune responses of macrophages and monocytes by regulating intracellular signaling.

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.82-83
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• 2003

In the past it was thought that autoimmunity is mediated by antibodies and immune complexes. It has now become clear that many diseases, especially tissue specific, are T cell mediated or at least T cell dependent. The pathogenesis of cell-mediated autoimmune diseases, such as multiple sclerosis, uveitis, diabetes, arthritis, and others, is thought to be in a large measure driven by interferon-gamma-producing antigen-specific T cells polarized toward the Th1 phenotype. (omitted)