• Investigative Magnetic Resonance Imaging
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• v.2 no.1
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• pp.73-82
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• 1998

Purpose : A precise NMR technique for measuring the rate of water exchange and cell membrane permeability across the hepatocyte membrane using liver-specific MR contrast agent is described. Materials and Methods : The rat hepatocytes isolated by perfusion of the livers were used for the NMR measurements. All experiments were performed on an IBM field cycling relaxometer operating from 0.02MHz to 60 MHz proton Larmor frequency. spin-echo pulse sequence was empolyed to measure spin-lattice relaxation time, T1. The continuous distribution analysis of water proton T1 data from rat hepatocytes containing low concentrations of the liver specific contrast agent, Gd-EOB-DTPA, modeled by a general two compartment exchange model. Results : The mean residence time of water molecule inside the hepatocyte was approximately 250 msec. The lower limit for the permeability of the hepatocyte membrane was $(1.3{\pm}0.1){\;}{\times}{\;}10^{-3}cm/sec$. The CONTIN analysis, which seeks the natural distribution of relaxation times, reveals direct evidence of the effect of diffusive exchange. the diffusive water exchange is not small in the intracellular space in the case of hepatocytes. Conclusions : Gd-EOB-DTPA, when combined with continuous distribution analysis, provides a robust method to study water exchange and membrane permeability in hepatocytes. Water exchange in hepatocyte is much slower thatn that in red blood cells. Therefore, tissue-specific contrast agent may be used as a functional agent to give physiological information such as cell membrane permeability.

• Environmental Mutagens and Carcinogens
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• v.21 no.2
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• pp.164-168
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• 2001

Abel et al. in Germany discovered a new dioxin-responsive gene, which has later been identified as rpt-1 (regulatory protein T-lymphocyte 1). While it is speculated that rpt-1 may play a role in signal transduction and carcinogenesis, its roles and functions remain unknown. The present study attempted to analyze functions of rpt-1 in human epithelial cells following the xenobiotic exposures. While German counterpart analyzed expressionn of rpt-1 in spleen and thymus cells from mouse and rat and characterizes molecular properties of the gene, our work mainly focused on analyzing function of rpt-1 in human skin cells. Expression of rpt-1 in human cells were analyzed by western and northern blot RT-PCR analysis. Expression of rpt-1 as well as Staf-50 in human cells with or without exposure to environmental pollutants were also analyzed by northern blot analysis, since Staf-50 is homologous with rpt-1 and found in human cells. To help study roles of rpt-1 in human cell system, retroviral vector system carrying rpt-1 gene under the CMV promoter were constructed and transfected. Cells overexpressing the gene after the transfection showed an increase of cell density and soft agar colony formations, as compared to the control cells, suggesting that rpt-1 may play a certain role in the transformation processes of human cells. While the expression of rpt-1 in spleen and thymus is known to be strong in the laboratory animals, both the basal and TCDD-induced expression of rpt-1 in the current cellular system remained insignificant. It is speculated that the expression pattern of rpt-1 may be tissue- and species-specific. The present study demonstrated a strong expression of rpt-1 protein in the brain of SD rat model. Since there is no previous report on the expression of rpt-1 in the brain tissue, the result may play a significant role in understanding dioxin-induced neurotoxicities in the future. The present study provides an opportunity to understand a role of rpt-1 in human cell system and suggest a possible lead and basis for the future study of dioxin-induced neurotoxicities.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.87-94
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• 2017

Tissue engineering (TE) has been developed to create functional organs and tissue by combining 3D matrix and cells in vitro. Vascularization and angiogenesis are utmost important for supply of nutrients and oxygen in tissue engineered organs. The present study was performed to isolate and characterize primary endothelial cells (EC) from aorta of alpha 1, 3-enzyme galactosyltransferase knock out (GalT KO) pig, to minimize immune rejection and analyze body immune system for future xenotransplantation studies. Isolation of primary EC from aorta were performed by incubation with dispase for 8-10 min at $37^{\circ}C$. Primary EC were cultured in EC growth medium on different extra cellular matrix (ECM), either collagen or gelation. Primary EC exhibits morphological characteristics and showed positive expressions of EC specific marker proteins i.e. PECAM1, KDR and VWF despite of their ECM surface; however, on collagen based surface they showed increase in mRNA level analyzed by qPCR. Primary EC cultured on collagen were sorted by flow cytometer using KDR marker and cultured as KDR positive cells and KDR negative cells, respectively. KDR positive cells showed dramatically increased in PECAM1 and VWF level as compared to KDR negative cells. Based on the above results, primary EC derived from GalT KO are successfully isolated and survived continuously in culture without becoming overgrown by fibroblast. Therefore, they can be utilize for xeno organ transfer, tissue engineering, and immune rejection study in future.

• Journal of Acupuncture Research
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• v.22 no.2
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• pp.71-81
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• 2005

Objective : Dear antler (Cervus korean TEMMINCK var. mantchuricus Swinhoe) used for traditional immunosuppressive and immuno-activating action. The effect of deer antler herbal-acupuncture(DAH) solution, prepared by water extract method, on cathepsin activities in bone tissues (cartilage and synovial) cells from mouse rheumatoid arthritis (RA) model was studied. The cysteine endoprotease cathepsin mediates degradation of the MHC class II invariant chain (Ii) in human and mouse antigen-presenting cells. The studies described here examine the functional significance of cathepsin inhibition on autoantigen presentation and organ-specific autoimmune diseases in a murine model for RA. Methods : An animal model for RA in BALB/c mice thymectomized 3 days after birth (3d-Tx) was constructed All 3d-Tx BALB/c mice developed autoimmune lesions in the bone tissue cells, starting at 3 weeks of age, and the disease mediated by CD4+ T cells was chronic and progressive. Significant inhibitory effects of DAH solution on cathepsin S and L were observed in each organ in a dose-dependent manner. Moreover, we confirmed that cathepsin S and L activity in each organ were clearly inhibited by DAB solution. When we examined the inhibitory effects of DAH solution against autoantigen-specific T cell responses in vitro, in regional lymph node cells, but not in spleens, from model mice, a significant inhibitory effect of DAB solution was observed in a dose-dependent manner. DAH solution do not block T cell proliferation to Con A, indicated that the dose of DAB solution 10 to $20\;{\mu}g/m{\ell}$ was sufficient to inactivate the autoantigen-specific T cell responses in vitro. In vivo therapeutic effects of DAB solution were examined in a murine model for RA, autoantigen-specific (C-II-specific) T cell response were significantly inhibited in LNCs from DAH solution-treated mice. Results : Iinhibition of cathepsin S and L in vivo alters autoantigen presentation and development of organ-specific autoimmunity in RA model. Conclusion : These data identify selective inhibition of cysteine protease cathepsin S and L as a potential therapeutic strategy for autoimmune disease process such RA. Thus, DAH solution will served as a potent anti-inflammatory and anti-arthritic agents for treatment of human RA.

• Medical Lasers
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• v.9 no.2
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• pp.134-141
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• 2020

The use of stem cell therapy to treat various diseases has become a promising approach. The ability of stem cells to self-renew and differentiate can contribute significantly to the success of regenerative medical treatments. In line with these expectations, there is a great need for an efficient research methodology to differentiate stem cells into their specific targets. Photobiomodulation (PBM), formerly known as low-level laser therapy (LLLT), is a relatively non-invasive technique that has a therapeutic effect on damaged tissue or cells. Recent advances in adapting PBM to stem cell therapy showed that stem cells and progenitor cells respond favorably to light. PBM stimulates different types of stem cells to enhance their migration, proliferation, and differentiation in vitro and in vivo. This review summarizes the effects of PBM on targeted differentiation across multiple stem cell lineages. The analytical expertise gained can help better understand the current state and the latest findings in PBM and stem cell therapy.

• Archives of Reconstructive Microsurgery
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• v.21 no.1
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• pp.34-40
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• 2012

Prevention of acute rejection in composite tissue allotransplantation without continuous immunosuppression lacks reports in worldwide literature. Recently dendritic cells (DC) gained considerble attention as antigen presenting cells that are also capable of immunologic tolerance induction. This study assesses the effect of alloantigen-pulsed dendritic cells in induction of survival in a rat hindlimb allograft. We performed hindlimb allotransplantation between donor Sprague-Dawley and recipient Fischer344 rats. Recipient derived dendritic cells were harvested from rat whole blood and cultured with anti-inflammatory cytokine IL-10. Then donor-specific alloantigen pulsed dendritic cells were reinjected into subcutaneous tissue before limb transplantation. Groups: I) untreated (n=6), II) DC injected (n=6), III) Immunosuppressant (FK-506, 2 mg/Kg) injected (n=6), IV) DC and immunouppressant injected (n=6). Graft appearance challenges were assessed postoperatively. Observation of graft appearance, H-E staning, immunohistochemical (IHC) study, and confocal immunofluoreiscece were performed postoperatively. Donor antigen pulsed host dendritic cell combined with short-term immunosuppression showed minimal mononuclear cell infiltration, regulator T cell presence, and could prolong limb allograft survival.

• Molecules and Cells
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• v.38 no.5
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• pp.426-431
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• 2015

Odin has been implicated in the downstream signaling pathway of receptor tyrosine kinases, such as the epidermal growth factor and Eph receptors. However, the physiologically relevant function of Odin needs to be further determined. In this study, we used Odin heterozygous mice to analyze the Odin expression pattern; the targeted allele contained a ${\beta}$-geo gene trap vector inserted into the 14t intron of the Odin gene. Interestingly, we found that Odin was exclusively expressed in ependymal cells along the brain ventricles. In particular, Odin was highly expressed in the subcommissural organ, a small ependymal glandular tissue. However, we did not observe any morphological abnormalities in the brain ventricles or ependymal cells of Odin null-mutant mice. We also generated BAC transgenic mice that expressed the PTB-deleted Odin (dPTB) after a floxed GFP-STOP cassette was excised by tissue-specific Cre expression. Strikingly, Odin-dPTB expression played a causative role in the development of the hydrocephalic phenotype, primarily in the midbrain. In addition, Odin-dPTB expression disrupted proper development of the subcommissural organ and interfered with ependymal cell maturation in the cerebral aqueduct. Taken together, our findings strongly suggest that Odin plays a role in the differentiation of ependymal cells during early postnatal brain development.

• IMMUNE NETWORK
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• v.1 no.1
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• pp.61-69
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• 2001

Background: To determine the molecular structure of type II collagen-specific T-cell receptors associated with rheumatoid arthritis (RA). Methods: We generated CII-specific T-cell lines of 8 RA patients by prolonged in vitro culture with bovine CII (bCII) and the immunogenic peptide (256-270) of human CII. The proliferation response towards CII stimulation was measured from the uptake of 3H-thymidine. Changes in the secretion of Th 1 and Th2 cytokines in the culture supernatent were measured by ELISA. The TCR clonotypes of these T-cells were examined by RT-PCR/SSCP analyses of all 22 $V_{\beta}$ chains. Results: T-cells from patients' tissue exhibited strong proliferation index upon CII stimulation, which was maintained up to 6 months in the culture. The secretion of INF-$\gamma$from these T-cells increased along with the duration of culture time, while the amount of IL-4 production did not show significant changes. The SSCP band patterns of patients' T-cells appear as discrete bands unlike the smeary streak produced from normal samples. Some SSCP bands, each representing selected expansion of a TCR containing certain subtype of $V_{\beta}$ peptides, appeared to be identical in more than one patients. Among these, the expansion of SSCP band representing the $V_{\beta}$ 14 CDR3 region persisted after switching the antigen to the immunogenic human peptide (256-270). Conclusion: CII-reactive T-cells expressing distinct CDR3 motifs are selectively expanded in the peripheral blood and synovial fluid of RA patients, and their persistent proliferation upon CII stimulation, as well as the production Th 1-type cytokines, may play pivotal roles in RA pathogenesis.

• Journal of Life Science
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• v.18 no.10
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• pp.1348-1354
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• 2008

mi transcription factor (MITF) is important in regulating the differentiation of mast cells. In particular, MITF regulates the transcription of the mouse mast cell-specific serine protease (mMCP)-6 gene, which is generally expressed by the connective tissue-type of mast cells. In this study, we investigated alternative isoforms of MITF that regulate transcription of the mMCP-6 gene in bone marrow-derived cultured mast cells in mice. The expression of MITF isoforms was examined by RT-PCR. We observed that MITF-A, -E, -H and -Mc were expressed by mucosal-type mast cells cultured in the presence of IL-3, whereas the connective tissue-type mast cells cultured in the presence of stem cell factor (SCF) expressed MITF-A. Overexpression of MITF isoforms increased luciferase activity through the mMCP-6 promoter in NIH-3T3 cells and elevated the level of mMCP-6 expression in the MC/9 mast cell line. Moreover, mMCP-6 expression in mast cells was significantly inhibited by the depletion of MITF. The transcriptional activity and DNA binding of MITF-A was comparable to that of MITF isoforms, including MITF-E, -H, and -Mc. Our results therefore suggest that MITF-A may be an important isoform of MITF in regulating the transcription of mMCP-6 in mouse connective tissue mast cells.

• Food Science and Biotechnology
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• v.18 no.1
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• pp.84-88
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• 2009

Cathepsin S is a cysteine protease that affects extracellular matrix remodeling. Recently, several studies have reported that cathepsin S is involved in obesity. Both mouse and human adipose cells produce this enzyme in the early phase of adipocyte differentiation, where it degrades fibronectin. Cathepsin S gene expression is elevated in the adipose tissue of obese mice as compared to that of lean mice. Paecilomyces tenuipes water extracts (PTW) are shown to have an inhibitory effect on cathepsin S activity. In this study, Z-Val-Val-Arg-MCA was used as a cathepsin S-specific substrate in order to examine inhibitory effect of PTW. Supplementing 3T3-L1 cell media with PTW clearly reduced lipid droplet accumulation and cathepsin S-induced adipogenesis. Furthermore, PTW decreased weight gain, subcutaneous adipose tissue growth, the level of serum triglyceride, and total cholesterol in mice fed a high-fat diet. These data suggest that PTW work against adipose cathepsin S and presumably contribute to anti-obese activities.