Browsing by Author "Chu, Rea-Min"

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Canine CD8 T cells showing NK cytotoxic activity express mRNAs for NK cell-associated surface molecules
(2010-02-15)
Lin, Yi-Chun
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Huang, Yi-Chun
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Wang, Yu-Shan
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Juang, Rong-Huay
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Liao, Kuang-Wen
Natural killer (NK) cells have been considered to be a group of lymphocytes lacking clonally distributed receptors for antigens typical of T cells and B cells. in some mammalian species, including humans, a subpopulation of CD8(+) peripheral blood lymphocytes (PBLs) exhibits NK activity. This NK subpopulation has not been well characterized in mammals and its characterization is particularly poor in the dog. In this study, we demonstrated that a subset of canine CD8(+) cells derived from PBLs and lymphokine (IL-2)-activated killers (LAKs) of PBLs that was CD3(+), CD4(-), CD21(-), CD5(lo), alpha/beta TCR(+), and gamma/delta TCR(-) contained substantially higher levels of mRNAs for NK cell-related receptors (NKp30, NKp44, NKG2D, 2B4, and CD16 for PBL, and NKG2D and CD56 for LAK) than the corresponding CD8(-) cells. This subset of CD8(+) lymphocytes derived from LAKs also displayed significantly higher NK cytotoxic activity than the corresponding CD8(+) cells. In contrast, CD8(+) cells derived from nonstimulated PBLs showed very low levels of NK cytotoxic activity. Our results indicate that, in IL-2-stimulated PBLs, canine CD8(+) cells are an important subset associated with NK cytotoxic activity. (C) 2009 Elsevier B.V. All rights reserved.
Characterization of canine monocyte-derived dendritic cells with phenotypic and functional differentiation
(2007-07-01)
Wang, Yu-Shan
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Chi, Kwan-Hwa
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Liao, Kuang-Wen
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Liu, Cheng-Chi
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Cheng, Chiao-Lei
For therapeutic purposes, large numbers of dendritic cells (DCs) are essential. In this study, we used 2% autologous canine plasma, granulocyte/macrophage colony-stimulating factor (GM-CSF), fms-like tyrosine kinase 3 ligand (Flt3L), and interleukin 4 (IL-4) in generating monocyte-derived DCs from peripheral blood mononuclear cells of dogs. The plasma enriched the population of CD14-positive monocytes by greatly enhancing the efficiency of monocyte adherence, the proportion of adherent cells increasing from 6.6% with 10% fetal bovine serum to 15.3% with 2% autologous canine plasma. Culturing the adherent monocytes for 6 d with human GM-CSF, canine IL-4, and human Flt3L significantly increased the yield of DCs, more than 90% of which were CD14-negative. Because, in the presence of lipopolysaccharide (LPS), monocytes that were CD14-positive expressed tumor necrosis factor alpha much more than DCs with low levels of CD14, it is important to decrease the numbers of CD14-positive cel, Is in generating monocyte-derived DCs. With flow cytometry and real-time reverse-transcriptase-mediated polymerase chain reaction assays, we found that in canine immature DCs (iDCs) the expression of DLA class 11 molecules, CD1a, CD11c, CD40, and CD86 was high and the expression of CD80, CD83, and CD14 either low or negative. During maturation (stimulated by LPS), the expression of CD1a, CD40, CD83, and CD80 was upregulated. However, the expression of DLA class 11 molecules, CD11c, and CD86 was not increased in mature DCs. Incubating the iDCs with LPS decreased antigen uptake and increased the cells' immunostimulatory capacity (assessed by the allogeneic mixed-lymphocyte reaction), indicating that LPS accelerates the functional maturation of DCs. This protocol may facilitate the use of DCs in cellular immunotherapy.
Combined immunogene therapy of IL-6 and IL-15 enhances anti-tumor activity through augmented NK cytotoxicity
(2008-12-18)
Lin, Ching-Yi
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Chuang, Tien-Fu
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Liao, Kuang-Wen
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Huang, Yi-Jun
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Pai, Chien-Chun
Many tumors evade host immunity by lowering expression of major histocompatibility complex (MHC) molecules. Theoretically, low MHC expression should activate natural killer (NK) cells and in some cases suppress tumor growth; nevertheless, some tumors also produce high concentrations of immunosuppressive cytokines, such as transforming growth factor (TGF)-beta, to inhibit the activity of NK cells. Using a canine transmissible venereal tumor (CTVT) model, we have previously demonstrated that IL-6 is a strong antagonist for TGF-beta. Herein, we found that IL-6 alone was unable to significantly promote TGF-beta-inhibited NK activities. Conversely, IL-15 alone strongly promoted NK activities; however, NK activities were inhibited to baseline levels following the addition of TGF-beta. Therefore, a new strategy using combined immunogene therapy of both IL-6 and IL-15 mediated by electroporation was used in this study. This combined IL-6 and IL-15 treatment effectively relieved the inhibitory effect of TGF-beta and activated NK cell cytotoxicity of lymphokine-activated killer (LAK) cells. Similarly, in isolated DX5(+) NK cells, only IL-6 and IL-15 in combination significantly overcame the inhibitory effect of TGF-beta and promoted NK cytotoxicity. The group of BALB/c mice injected with plasmids with IL-6 and IL-15 genes (pIL-6/pIL-15) had the highest percentages of DX5(+) NK cells as compared with either the pIL-6 or pIL-15 groups. Further, in SCID mice inoculated with CTVT, electroporation-mediated delivery of pIL-6/pIL-15 was significantly more efficient in suppressing both tumor establishment and tumor growth as compared with pIL-6 or pIL-15 inoculation alone. In addition, the anti-asialo GM-1 antibody abolished NK activities in SCID mice and resulted in outgrowth of the tumors. Together, these results suggest that the TGF-beta-associated inhibition of NK cytotoxicity cannot be adequately restored by simply antagonizing TGF-beta with IL-6: the co-existence of NK activating factors such as IL-15 is also important in restoring TGF-beta-inhibited cytotoxicity. This study highlights the therapeutic potential of the pIL-6/pIL-15 combination by inhibiting TGF-beta activity and enhancing NK cytotoxicity. (c) 2008 Elsevier Ireland Ltd. All rights reserved.
Immunopathogenic behaviors of canine transmissible venereal tumor in dogs following an immunotherapy using dendritic/tumor cell hybrid
(2011-02-15)
Pai, Chien-Chun
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Kuo, Tzong-Fu
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Mao, Simon J. T.
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Chuang, Tien-Fu
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Lin, Chen-Si
Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor that can be transmitted between dogs via live tumor cell inoculation. It is also a spontaneous self-regression tumor and its behavior is closely related to host immune responses. Since CTVT had been widely used for tumor models in canine cancers, whether this self-regression may overtake the immunity elicited from an exogenous tumor vaccine remains unclear and certainly worthwhile to be investigated. In this study, we used DCs/tumor hybrids as a tumor vaccine to evaluate the CTVT model. We prepared mature allogeneic dendritic cells from bone marrow and then assessed their phenotype (CD80, CD83, CD86, CD1a, CD11c, CD40 and MHC II), antigen uptake and presenting abilities. Fused dendritic cell/CTVT hybrids were then used as a vaccine, administered three times at two-week intervals via subcutaneous injection near the bilateral auxiliary and inguinal lymph nodes. In comparison with unvaccinated dogs (spontaneous regressed group), within a period of 2.5 months, the vaccinations substantially inhibited tumor progression (p<0.05) and accelerated the rate of regression by a mechanism involving amplification of the host tumor-specific adaptive immune responses and NK cytotoxicity (p<0.001). Pathologic examination revealed early massive lymphocyte infiltration resulting in final tumor necrosis. In addition, there are not any detectable effects on routine physical, body temperature or blood chemistry examinations. In conclusion, our data furnishes a reference value showing that CTVT is a model of potential use for the study of immunity elicited by vaccines against tumors, and also enable early-phase evaluation of the dendritic cell/tumor vaccine in terms of raising host immunity. (C) 2010 Elsevier B.V. All rights reserved.
Interactions of host IL-6 and IFN-gamma and cancer-derived TGF-beta 1 on MHC molecule expression during tumor spontaneous regression
(2008-07-01)
Hsiao, Ya-Wen
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Liao, Kuang-Wen
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Chung, Tien-Fu
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Liu, Chen-Hsuan
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Hsu, Chia-Da
Many tumors down-regulate major histocompatibility complex (MHC) antigen expression to evade host immune surveillance. However, there are very few in vivo models to study MHC antigen expression during tumor spontaneous regression. In addition, the roles of transforming growth factor beta1 (TGF-beta 1), interferon gamma (IFN-gamma), and interleukin (IL)-6 in modulating MHC antigen expression are ill understood. We previously reported that tumor infiltrating lymphocyte (TIL)-derived IL-6 inhibits TGF-beta 1 and restores natural killing (NK) activity. Using an in vivo canine-transmissible venereal tumor (CTVT) tumor model, we presently assessed IL-6 and TGF-beta involvement associated with the MHC antigen expression that is commonly suppressed in cancers. IL-6, IFN-gamma, and TGF-beta 1, closely interacted with each other and modulated MHC antigen expression. In the presence of tumor-derived TGF-beta 1, host IFN-gamma from TIL was not active and, therefore, there was low expression of MHC antigen during tumor progression. TGF-beta 1-neutralizing antibody restored IFN-gamma-activated MHC antigen expression on tumor cells. The addition of exogenous IL-6 that has potent anti-TGF-beta 1 activity restored IFN-gamma activity and promoted MHC antigen expression. IFN-gamma and IL-6 in combination acted synergistically to enhance the expression of MHC antigen. Thus, the three cytokines, IL-6, TGF-beta 1, and IFN-gamma, closely interacted to modulate the MHC antigen expression. Furthermore, transcription factors, including STAT-1, STAT-3, IRF-1, NF-kappa B, and CREB, were significantly elevated after IL-6 and IFN-gamma treatment. We conclude that the host IL-6 derived from TIL works in combination with host IFN-gamma to enhance MHC molecule expression formerly inhibited by TGF-beta 1, driving the tumor toward regression. It is suggested that the treatment of cancer cells that constitutively secrete TGF-beta 1 should incorporate anti-TGF-beta activity. The findings in this in vivo tumor regression model have potential applications in cancer immunotherapy.
Utilization of I kappa B-EGFP Chimeric Gene as an Indicator to Identify Microbial Metabolites with NF-kappa B Inhibitor Activity
(2010-12-01)
Lin, Yu-Ling
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Chen, Yen-Shun
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Hsieh, Jui-Hung
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Lin, Ching-Min
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Wu, Hsin-Yi
NF-kappa B regulates several important expressions, such as cytokine release, anti-apoptosis, adhesion molecule expression, and cell cycle processing. Several NF-kappa B inhibitors have been discovered as an anti-tumor or anti-inflammatory drug. The activity of NF-kappa B transcription factor is negatively regulated by I kappa B binding. In this study, I kappa B assay system was established and I kappa B-EGFP fusion protein was used as an indicator to monitor the effects of substances on the I kappa B degradation. The results indicated that the chosen hydroquinone could inhibit the I kappa B degradation and cause the cell de-attachment from the bottom of culture plate. In addition, this system could also monitor the I kappa B degradation of microbial metabolite of natural mixtures of propolis. Thus, the I kappa B assay system may be a good system for drug discovery related to microbial metabolite.