鼻腔途徑接種疫苗的優點，在於能同時誘發黏膜與全身部位之抗原專一 性免疫反應，藉此提供呼吸道對抗病原感染的能力。然而，由於黏膜屏障和免疫 耐受性等因素，使得給予抗原於黏膜時，僅能誘發出微乎其微的免疫反應，無法 達到保護效果。為了增強疫苗的臨床功效，我們希望能夠藉由乳液佐劑的使用， 透過黏膜免疫系統的辨認而產生廣效免疫反應。於此，我們針對:乳液顆粒尺寸、 核心油脂以及接種途徑的選擇，開啟了作用機制的探討。首先，我們勘測乳液顆 粒尺寸，是否能夠影響小鼠的抗原專一性免疫反應，而奈米級乳液顆粒，能藉由 通過擠壓器薄膜製備;其二，我們比較乳液的核心油脂(角鯊烷、角鯊烯)的差 異，能否影響免疫反應;其三，我們研究，經不同給予途徑所誘發之免疫反應。 於小鼠實驗選用卵白蛋白作為模擬抗原，以鼻腔給予途徑接種抗原或佐以候選佐 劑之抗原。結果顯示，當小鼠接種佐以角鯊烷次微米乳液之抗原，於脾臟細胞分 泌上清液中，能測得濃度較高之 IFN-𝛾、IL-4、IL-5 與 IL-17，另一方面，當小鼠 接種，佐以角鯊烯奈米乳液之抗原，於血清中，能測得較高力價之 IgG1 與 IgG2c; 於鼻腔沖洗液中，能測得較高力價之 IgA。這些結果指出，次微米乳液較能夠誘 發細胞免疫反應，反之，奈米乳液則較能夠誘發體液免疫反應。相較於肌肉注射， 鼻腔給予更適用奈米乳液來誘發免疫反應。最終，組織切片數據顯示此類乳液並 不會在接種部位造成傷害。本研究將揭示，選擇含乳液佐劑疫苗之最佳接種途徑 方面的應用潛力。

Intranasal (i.n.) vaccination provides major respiratory route defections in the form of both antigen-specific mucosal and systemic immunity against pathogen infection; however, antigen applied to mucosal membrane generally induces miniscule immune responses owing to the mucosal membrane barriers and immune tolerance. Toward that goal, it is therefore required the aid of a potent mucosal adjuvant with high safety and efficacy for antigen recognition by mucosal immune systems and generating broad- spectrum immune responses. Here we launch a mechanistic study on how particle size, core oil as well as the administration route, on mucosal immunity. First of all, we investigated whether the size matter can affect the antigen-specific immune response in mice. The dimensions of emulsions could be processed to nanoscale by passing through the extruder membrane. Second, we compared the immune response elicited by squalane- cored emulsion with those induced by squalene-cored emulsion. Third, we studied the immune responses following intramuscular and intranasal vaccinations. Mice were vaccinated intranasally or intramuscularly with a model antigen ovalbumin (OVA), alone or formulate with candidate adjuvants. The data demonstrated that significantly elevated levels of IFN-𝛾, IL-4, IL-5, IL-17 secretion were detected in splenocyte supernatants collected from mice treated with squalane-formulated emulsions at submicron scale. On the other hand, mice treated with squalene-formulated nanoemulsions increased OVA- specific IgG1, IgG2c titers in sera and IgA titers in nasal washes. These results indicated that emulsified particles at submicron scale are better able than the ones at nanoscale to enhance cell-mediated immune response. By contrast, those at nano scale showed a high potential to induce antibody responses. Such studies are indicated that intranasal vaccination is the optimal route for inducing immune response, while intramuscular vaccination is less suitable for this purpose. Finally, histological examination indicated that these emulsions do not cause tissue damage at immunization site. These results suggest the potential application of adjuvanted vaccines in optimizing alternative vaccination routes.

中文摘要 ........................................................................................................................... i Abstract............................................................................................................................ ii 致謝 ................................................................................................................................... iii
目錄................................................................................................................................... iv 圖目次 ............................................................................................................................. vii 表目次 ............................................................................................................................... x
第 1 章 緒論 .................................................................................................................... 1 1.1 研究動機................................................................................................................... 1 1.2 現今概況................................................................................................................... 2
1.2.1 黏膜免疫 ........................................................................................................... 2 1.2.2 黏膜佐劑 ........................................................................................................... 3 1.2.3 乳液佐劑 ........................................................................................................... 4
1.2.4 粒徑尺寸對黏膜免疫之影響 .......................................................................... 9 1.2.5 PELA................................................................................................................ 10 1.2.6 設計理念 ......................................................................................................... 11
1.3 研究目的................................................................................................................. 12 第 2 章 材料與方法.................................................................................................... 13
2.1 材料 ......................................................................................................................... 13 iv

2.1.1 化學性實驗藥品 ............................................................................................. 13
2.1.2 生物性實驗藥品 ............................................................................................. 14 2.1.3 實驗器具.............................................................................................................. 16 2.1.4 實驗儀器 ......................................................................................................... 18 2.1.5 實驗動物 ......................................................................................................... 19 2.2 方法 ......................................................................................................................... 20 2.2.1 反應物的純化 ................................................................................................. 20 2.2.2 合成 PEG-b-PLA ............................................................................................ 21 2.2.2 PEG-b-PLA 鑑定............................................................................................. 23 2.2.3 乳液的製備 ..................................................................................................... 24 2.2.4 乳液之性質 ..................................................................................................... 25 2.2.5 不同乳液搭配卵白蛋白所誘發之免疫反應 ............................................... 26 2.2.6 不同接種配方對於接種部位之組織切片與染色 ....................................... 36
第 3 章 結果與討論.................................................................................................... 37 3.1 高分子型乳化劑 PEG-b-PLA 之合成.................................................................. 37 3.2 PEG-b-PLA 鑑定.................................................................................................... 38 3.3 乳液的製備............................................................................................................. 42 3.4 乳液之性質............................................................................................................. 43 3.5 不同乳液搭配卵白蛋白所誘發之免疫反應評估............................................... 46
v
3.6 不同接種配方對於接種部位之安全性評估....................................................... 59 第 4 章 結論 .................................................................................................................. 61 第 5 章 參考文獻......................................................................................................... 62
附錄.................................................................................................................................. 71 A.1 不同乳液搭配卵白蛋白所誘發之免疫反應...................................................... 71 A.2 不同接種配方對於接種部位之組織切片與染色.............................................. 73 A.3 不同乳液搭配卵白蛋白所誘發之免疫反應評估 ............................................. 74 A.4 不同接種配方對於接種部位之安全性評估...................................................... 86

1.Delany I, Rappuoli R, De Gregorio E. Vaccines for the 21st century. EMBO Molecular Medicine. 2014; 6(6):708–20.
2.Nir, Y., Paz, A., Sabo, E. & Potasman, I. Fear of injections in young adults: prevalence and associations. The American Journal of Tropical Medicine and Hygiene. 2003; 68; 341–4.
3.Breau LM, McGrath PJ, Craig KD, Santor D, Cassidy KL, Reid GJ. Facial expression of children receiving immunizations: a principal components analysis of the child facial coding system. The Clinical Journal of Pain. 2001; 17(2): 178–86.
4.Slütter B, Hagenaars N, Jiskoot W. Rational design of nasal vaccines. Journal of Drug Targeting. 2008; 16(1): 1–17.
5.Safe injection global network SIGN—advocacy booklet; 2001.
6.Varmus H, Klausner R, Zerhouni E, Acharya T, Daar AS, Singer PA. Grand challenges
in global health. Science. 2003; 302(5644):398–9.
7.Mitragotri S, Immunization without needle. Nature Reviews Immunology. 2005; 5:
905–16.
8.Rhee JH, Lee SE, Kim SY. Mucosal vaccine adjuvants update. Clinical and
Experimental Vaccine Research. 2012; 1(1): 50–63.
9.Lycke N, Recent progress in mucosal vaccine development: potential and limitations.
Nature Reviews Immunology. 2012; 12: 592–605.
10.Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nature Medicine. 2005;
11: S45–S53.
11.Brandtzaeg P. Function of mucosa-associated lymphoid tissue in antibody formation.
Immunological Investigations. 2010; 39: 303–55.
12.Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nature
Reviews Immunology. 2006; 6: 148–58.
13.Garmise RJ, Mar K, Crowder TM, Hwang CR, Ferriter M, Huang J, Mikszta JA,
Sullivan VJ, Hickey AJ. Formulation of a dry powder influenza vaccine for nasal
delivery. American Association of Pharmaceutical Scientists. 2006; 7(1): E131–7. 14.Zaman M, Chandrudu S, Toth I. Strategies for intranasal delivery of vaccines. Drug
Delivery and Translational Research. 2013; 3(1) :100–9.
15.Zuercher AW, Coffin SE, Thurnheer MC, Fundova P, Cebra JJ. Nasal-associated
lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular
immune responses. The Journal of Immunology. 2002; 168(4): 1796–803.
16.Merkus FW, Verhoef JC, Schipper NG, Marttin E. Nasal mucociliary clearance as a factor in nasal drug delivery. Advanced Drug Delivery Reviews. 1998; 5:29(1-2): 13–
38.
62
17.Ozsoy Y, Gungor S, Cevher E. Delivery of high molecular weight drugs. Molecules. 2009; 14(9): 3754–79.
18.Sasaki S, Okuda K. The use of conventional immunologic adjuvants in DNA vaccine preparations. Methods in Molecular Medicine. 2000; 29: 241–9.
19.Singh M, O'Hagan D. Advances in vaccine adjuvants. Nature Biotechnology. 1999; 17(11): 1075–81.
20.Davis HL, McCluskie MJ. DNA vaccines for viral diseases. Microbes Infect. 1999; 1(1): 7–21.
21.Woodrow KA, Bennett KM, Lo DD. Mucosal vaccine design and delivery. Annual Review of Biomedical Engineering. 2012; 14: 17–46.
22.Pizza M, Giuliani MM, Fontana MR, Douce G, Dougan G, Rappuoli R. LTK63 and LTR72, two mucosal adjuvants ready for clinical trials. International Journal of Medical Microbiology. 2000; 290(4-5): 455–61.
23.Sánchez J, Holmgren J. Cholera toxin structure, gene regulation and patho- physiological and immunological aspects. Cellular and Molecular Life Sciences. 2008; 65(9): 1347–60.
24.Lewis DJ, Huo Z, Barnett S, Kromann I, Giemza R, Galiza E, Woodrow M, Thierry- Carstensen B, Andersen P, Novicki D, Del Giudice G, Rappuoli R. Transient facial nerve paralysis (Bell’s palsy) following intranasal delivery of a genetically detoxified mutant of escherichia coli heat labile toxin. PLoS ONE. 2009; 4(9): e6999.
25.Hajishengallis G, Lambris JD. Crosstalk pathways between toll-like receptors and the complement system. Trends Immunology. 2010; 31(4): 154–63.
26.Fischetti L, Zhong Z, Pinder CL, Tregoning JS, Shattock RJ. The synergistic effects of combining TLR ligand based adjuvants on the cytokine response are dependent upon p38/JNK signalling. Cytokine. 2017; 99: 287–96.
27.Millan CLB, Weeratna R, Krieg AM, Siegrist C-A, Davis HL. CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice. Proceedings of the National Academy of Sciences of the United States of America. 1998; 95(26): 15553–8.
28.Meng W, Yamazaki T, Nishida Y, Hanagata N. Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists. BMC Biotechnology. 2011; 11: 88.
29.Ichinohe T, Ainai A, Tashiro M, Sata T, Hasegawa H. PolyI:polyC12U adjuvant- combined intranasal vaccine protects mice against highly pathogenic H5N1 influenza virus variants. Vaccine. 2009; 27(45): 6276-9.
30.Temizoz B, Kuroda E, Ishii KJ. Vaccine adjuvants as potential cancer immune- therapeutics. International Immunology. 2016; 28(7): 329–38.
63
31.Maijó M, Miró L, Polo J, Campbell J, Russell L, Crenshaw J, Weaver E, Moretó M, Pérez-Bosque A. Dietary plasma proteins modulate the adaptive immune re- sponse in mice with acute lung inflammation. Journal of Nutrition. 2012; 142(2): 264–70.
32.Ogawa Y, Kanoh S. Involvement of central action of lipopolysaccharide in pyrogen fever. The Japanese Journal of Pharmacology. 1984; 36(3): 389–-95.
33.Baldridge JR, Yorgensen Y, Ward JR, Ulrich JT. Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration. Vaccine. 2000; 18(22): 2416–25.
34.Qureshi N, Mascagni P, Ribi E, Takayama K. Monophosphoryl lipid A obtained from lipopolysaccharides of Salmonella minnesota R595: purification of the dimethyl derivative by high performance liquid chromatography and complete structural determination. The Journal of Biological Chemistry. 1985; 260: 5271–8.
35.Chen J, Tao G, Wang X. Construction of an escherichia coli mutant producing monophosphoryl lipid A. Biotechnology Letters. 2011; 33(5): 1013–9.
36.Stefan H.E. Kaufmann, Paul-Henri Lambert. The Grand Challenge for the Future Vaccines for Poverty-Related Diseases from Bench to Field. Springer Science & Business Media, Berlin, Heidelberg. 2005; 99-118.
37.Sasaki S, Sumino K, Hamajima K, Fukushima J, Ishii N, Kawamoto S, Mohri H, Kensil CR, Okuda K. Induction of systemic and mucosal immune responses to human immunodeficiency virus type 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular and intranasal routes. Journal of Virology. 1998; 72(6): 4931–9.
38.Ragupathi G, Damani P, Deng K, Adams MM, Hang J, George C, Livingston PO, Gin DY. Preclinical evaluation of the synthetic adjuvant SQS-21 and its constituent isomeric saponins. Vaccine. 2010; 28(26): 4260–7.
39.Kuroda E, Coban C, Ishii KJ. Particulate adjuvant and innate immunity: past achievements, present findings, and future prospects. International Reviews of Immunology. 2013; 32(2): 209–20.
40.Yu H, Chen X, Lu T, Sun J, Tian H, Hu J, Wang Y, Zhang P, Jing X. Poly(L-lysine)- graft-chitosan copolymers: synthesis, characterization, and gene transfection effect. Biomacromolecules. 2007; 8(5): 1425–35.
41.Sinha VR, Singla AK, Wadhawan S, Kaushik R, Kumria R, Bansal K, Dhawan S. Chitosan microspheres as a potential carrier for drugs. International Journal of Pharmaceutics. 2004; 274(1-2):1–33.
42.Zheng M, Qu D, Wang H, Sun Z, Liu X, Chen J, Li C, Li X, Chen Z. Intranasal administration of chitosan against influenza A (H7N9) virus infection in a mouse model. Scientific Reports. 2016; 6: 28729.
64
43.Aspden TJ, Mason JD, Jones NS, Lowe J, Skaugrud O, Illum L. Chitosan as a nasal delivery system: the effect of chitosan solutions on in vitro and in vivo mucociliary transport rates in human turbinates and volunteers. Journal of Pharmaceutical Sciences. 1997; 86(4): 509-13.
44.Li X, Min M, Du N, Gu Y, Hode T, Naylor M, Chen D, Nordquist RE, Chen WR. Chitin, chitosan, and glycated chitosan regulate immune responses: the novel adjuvants for cancer vaccine. Clinical & Developmental Immunology. 2013; 2013: 387023.
45.Material Safety Data Sheet Chitosan MSDS. Available: http://www.sciencelab.com/msds.php?msdsId=9927494.
46.Makadia HK, Siegel SJ. Poly Lactic-co-Glycolic Acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers. 2011; 3(3): 1377–97.
47.Jaganathan KS, Vyas SP. Strong systemic and mucosal immune responses to surface- modified PLGA microspheres containing recombinant hepatitis B antigen administered intranasally. Vaccine. 2006; 24(19): 4201–11.
48.Sahdev P, Ochyl LJ, Moon JJ. Biomaterials for nanoparticle vaccine delivery systems. Pharmaceutical Research. 2014; 31(10): 2563–82.
49.Henderson A, Propst K, Kedl R, Dow S. Mucosal immunization with liposome-nucleic acid adjuvants generates effective humoral and cellular immunity. Vaccine. 2011; 29(32): 5304–12.
50.Nandini V Katre. Liposome-based depot injection technologies: how versatile are they? American Journal of Drug Delivery. 2004; 4(2): 213–27.
51.Sjölander S, Drane D, Davis R, Beezum L, Pearse M, Cox J. Intranasal immunesation with influenza-ISCOM induces strong mucosal as well as systemic antibody and cytotoxic T-lymphocyte responses. Vaccine. 2001; 19(28-29): 4072–80.
52.Srivastava A, Gowda DV, Madhunapantula SV, Shinde CG, Iyer M. Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles. Acta Pathologica, Microbiologica, et Immunologica Scandinavica. 2015; 123(4): 275–88.
53.Atta-ur-Rahman. Bioactive Natural Products (Part E). Elsevier, Amsterdam, Netherlands. 2000; chpter3; 131-174.
54.W. John W. Morrow, Nadeem A. Sheikh, Clint S. Schmidt, D. Huw Davies. Vaccinology: Principles and Practice. John Wiley & Sons, Hoboken, New Jersey. 2012; part2 (chapter2).
55.Mohan T, Verma P, Rao DN. Novel adjuvants & delivery vehicles for vaccines development: A road ahead. The Indian Journal of Medical Research. 2013; 138(5): 779–95.
65
56.Liu XS, Abdul-Jabbar I, Qi YM, Frazer IH, Zhou J. Mucosal immunisation with papillomavirus virus-like particles elicits systemic and mucosal immunity in mice. Virology. 1998; 252(1): 39–45.
57.Vicente T, Roldão A, Peixoto C, Carrondo MJ, Alves PM. Large-scale production and purification of VLP-based vaccines. Journal of Invertebrate Pathology. 2011; 107 Suppl: S42–8.
58.Makidon PE, Belyakov IM, Janczak KW, et al. Nanoemulsion mucosal adjuvant uniquely activates cytokine production by nasal ciliated epithelium and induces dendritic cells trafficking. European Journal of Immunology. 2012; 42(8): 2073–86.
59.Wong PT, Wang SH, Ciotti S, Makidon PE, Smith DM, Fan Y, Schuler CF 4th, Baker JR Jr. Formulation and characterization of nano-emulsion intranasal adjuvants: effects of surfactant composition on mucoadhesion and immuno-genicity. Molecular Pharmaceutics. 2014; 11(2): 531–44.
60.Bielinska AU, O'Konek JJ, Janczak KW, Baker JR Jr. Immunomodulation of TH2 biased immunity with mucosal administration of nanoemulsion adjuvant. Vaccine. 2016; 34(34): 4017–24.
61.Shahiwala A, Amiji MM. Enhanced mucosal and systemic immune response with squalane oil-containing multiple emulsions upon intranasal and oral administration in mice. Journal of Drug Targeting. 2008; 16(4): 302–10.
62.Makidon PE, Bielinska AU, Nigavekar SS, Janczak KW, Knowlton J, Scott AJ, Mank N, Cao Z, Rathinavelu S, Beer MR, Wilkinson JE, Blanco LP, Landers JJ, Baker JR Jr. Pre-clinical evaluation of a novel nanoemulsion-based hepatitis B mucosal vaccine. PLoS One. 2008; 3(8): e2954.
63.Bielinska AU, Gerber M, Blanco LP, Makidon PE, Janczak KW, Beer M, Swanson B, Baker JR Jr. Induction of Th17 cellular immunity with a novel nanoemulsion adjuvant. Critical Reviews in Immunology. 2010; 30(2): 189–99.
64.Myc A, Kukowska-Latallo JF, Bielinska AU, Cao P, Myc PP, Janczak K, Sturm TR, Grabinski MS, Landers JJ, Young KS, Chang J, Hamouda T, Olszewski MA, Baker JR Jr. Development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza A virus and nanoemulsion. Vaccine. 2003; 21(25-26):3801–14.
65.Huang MH, Huang CY, Lien SP, Siao SY, Chou AH, Chen HW, Liu SJ, Leng CH, Chong P. Development of multi-phase emulsions based on bioresorbable polymers and oily adjuvant. Pharmaceutical Research. 2009; 26(8): 1856–62.
66.Allison AC. Squalene and squalane emulsions as adjuvants. Methods. 1999; 19(1) :87– 93.
67.Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunology & Cell Biology. 2004; 82(5): 488–96.
66
68.Arunachalam G. Basics and potential applications of surfactants - a review. International Journal of PharmTech Research. 2009; 1354–64.
69.Sylvia S. Talmage. Environmental and Human Safety of Major Surfactants: Alcohol Ethoxylates and Alkylphenol Ethoxylates. CRC Press, Boca Raton, Florida.1993; chapter4(A.8): 59-94.
70.Shalel S, Streichman S, Marmur A. The mechanism of hemolysis by surfactants: effect of solution composition. Journal of Colloid and Interface Science. 2002; 252(1): 66– 76.
71.Torchilin VP. Nanoparticulates as Drug Carriers. Imperial College Press, London. 2006; chapter7(3.1): 125-172.
72.ICI Americas, inc. The HLB System: A Time-saving Guide to Emulsifier Selection. ICI Americas, Incorporated, Wilmington, Delaware.1984; chapter1: 2-4.
73.Chen WL, Liu SJ, Leng CH, Chen HW, Chong P, Huang MH. Disintegration and cancer immunotherapy efficacy of a squalane-in-water delivery system emulsified by bioresorbable poly(ethylene glycol)-block-polylactide. Biomaterials. 2014; 35(5): 1686–95.
74.Aucouturier J, Dupuis L, Ganne V. Adjuvants designed for veterinary and human vaccines. Vaccine. 2001; 19(17-19): 2666–72.
75.Huang MH, Chou AH, Lien SP, Chen HW, Huang CY, Chen WW, Chong P, Liu SJ, Leng CH. Formulation and immunological evaluation of novel vaccine delivery systems based on bioresorbable poly(ethylene glycol)-block-poly(lactide-co-epsilon- caprolactone). Journal of Biomedical Materials Research. Part B, Applied Biomaterials. 2009; 90(2): 832–41.
76.Siao SY, Lin LH, Chen WW, Huang MH, Chong P. Characterization and emulsifying properties of block copolymers prepared from lactic acid and poly(ethylene glycol). Journal of Applied Polymer Science. 2009 ;114: 509–16.
77.Material Safety Data Sheet POLYSORBATE 80 MSDS. Available: http://www.sciencelab.com/msds.php?msdsId=9926645.
78.Torres-Arraut E, Singh S, Pickoff AS. Electrophysiologic effects of Tween 80 in the myocardium and specialized conduction system of the canine heart. Journal of Electrocardiology. 1984; 17(2): 145–51.
79.Benjamin L. Cells. Jones & Bartlett Learning, Burlington, Massachusetts. 2007; chapter4: 160.
80.Oyewumi MO, Kumar A, Cui Z. Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses. Expert Review of Vaccines. 2010; 9(9): 1095–107.
81.Mariusz Skwarczynski, stvan Toth. Micro- and Nanotechnology in Vaccine Development. William Andrew, New York, Norwich. 2016; chpter 9(1.5):171-83
67
82.Iyer V, Cayatte C, Guzman B, Schneider-Ohrum K, Matuszak R, Snell A, Rajani GM, McCarthy MP, Muralidhara B. Impact of formulation and particle size on stability and immunogenicity of oil-in-water emulsion adjuvants. Human Vaccines & Immunotherapeutics. 2015; 11(7): 1853–64.
83.Thakur VK, Thakur MK. Handbook of Polymers for Pharmaceutical Technologies, Structure and Chemistry. John Wiley & Sons, Hoboken, New Jersey. 2015; chapter14(4.2.5).
84.Avanti Lipids Polar, Inc. The Mini-extruder Instruction. Available: https://www.ncnr.nist.gov/userlab/pdf/E134extruder.pdf.
85.Kowert BA, Watson MB, Dang NC. Diffusion of squalene in n-alkanes and squalane. The Journal of Physical Chemistry B (ACS Publications). 2014; 118(8): 2157–63.
86.Christopher B. Fox. Final report on the safety assessment of squalane and squalene. Molecules. 2009; 14: 3286–312.
87.Gerhardt WW, Noga DE, Hardcastle KI, García AJ, Collard DM, Weck M. Functional lactide monomers: methodology and polymerization. Biomacromolecules. 2006; 7(6): 1735–42.
88.Su WF. Principles of Polymer Design and Synthesis. Springer Science & Business Media, Berlin, Heidelberg. 2013; chapter11(2): 267-300.
89.boua-in K, chaiyut N, ksapabutr B. Preparation of polylactide by ring-opening polymerisation of lactide. Optoelectronics and Advanced Materials-Rapid Communications. 2010; 1404–7.
90.Prendergast GC, Jaffee EM. Cancer Immunotherapy Immune Suppression and Tumor Growth, 2ed Ed. Academic Press, Cambridge, Massachusetts. 2013; chapter5: 55–70.
91.Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Pa- lucka K. Immunobiology of dendritic cells. Annual Review of Immunology. 2000; 18: 767-811.
92.Lecoanet-Henchoz S, Gauchat JF, Aubry JP, Graber P, Life P, Paul-Eugene N, Ferrua B, Corbi AL, Dugas B, Plater-Zyberk C, Bonnefoy JY. CD23 regulates monocyte activation through a novel interaction with the adhesion molecules CD11b-CD18 and CD11c-CD18. Immunity. 1995; 3(1): 119–25.
93.Ma DY, Clark EA. The role of CD40 and CD40L in dendritic cells. Seminars in immunology. 2009; 21(5): 265–72.
94.Grewal IS, Flavell RA. The role of CD40 ligand in costimulation and T-cell activation. Immunological Reviews. 1996; 153: 85–106.
95.Bishop GA, Hostager BS. Signaling by CD40 and its mimics in B cell activation. Immunologic Research. 2001; 24(2): 97–109.
96.Dilioglou S, Cruse JM, Lewis RE. Function of CD80 and CD86 on monocyte- and
68
stem cell-derived dendritic cells. Experimental and Molecular Pathology. 2003; 75(3): 217–27.
97.Mukherjee S, Maiti PK, Nandi D. Role of CD80, CD86, and CTLA4 on mouse CD4(+) T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin. Journal of Leukocyte Biology. 2002; 72(5): 921–31.
98.Vasilevko V, Ghochikyan A, Holterman MJ, Agadjanyan MG. CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA. DNA and Cell Biology. 2002; 21(3): 137–49.
99.Huang CH, Huang CY, Cheng CP, Dai SH, Chen HW, Leng CH, Chong P, Liu SJ, Huang MH. Degradable emulsion as vaccine adjuvant reshapes antigen-specific immunity and thereby ameliorates vaccine efficacy. Scientific Reports. 2016; 6: 36732.
100.van der Merwe PA, Davis SJ. Molecular interactions mediating T cell antigen recognition. Annual Review of Immunology. 2003; 21: 659–84.
101.Eskelinen EL. Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Molecular Aspects of Medicine. 2006; 27(5-6): 495–502.
102.Schulte, Ina Zhang, Enlu Meng, Pei ZJ, Lu RJ, Mengji Roggendorf, Michael. Recent advances in research on hepadnaviral infection in the woodchuck model. Virologica Sinica. 2008; 23: 107–115.
103.Kidd P. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Alternative Medicine Review. 2003; 8(3): 223–46.
104.Gaffen SL, Liu KD. Overview of interleukin-2 function, production and clinical applications. Cytokine. 2004; 28(3): 109–23.
105.Economou JS, McBride WH, Essner R, Rhoades K, Golub S, Holmes EC, Morton DL. Tumour necrosis factor production by IL-2-activated macrophages in vitro and in vivo. Immunology. 1989; 67(4): 514–19.
106.Schoenborn JR, Wilson CB. Regulation of interferon-gamma during innate and adaptive immune responses. Advances in Immunology. 2007; 96: 41–101.
107.Hofman FM, Brock M, Taylor CR, Lyons B. IL-4 regulates differentiation and proliferation of human precursor B cells. The Journal of Immunology. 1988; 141: 1185–90.
108.Lombard-Platet S, Meyer V, Ceredig R. Both IFN-γ and IL-4 induce MHC Class II expression at the surface of mouse pro-B cells. Developmental Immunology. 1997; 5(2): 115–20.
109.Horikawa K, Takatsu K. Interleukin-5 regulates genes involved in B-cell terminal maturation. Immunology. 2006; 118(4): 497–508.
69
110.Itoh K, Hirohata S. The role of IL-10 in human B cell activation, proliferation, and differentiation. The Journal of Immunology. 1995; 154(9): 4341–50.
111.Jin W, Dong C. IL-17 cytokines in immunity and inflammation. Emerging Microbes & Infections. 2013; 2(9): e60.
112.Murcia RY, Vargas A, Lavoie JP. The interleukin-17 induced activation and increased survival of equine neutrophils is insensitive to glucocorticoids. PLoS One. 2016; 11(5): e0154755.
113.Strelkauskas A, Edwards A, Fahnert B, Pryor G, Strelkauskas J. Microbiology: A Clinical Approach, 2ed Ed., Garland Science, New York. 2015; part IV(chapter16): 383-406.
114.Paul, William E. Fundamental Immunology, Sixth Ed., Lippincott Williams & Wilkins, Philadelphia. 2008; section3(chapter4): 125-51.
115.Klein-Schneegans AS, Kuntz L, Fonteneau P, Loor F. Serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL/6 mice and their congenics at the lpr (lymphoproliferation) locus. Journal of Autoimmunity. 1989; 2(6): 869-75.
116.Martin R, Brady J, Lew A. The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice. Journal of Immunological Methods. 1998; 212: 187-92.
117.Mantis NJ, Rol N, Corthésy B. Secretory IgA’s complex roles in immunity and mucosal homeostasis in the gut. Mucosal Immunology. 2011; 4(6): 603-11.
118.Fischer AH, Jacobson KA, Rose J, Zeller R. Hematoxylin and Eosin Staining of Tissue and Cell Sections. Cold Spring Harbor Protoc. 2008; pdb.prot4986.