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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 伍安怡(Betty A. Wu-Hsieh) | |
dc.contributor.author | Catherine Wenshane Fu | en |
dc.contributor.author | 傅文萱 | zh_TW |
dc.date.accessioned | 2021-06-15T04:45:55Z | - |
dc.date.available | 2015-09-09 | |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-06 | |
dc.identifier.citation | Allendoerfer, R., and Deepe, G.S., Jr. (1998). Blockade of endogenous TNF-alpha exacerbates primary and secondary pulmonary histoplasmosis by differential mechanisms. The Journal of Immunology 160, 6072-6082.
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(1992) Iron modulates interferon-g effects in the human myelomonocytic cell line THP-1. Experimental hematology 20,605–10. Weiss, G., Werner-Felmayer, G., Werner, E.R., Grunewald, K., Wachter, H., Hentze, M.W. (1994). Iron regulates nitric oxide synthase activity by controlling nuclear transcription. The Journal of Experimental Medicine 180, 969–76. Weiss G, Wachter H, Fuchs D. (1995). Linkage of cell-mediated immunity to iron metabolism. Immunology Today 6,417. Wu-Hsie, B.A., Lee, G.S., Franco, M., and Hofman, F.M. (1992). Early activation of splenic macrophage by tumor necrosis fector alpha is important in determining the outcome of experimental histoplasma in mice. Infection and Immunity 60, 4230-4238. Zanella, A., Fermo, E., Bianchi, P., and Valentini, G. (2005). Red cell pyruvate kinase deficiency: molecular and clinical aspects. British Journal of Haematology 130, 11–25 Zanella, A., Berzuini, A., Colombo, M.B., Guffanti, A., Lecchi, L., Poli, F., Cappellini, M.D., and Barosi, G. (1993). Iron status in red cell pyruvate kinase deficiency: study of Italian cases. British Journal of Haematology 83, 485–490. Zhou, P., Miller, G. and Seder, R.A. (1998). Factors involved in regulating primary and secondary immunity to infection with Histoplasma capsulatum : TNF-alpha plays a critical role in maining secondary immunity in the absence of IFN-gamma. The Journal of Immunology 160, 1359-1368. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45774 | - |
dc.description.abstract | 鐵在宿主與病菌間的交互關係中扮演兩種角色:一方面可以調控宿主的免疫反應,另一方面為病菌養分的提供者。本篇研究主要利用組織胞漿菌的感染模式探討丙酮酸激酶缺乏的小鼠免疫細胞的功能。在糖酵解循環中,丙酮酸激酶是一種常見的酵素並且缺乏此酵素會導致慢性非溶血性貧血以及肝臟和脾臟有鐵沉積的情形。至於丙銅酸激酶缺乏是如何影響免疫細胞的功能這個問題目前還未被討論。在這邊我使用中研院所提供的基因突變小鼠,這是經由ENU所導致pklr基因點突變形成丙銅酸激酶缺乏的小鼠P354,藉由此方式去了解丙銅酸激酶缺乏在感染的模式中是如何影響免疫反應。
本篇研究發現經由流式細胞儀分析得到P354小鼠脾臟細胞中TER119 紅血球前驅物占了很高的比例,但B淋巴球和T淋巴球占的比例卻比正常小鼠低了很多。然而P354小鼠T淋巴球在胸腺和淋巴結所占的比例是和正常小鼠沒有差異性的。此篇研究也證實P354小鼠的脾臟和肝臟有明顯的鐵沉積情形並且發現在感染組織胞漿菌後的不同天數,P354小鼠真菌數量在肝臟和脾臟而非肺臟都高於正常小鼠。更進一步地,P354小鼠在感染組織胞漿菌之後脾臟所產生TNF-alpha、IL-6、IFN-gamma、IL-4、IL-10、IL-17和T細胞所產生的IFN-gamma 量都比起正常小鼠低很多。除此之外,有趣地是我還發現雖然P354小鼠腹腔巨噬細胞的功能和正常小鼠沒有差異,但脾臟巨噬細胞在吞噬組織胞漿菌的功能上和產生TNF-alpha有明顯的缺失現象。以上實驗結果顯示,P354小鼠無法有效清除脾臟和肝臟中的組織胞漿菌而這兩個器官也剛好有鐵沉積的情形。脾臟清除能力下降其原因為細胞激素產量較低以及巨噬細胞的功能上有缺陷。雖然P354突變小鼠腹腔巨噬細胞的功能和正常小鼠沒有差異,但若外加全轉鐵蛋白給正常小鼠的腹腔巨噬細胞就會降低TNF-alpha產生的量。即使無法排除丙酮酸激酶缺乏的小鼠脾臟巨噬細胞吞入太多紅血球而造成免疫功能上的缺陷但我的研究結果可以得知是因為脾臟和肝臟鐵沉積而導致巨噬細胞功能上有缺陷因此直接或間接的影響清除細胞內病原的能力。 | zh_TW |
dc.description.abstract | Iron is a critical element in host-pathogen interaction. It is both a modifier of cellular immune function and an essential nutrient for microbes. Here, I investigated immune cell functions in pyruvate kinase deficient mice following infection by Histoplasma capsulatum (HC). Pyruvate kinase (PK) deficiency is the most common enzyme abnormality of glycolysis causing chronic non-spherocytic haemolytic anemia and accumulation of iron in the liver and spleen. Whether pyruvate kinase deficiency affects immune cell functions has never been addressed. The availability of ENU-mutagenized mice P354 with point-mutated pklr gene presents an opportunity to study how pyruvate kinase deficiency affects immune response to infection.
In this study, cytometric analysis of the splenic cell populations revealed elevated numbers of TER119-positive erythroid precursor cells and lower percentages of T cells and B cells in P354 mutant mice compared to wild-type mice. However, the T cell populations in the thymus and lymph nodes of P354 mutant mice were comparable to that in wild-type mice. I demonstrated in this study that there was iron accumulation in the spleen and liver of P354 mutant mice and found that fungal burdens in the spleen and liver but not that in the lung of P354 mutant mice were higher than in wild-type mice at different time points after Histoplasma infection. Furthermore, the levels of cytokines (TNF-α, IL-6, IFN-γ, IL-4, IL-10, and IL-17) and the numbers of IFN-γ-producing T cells were lower in P354 mutant mice than in wild-type mice. Interestingly, while peritoneal macrophages from P354 mutant mice were functionally comparable to wild-type mice, splenic macrophages obtained from P354 mutant mice were less competent in phagocytosis of Histoplasma and production of TNF-α. These results showed that mice with pyruvate kinase deficiency were inefficient to clear Histoplasma from the spleen and liver, coinciding with increased iron contents in these organs. Inefficiency to rid the spleen of fungus also correlated with lower cytokine production and impaired macrophage function. While the function of peritoneal macrophages from P354 mutant mice was comparable to that of wild-type mice, the addition of holo-transferrin to peritoneal macrophages from wild-type mice impaired their ability to produce TNF-α. Although the possibility of overcrowding effect has not been excluded, the results of my study suggest that iron overload as observed in the spleen and liver of mice with pyruvate kinase deficiency impairs the function of macrophages, thereby directly or indirectly, affects their ability to rid an intracellular pathogen. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:45:55Z (GMT). No. of bitstreams: 1 ntu-99-R97449001-1.pdf: 2537298 bytes, checksum: c7e9adafef140dfda2e0ebe84f1628b2 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | Abstract ………………………………………………………………………………i
Abstract (Chinese) …………………………………………………………………iii Abbreviation…………………………………………………………………………iv Table of Contents……………………………………………………………………v Chapter I. Introduction ………………………………………………………………1 1.Pyruvate kinase deficiency and infectious disease…………………………2 2.Iron overload and Immunity…………………………………………………………3 2.1 Iron overload and innate immunity……………………………………………4 2.2 Iron overload and adaptive immunity…………………………………………...5 3.ENU- mutagenized mice-Pedigree 354……………………………………………...5 4.Histoplama capsulatim………………………………………………………………6 4.1 Histoplasma capsulatum infection………………………………………………6 4.2 Histoplasma capsulatum requires iron for growth………………………………6 4.3 Host defense against Histoplasma capsulatum………………………………….7 Chapter II. Aims of The Study ………………………………………………………..9 1.To characterize the phenotype of ENU-mutagenized mice P354……………….10 2.Employing Histoplasma to understand how iron modulates immune functions……10 3.The correlation between iron and immune function……………………………11 Chapter III. Materials and Methods………………………………………………12 Part I. Materials……………………………………………………………………...13 1.Mice………………………………………………………………………………..13 2.Antibodies………………………………………………………………………….13 3.Solutions …………………………………………………………………………..14 4.Chemical and reagents……………………………………………………………..18 5.Equipments ……………………………………………………………………….21 Part II. Methods………………………………………………………………………22 1.Fungus and infection ………………………………………………………………22 2.Quantitation of fungal load…………………………………………………………22 3.Mouse lymphocytes isolation………………………………………………………22 3.1 Preparation of single cell suspension from mouse spleen……………………22 3.2 Preparation of single cell suspension from thymus and lymph nodes…………23 4. Cell surface and intracellular cytokine staining for flow cytometric analysis …….23 4.1 Cell surface marker staining……………………………………………………23 4.2 Intracellular cytokine staining…………………………………………………23 4.3 Flow cytometric analysis………………………………………………………24 5 Blood smears………………………………………………………………………24 6 Hematoxylin and Eosin staining…………………………………………………..24 7 Perl's iron stain…………………………………………………………………….25 8 MRI T1 technique to measure iron loading in tissue……………………………25 9 Cytokine ELISA assay…………………………………………………………….25 10 Preparation of heat-killed Histoplasma capsulatim………………………………26 11 Isolation of splenic macrophage………………………………………………….26 11.1 Phagocytosis of Histoplasma by splenic macrophage………………………..27 11.2The cytokine ELISA assay………………………………………………….27 12. Isolation of mouse peritoneal macrophage…………………..................................28 13. Labeling of Histoplasma yeasts with fluorescein isothiocyanate (FITC)…………28 14. Phagocytosis of Histoplasma by peritoneal macrophage…………………………..28 15.Fungus replication rate and the ability to inhibit fungus growth in macrophage…..29 15.1 Fungus replication rate……………………………………………………….29 15.2 The ability of inhibition fungus growth in macrophage……………………..30 16. Treatment of peritoneal macrophages with human holo-transferrin……………….30 17.Statistics………………………………………………………………………….30 Chapter IV. Results…………………………………………...……………………….31 1. Characterization of P354 mice……………………………………………………32 1.1 P354 mice have RBC defects, disorganized splenic architecture and high iron contents in the spleen……………………………………32 1.2 Immune cell populations in the spleen, thymus and lymph nodes of P354 mice..……………………33 2. Employing Histoplasma infection model to understand how iron modulate immune cell functions……………………………………………………34 2.1 The ability of P354 mice to clear infection by Histoplasma…………………………………………34 2.2 Comparing the iron contents in P354 and wild-type mice before and after infection with Histoplasma………………………………………………35 3.To study how iron affects immune cell functions …………………………………35 3.1 Production of poinflammatory cytokines by P354 mice in response to Histoplasma infection………………………………………………………35 3.2To compare the function of peritoneal macrophage and splenic macrophage…………………………………………….……………………..36 3.3 To investigate the cell population, the T cell cytokines and T cell function in spleen of P354 mice after infection………………………………38 Chapter V. Discussion…………………………………………………………………41 1.The phenotype of P354 mutant mice…………………………………………………42 2.Fungal clearance and splenic macrophage functions in P354 mice…………………………43 3.RBC defect will cause erythrophagocytosis and iron overloading in macrophage and affect the immune effector function…………………………………………….45 4.The kinetics of T cell and B cell expansion after infection………………………47 5.Iron overload and T cell response……………………………………………………………47 6.Conclusion………………………………………………………………………….48 Reference………………………………………………………………………………49 Figures…………………………………………………………………………………56 Figure 1. P354 mutant mice have higher percentage of reticulocytes, splenomegaly and abnormal splenic architectures. ……………………………………57 Figure 2. Iron overload in the spleen and liver but not the lungs of P354 mutant mice…………………………………………………………………………………59 Figure 3. Comparing the splenic T cell, B cell and macrophage populations between P354 mutant and wild-type mice.………………………………………………62 Figure 4. The T cell populations in the thymus and lymph nodes in P354 mutant mice are comparable to wild-type mice……………………………………………64 Figure 5. The fungal burdens in the spleen and liver but not those in the lung of P354 mice are higher than in wild-type mice after Histoplasma infection……..66 Figure 6. Iron remains detectable in the spleen and liver of P354 mutant mice after infection with Histoplasma………………………………………………….69 Figure 7. The levels of TNF-α and IL-6 in the spleen of P354 mutant mice are lower than in wild-type mice after Histoplasma infection………………………71 Figure 8. The splenic macrophages of P354 mutant mice are functionally impaired………………………………………………………………………………………………73 Figure 9. The peritoneal macrophages of P354 mutant mice are functionally comparable to the macrophages of wild-type mice……………………………………….75 Figure 10. Transferrin-bound iron inhibits the production of TNF-α and IL-6 by Histoplasma infected macrophages…………………………………78 Figure 11. Comparing the spleen cellularity between P354 mutant and wild-type mice after Histoplasma infection………………………………………………80 Figure 12. The level of IFN-γ in the spleen of P354 mutant mice is lower than in wildtype mice after Histoplasma infection……………………………………82 Figure 13. The levels of IL-4 and IL-10 in the spleen of P354 mutant mice are lower than in wild-type mice after Histoplasma infection…………………………84 Figure 14. The level of IL-17 in the spleen of P354 mutant mice is lower than in wildtype mice after Histoplasma infection……………………………………86 Figure 15. IFN-γ-producing CD4 and CD8 T cells are lower in P354 mutant and wild-type mice after Histoplasma infection…………………………… 88 | |
dc.language.iso | en | |
dc.title | 丙酮酸激酶缺乏導致鐵含量過剩影響脾臟巨噬細胞對真菌的反應 | zh_TW |
dc.title | Iron overload in pyruvate kinase mutant mice affects the
response of splenic macrophage to fungal infection | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 孔祥智(John Kung),顧家綺(Chia-Chi Ku),黃佩欣(Pei-Hsin Huang) | |
dc.subject.keyword | 組織胞漿菌,丙酮酸激酶,ENU,P354, | zh_TW |
dc.subject.keyword | Histoplasma capsulatum(Hc),Pyruvate kinase(PK),N-ethyl-N-nitrosourea(ENU),Pyruvate kinase, liver and RBC(PKLR),Pedigree 354(P354),Antigen-presenting cell (APC),Wild-type(WT), | en |
dc.relation.page | 89 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-06 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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