Target Discovery and Validation Reviews and Protocols

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Overview of Immune System 311 Fig. 14. Immunostimulatory small-interfering RNA (siRNA)-liposomes can be highly effective adjuvant for bridging between type 1 innate and acquired immunity. Administration of double-stranded siRNA or single-stranded siRNAs in liposomes induced the activation of Toll-like receptor 7- and 8-expressing monocytes and dendritic cells (DCs) to produce cytokines (e.g. tumor necrosis factor [TNF]α, IL-12) and interferons (IFNα), which can activate natural killer (NK) cells and natural killer T-cells (NKT) (97). The produced cytokines induce the activation DCs and potential maturation of tumor monocytes into dendritic cells, which will activate T- and B-cells, leading to T-helper (Th) type 1 response. Activated cytoxic T-cells (CTL) migrate into the tumor site from the draining lymph nodes and eventually will eradicate tumor cells. which enables them to migrate to the lymph nodes and to present peptides derived from the acquired antigens to lymph node-residing T-cells, leading to a cellular immune response that involves both CD4+ and CD8+ T-cells (Fig. 7).
312 Sioud Because of their important role in immunity, ex vivo-generated antigen-loaded DCs have now been used as vaccines to improve immunity. Although DCs can be obtained from several sources, for clinical trials monocyte-derived DCs are the most widely used DCs. Monocytes can be differentiated into either macrophages, which function as scavengers, or DCs, which induce specific immune responses. Different cytokines skew the in vivo differentiation of monocytes into DCs with different phenotypes and functions. Alteration of DC function can be achieved by transfection with immunomodulatory molecules. Endogenous signal also can be inhibited by several techniques, such as the use of antisense oligonucleotides and siRNAs. It is worth noting that the inherent immunogenicity of DCs can be unregulated by stimulation of these cells through various receptors, such as the TLRs. More recently, we have found that certain siRNA sequences activated monocytes and DCs via endosomal TLRs, leading to the production of TNFα and IFNα (98). Internalization of siRNAs was a prerequisite to activate TLR7 and -8 signaling pathways, suggesting that encapsulation of immunostimulatory siRNA in liposomes may increase their recognition by the innate effector cells such DCs and macrophages. Besides the activation of DC maturation by immunostimulatory siRNAs, the inhibition of immunosuppressive cytokines and host factors by siRNAs should lead to successful cancer vaccines. Notably, IL-10–producing Treg cells and IL-10–producing regulatory DCs suppress cellular immune responses. By targeting IL-10 with an immunostimulatory siRNA, we hope to both downregulate the expression IL- 10 and activate innate type 1 response against tumors, as illustrated in Fig. 14. Several recent studies have documented the utility of siRNA-mediated gene silencing in vitro and in vivo (90). Therapeutic vaccination combined with strategies that seek to inactivate the function of Treg cells via the use the RNA interference should lead to the design of better vaccines. References 1. Burnet, F. M. (1971) Immunological surveillance in neoplasia. Transplant. Rev. 7, 3–25. 2. Diefenbach, A. and Roulette, D. H. (2002) The innate immune response to tumors and its role in the induction of T-cell immunity. Immunol. Rev. 188, 9–21. 3. Rosenberg, S. A. (1999) A new era for cancer immunotherapy based on the genes that encodes cancer antigens. Immunity 10, 281–287. 4. Vose, B. M. and Moore, M. (1985) Human tumor-infiltrating lymphocytes: a marker of host response. Semin. Hematol. 22, 27–40. 5. Østenstad, B., Sioud, M., Schlichting, E., Lea, T., and Harboe, M. (1995) Freshly isolated tumour-infiltrating T-lymphocytes have a high cytotoxic potential, as
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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204 Vijayaraj, Söhl, and Magin 1.
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12 The HUVEC/Matrigel Assay An In V
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258 Skovseth, Küchler, and Haralds
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Page 332 and 333: 15 Potential Target Antigens for Im
Page 334 and 335: Tumor Antigen Discovery 321 develop
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Page 340 and 341: 16 Identification of Tumor Antigens
Page 342 and 343: Immunoproteomics 329 by “shot gun
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Page 346 and 347: Immunoproteomics 333 3. 2D-PAGE is
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Page 356 and 357: Protein Arrays 343 The ideal proteo
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Page 360 and 361: Protein Arrays 347 49. Yuko Kawahas
Page 362 and 363: Index 349 Index A Acetyl-CoA carbox
Page 364 and 365: Index 351 conditional by using FRT
Page 366 and 367: Index 353 Recombinant tumor antigen
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