• Animal cells and systems
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• v.8 no.2
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• pp.125-133
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• 2004

We have extended our previous work that cross-linking CD4 molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$We have extended our previous work that cross-linking CD$4^+$ molecules using specific MAb induced antigen nonspecific, MHC unrestricted killing of virally infected target cells by CD$4^+$ T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD$4^+$T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4 molecules. The CD$4^+$cross-linking failed to induce effector cell proliferation or the transcription of TNF${\beta}$ Upregulation of TNF${\beta}$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF${\beta}$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased p$56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.T cells. The killing activity of antibody activated CD$4^+$T cells was completely blocked by herbimycin A, a protein tyrosine kinase (PTK) inhibitor, but not by bisindolylamaleimide, a protein kinase C (PKC) inhibitor. Herbimycin A treated human or bovine peripheral blood CD4T cells lacked PTK activity and failed to kill virally infected target cells even after cross-linking of CD4molecules. The CD4 cross-linking failed to induce effector cell proliferation or the transcription of TNF$\beta$. Upregulation of TNF$\beta$ was induced by incubating the antibody activated effector cells with BHV-1 infected D17 target cells for 10 h. Anti-TNF$\beta$ antibody partially abolished (13-44%) the direct effector cell-mediated antiviral cytotoxicity. However, this antibody neutralized 70 to 100% of antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on the effector and target cell ratio. These results support the hypothesis that increased $56^ICK enzyme activity in effector cells transduces a signal critical for effector cell recognition of viral glycoproteins expressed on the target cells. Following target cell recognition, lytic cytokines known to participate in target cell killing were produced. A better understanding of the killing activity displayed by CD$4^+$T lymphocytes following surface receptor cross-linking will provide insight into the mechanisms of cytotoxic activity directed toward virally-infected cells.

• IMMUNE NETWORK
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• v.13 no.5
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• pp.194-198
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• 2013

Recent papers have shown that the initial event in the pathogenesis of autoimmune type 1 diabetes (T1D) comprises sensing of molecular patterns released from apoptotic ${\beta}$-cells by innate immune receptors such as toll-like receptor (TLR). We have reported that apoptotic ${\beta}$-cells undergoing secondary necrosis called 'late apoptotic' ${\beta}$-cells stimulate dendritic cells (DCs) and induce diabetogenic T cell priming through TLR2. The role of other innate immune receptors such as TLR7 or TLR9 in the initiation of T1D has also been suggested. We hypothesized that TLR2 blockade could inhibit T1D at the initial step of T1D. Indeed, when a TLR2 agonist, $Pam3CSK_4$ was administered chronically, the development of T1D in nonobese diabetic (NOD) mice was inhibited. Diabetogenic T cell priming by DCs was attenuated by chronic treatment with $Pam3CSK_4$, indicating DC tolerance. For the treatment of established T1D, immune tolerance alone is not enough because ${\beta}$-cell mass is critically reduced. We employed TLR2 tolerance in conjunction with islet transplantation, which led to reversal of newly established T1D. Dipeptidyl peptidase 4 (DPP4) inhibitors are a new class of anti-diabetic agents that have beneficial effects on ${\beta}$-cells. We investigated whether a combination of DPP4 inhibition and TLR2 tolerization could reverse newly established T1D without islet transplantation. We could achieve normoglycemia by TLR2 tolerization in combination with DPP4 inhibition but not by TLR2 tolerization or DPP4 inhibition alone. ${\beta}$-cell mass was significantly increased by combined treatment with TLR2 tolerization and DPP4 inhibition. These results suggest the possibility that a novel strategy of TLR tolerization will be available for the inhibition or treatment of established T1D when combined with measures increasing critically reduced ${\beta}$-cell mass of T1D patients such as DPP4 inhibition or stem cell technology.

• Journal of Life Science
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• v.10 no.1
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• pp.57-63
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• 2000

${\beta}$-catenin, which plays a critical role in both the cytoskeleton and in transcriptional regulation in variousadherent cell types, undergoes degradation during adherent cell apoptosis. Although ${\beta}$-catenin has been reported to be present in Jurkat T-acute lymphoblastic leukemia cells, the regulation of ${\beta}$-catenin in hematologic malignancies have not been examined. The data presented here demonstrate that treatment of the T cell leukemia Jurkat iwht the apoptosis inducer anti-Fas induced proteolytic cleavage of ${\beta}$-catenin. ${\beta}$-catenin was cleaved at both the N- and C-terminus after anti-Fas treatment. Cleavage of intact ${\beta}$-catenin was completely inhibited by caspase selective protease inhibitors. These data demonstrate that ${\beta}$ -catenin proteolysis is triggered by the cross-linking of the Fas receptor on Jurkat cells and subsequent activation of caspase protease. There was a clear accumulatio of the large proteolytic fragment in Jurkat cells treated with lactacystin of ALLM. These are potent inhibitors of proteasome and calpain. these results suggest that both the proteasome and clapain may recognize the large ${\beta}$-catenin fragment as a substrate fot further degradation and that these pathewasy may act downstream of scapase in response to Fas receptor activation. Therefore, we suggest that ${\beta}$-catenin may play a role in promoting Jurkat survival.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.878-885
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• 2017

Objective: Glucose is an essential fuel in the energy metabolism and synthesis pathways of all mammalian cells. In lactating animals, glucose is the major precursor for lactose and is a substrate for the synthesis of milk proteins and fat in mammary secretory (alveolar) epithelial cells. However, clear utilization of glucose in mammary cells during lactogenesis is still unknown, due to the lack of in vitro analyzing models. Therefore, the objective of this study was to test the reliability of the mammary alveolar (MAC-T) cell as an in vitro study model for glucose metabolism and lactating system. Methods: Undifferentiated MAC-T cells were cultured in three types of Dulbecco's modified Eagle's medium with varying levels of glucose (no-glucose: 0 g/L, low-glucose: 1 g/L, and high-glucose: 4.5 g/L) for 8 d, after which differentiation to casein secretion was induced. Cell proliferation and expression levels of apoptotic genes, Insulin like growth factor-1 (IGF1) receptor, oxytocin receptor, ${\alpha}S1$, ${\alpha}S2$, and ${\beta}$ casein genes were analyzed at 1, 2, 4, and 8 d after differentiation. Results: The proliferation of MAC-T cells with high-glucose treatment was seen to be significantly higher. Expression of apoptotic genes was not affected in any group. However, expression levels of the mammary development related gene (IGF1 receptor) and lactation related gene (oxytocin receptor) were significantly higher in the low-glucose group. Expressions of ${\alpha}S1-casein$, ${\alpha}S2-casein$, and ${\beta}-casein$ were also higher in the low-glucose treated group as compared to that in the no-glucose and high-glucose groups. Conclusion: The results demonstrated that although a high-glucose environment increases cell proliferation in MAC-T cells, a low-glucose treatment to MAC-T cells induces higher expression of casein genes. Our results suggest that the MAC-T cells may be used as an in vitro model to analyze mammary cell development and lactation connected with precise biological effects.

• Journal of Microbiology and Biotechnology
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• v.23 no.7
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• pp.1023-1030
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• 2013

Lipoteichoic acid (LTA), uniquely expressed on gram-positive bacteria, is recognized by Toll-like receptor 2 (TLR2) on not only antigen-presenting cells but also activated T cells. Therefore, it is reasonable to assume that LTA is acting on T cells. However, little is known about the effect of LTA on T-cell regulation. In the present study, we investigated the immunomodulatory effects of LTA on $CD4^+$ T cells. Effector $CD4^+$ T cells, induced after co-culture with S. aureus-pulsed dendritic cells, produced high levels of interferon-${\gamma}$, CD25, CD69, and TLRs 2 and 4. When effector $CD4^+$ T cells were treated with LTA, the expressions of the membrane-bound form of transforming growth factor (TGF)-${\beta}$ and forkhead box P3 increased. Coincidently, the proliferation of effector $CD4^+$ T cells was declined after LTA treatment. When TGF-${\beta}$ signaling was blocked by the TGF-${\beta}$ receptor 1 kinase inhibitor, LTA failed to suppress the proliferation of effector $CD4^+$ T cells. Therefore, the present results suggest that LTA suppresses the activity of effector $CD4^+$ T cells by enhancing TGF-${\beta}$ production.

• Parasites, Hosts and Diseases
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• v.53 no.4
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• pp.431-438
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• 2015

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T ($T_{reg}$) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and $T_{reg}$ cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/$TIRAP^{-/-}$ MEF cells, and quite substantially decreased in $TRIF^{-/-}$ MEF cells. In contrast, IL-10 and $TGF-{\beta}$ expression levels were not elevated in MyD88/$TIRAP^{-/-}$ MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and $T_{reg}$ cell mediated immune responses, although additional data are needed to convincingly prove this observation.

• Archives of Reconstructive Microsurgery
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• v.18 no.1
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• pp.9-15
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• 2009

Whether a seven days course of anti-${\alpha}{\beta}$-T cell receptor-antibody (${\alpha}{\beta}$-TCRmAb) combined with FK506 therapy promotes survival of limb allografts in fully MHC-mismatched combination (Brown Norway $\rightarrow$ Lewis) was examined. Eight animals received 250 ${\mu}g$/kg/day of ${\alpha}{\beta}$-TCRmAb for 7 days and 2 mg/kg/day of FK506 postoperatively (Combination therapy group). Eight animals had FK506 only (Mono-therapy group) and five animals did not have treatment (Control group). Clinical signs of early rejection with edema or erythema in the skin occurred at an average of 8.6${\pm}$1.5 days postoperatively in Control group and 59.0${\pm}$8.3 days in Mono-therapy group, both of which proceeded to irreversible rejection with necrosis of the epidermis and finally mummification. In Combination therapy group, all animals showed evidence of early rejection at an average of 56.8${\pm}$12.6 days postoperatively, however, in 4 of 8 limbs, early rejection resolved without any treatment and limbs survived >1 year. At 9 months postoperatively, donor skin grafts were accepted and third-party skin grafts were rejected by all four survivors, demonstrating donor-specific tolerance. Little or no detectable chimerism was observed in any of the 4 surviving animals at one-year postoperatively. Combination therapy of ${\alpha}{\beta}$-TCRmAb and FK506 resulted in long-term survival in fully MHC-mismatched limb transplants.

• Biomedical Science Letters
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• v.15 no.3
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• pp.179-185
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• 2009

In our previous report, we showed that $PPAR{\gamma}$ does not influence adipogenesis in females with functioning ovaries, indicating that $PPAR{\gamma}$ activity on adipogenesis is associated with sex-related factors. Among the sex-related factors, estrogen has been recognized as a major factor in inhibiting adiposgenesis in females. Thus, we hypothensized that $17{\beta}$-estradiol (E) inhibits 3T3-L1 cell adipogenesis by preventing $PPAR{\gamma}$ activity. E decreased triglyceirde accumulation in differentiated 3T3-L1 cells compared with control group. E also decreased the expression of $PPAR{\gamma}$ mRNA as well as $PPAR{\gamma}$ dependent adipocyte-specific genes, such as adipocyte fatty acid binding protein and tumor necrosis factor $\alpha$. In addition, E not only decreased luciferase reporter activity by $PPAR{\gamma}$, but also transfection of estrogen receptor $\alpha$ ($ER{\alpha}$) or $ER{\beta}$ led to decreases in $PPAR{\gamma}$ reporter gene activation. Moreover, E-activated ERs significantly decreased the luciferase reporter gene activation induced by $PPAR{\gamma}$ transfection, suggesting that estrogen-activated ERs inhibit $PPAR{\gamma}$-dependent transactivation. Accordingly, our results demonstrate that E inhibits the action of $PPAR{\gamma}$ on adipogenesis through E activated ER, providing evidence that lack of estrogen may potentiate $PPAR{\gamma}$ action on adipogenesis.

• The Journal of Korean Medicine
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• v.22 no.3
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• pp.141-155
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• 2001

Objective : The aim of this study is to investigate the effect of Injin fractions on hepatic fibrosis induced by $TGF-{\beta}1$. Method : $TGF-{\beta}1$ mRNA, protein, $TGF-{\beta}1$ receptor, Smad family and PAI-I mRNA were studied in HepG2 cell, and the proliferation, connective tissue growth factor, fibronectin and collagen type I mRNA in T3891 fibroblast by quantitative RT-PCR, ELISA and thymidine incorporation assay. Results : On $TGF-{\beta}1$ mRNA and protein synthesis in HepG2, $H_2O$, butanol and hexane fractions of Injin showed inhibitory effect in a dose-dependent way. In the study on $TGF-{\beta}1$ receptor, Smad family and PAI-1 mRNA in HepG2, $H_2O$, butanol and hexane fraction of Injin showed inhibitory effect on the expression of PAI-1 in a dose-dependent way. On the proliferation of T3891 fibroblast induced by $TGF-{\beta}1$, $H_2O$, ethylacetate and butanol fractions of Injin showed inhibitory effect. In the study on the factors affected by $TGF-{\beta}1$, $H_2O$, ethylacetate and butanol fractions of Injin showed inhibitory effect on CTGF, and $H_2O$, butanol, chloroform and hexane fractions showed inhibitory effect on the expression of collagen type I, whereas no fraction showed inhibitory effect on the expression of fibronectin Conclusion : These results show that each fraction of Injin acts as a fibrosis inhibitory factor by itself or in combination, ultimately inhibiting liver cirrhosis.

• Journal of Korean Neurosurgical Society
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• v.64 no.4
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• pp.505-513
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• 2021

Objective : The adaptive immune response following subarachnoid hemorrhage (SAH) is not well understood. We evaluated and compared the T cell receptor (TCR) immune repertoire of good-grade and poor-grade SAH patients to elucidate the T cell immunology after ictus. Methods : Peripheral blood from six SAH patients was collected at two different times, admission and at the 7-day follow-up. Composition and variation of the TCR β-chain (TCRB) complimentary determining regions (CDR) 3 repertoire was examined using high-throughput sequencing; the analysis was based on sampling time and disease severity (good vs. poor-grade SAH). Results : Clonality at admission and follow-up were 0.059 (0.037-0.038) and 0.027 (0.014-0.082) (median, 25th-75th percentile). Poor-grade SAH (0.025 [0.011-0.038]) was associated with significantly lower clonality than good-grade SAH (0.095 [0.079-0.101]). Poor-grade SAH patients had higher diversity scores than good-grade SAH patients. CDR length was shorter in good-grade SAH vs. poor-grade SAH. Differences in clonotype distribution were more prominent in TCRBV gene segments than TCRBJ segments. TCRBV19-01/TCRBJ02-04 and TCRBV28-01/TCRBJ02-04 were the most increased and the most decreased V-J pairs in the 7-day follow-up compared to admission in good-grade SAH. The most increased and decreased V-J pairs in poor-grade SAH patients were TCRBV28-01/TCRBJ02-06 and TCRBV30-01/TCRBJ02-04, respectively. Conclusion : The TCRB repertoire is dynamic in nature following SAH. TCRB repertoire may facilitate our understanding of adaptive immune response according to SAH severity.