cef397ca0dbff2d4a2fba3f3d8a135b0 pmc_9865626.pdf 08a0e8edc12545d4a5524d74a53a3576df43cc54 pmc_9865626.pdf 93e598de654032916e385f291fde02968429f36665a5bc9d4aac0eba6ed10653 pmc_9865626.pdf Title: The Local Activation of Toll-like Receptor 7 (TLR7) Modulates Colonic Epithelial Barrier Function in Rats Subject: Toll-like receptors (TLRs)-mediated host–bacterial interactions participate in the microbial regulation of gastrointestinal functions, including the epithelial barrier function (EBF). We evaluated the effects of TLR7 stimulation on the colonic EBF in rats. TLR7 was stimulated with the selective agonist imiquimod (100/300 g/rat, intracolonic), with or without the intracolonic administration of dimethyl sulfoxide (DMSO). Colonic EBF was assessed in vitro (electrophysiology and permeability to macromolecules, Ussing chamber) and in vivo (passage of macromolecules to blood and urine). Changes in the expression (RT-qPCR) and distribution (immunohistochemistry) of tight junction-related proteins were determined. Expression of proglucagon, precursor of the barrier-enhancer factor glucagon-like peptide 2 (GLP-2) was also assessed (RT-qPCR). Intracolonic imiquimod enhanced the EBF in vitro, reducing the epithelial conductance and the passage of macromolecules, thus indicating a pro-barrier effect of TLR7. However, the combination of TLR7 stimulation and DMSO had a detrimental effect on the EBF, which manifested as an increased passage of macromolecules. DMSO alone had no effect. The modulation of the EBF (imiquimod alone or with DMSO) was not associated with changes in gene expression or the epithelial distribution of the main tight junction-related proteins (occludin, tricellulin, claudin-2, claudin-3, junctional adhesion molecule 1 and Zonula occludens-1). No changes in the proglucagon expression were observed. These results show that TLR7 stimulation leads to the modulation of the colonic EBF, having beneficial or detrimental effects depending upon the state of the epithelium. The underlying mechanisms remain elusive, but seem independent of the modulation of the main tight junction-related proteins or the barrier-enhancer factor GLP-2. Keywords: epithelial barrier function; host–bacterial interactions; imiquimod; intestinal permeability; tight junctions; TLR; Toll-like receptors Author: Javier Estévez and Vicente Martínez Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Jan 9 04:10:17 2023 CET ModDate: Mon Jan 9 04:16:22 2023 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 17 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2315242 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- NBMIUS+VnURWPalladioL Type 1 Custom yes yes no 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 15 0 JLOJZC+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 CTLYYG+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 EDOKCR+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 YSPQJI+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 50 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 53 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 75 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 83 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 91 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 95 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 99 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 103 0 Arial,Bold TrueType WinAnsi no no no 111 0 Symbol CID TrueType Identity-H yes no yes 115 0 BCDIEE+ARIAL NARROW,Bold TrueType WinAnsi yes yes no 123 0 Arial TrueType WinAnsi no no no 127 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 141 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 145 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 149 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 153 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 169 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 173 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 177 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 181 0 Arial,Bold TrueType WinAnsi no no no 190 0 Arial TrueType WinAnsi no no no 193 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 207 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 211 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 215 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 219 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 236 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 240 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 244 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 248 0 Arial TrueType WinAnsi no no no 256 0 Arial,Bold TrueType WinAnsi no no no 269 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 282 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 286 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 290 0 BCDHEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 294 0 Arial TrueType WinAnsi no no no 302 0 Arial,Bold TrueType WinAnsi no no no 306 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-10-19 02:23:33 CEST RepresentationInformation: pmc_9865626.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-01-26 02:11:32 CET Size: 2315242 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 519 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: The Acute Addition of Imiquimod to the Ussing Chamber Did Not Affect Colonic Electrical Parameters or Permeability to Macromolecules Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: The Over-Stimulation of Colonic TLR7 with Imiquimod Does Not Trigger an Inflammatory-like Response within the Colon Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Pretreatment with Imiquimod Altered the Colonic Epithelial Conductance and Reduced Paracellular Permeability In Vitro Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Imiquimod Enhances Colonic Permeability after Disruption of the Epithelial Barrier in In Vivo Conditions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Effects of Imiquimod on Tight Junction-Related Proteins and Barrier-Modulators Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Children: Item: Title: Chemicals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Animals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Tissue Sampling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Measurement of Electrophysiological Parameters (Ussing Chambers) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Epithelial Paracellular Permeability In Vitro Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: The In Vivo Colonic Permeability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Experimental Protocols Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Children: Item: Title: The Effects of Imiquimod on Epithelial Electrical Parameters Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: The Effects of Imiquimod on Epithelial Permeability In Vitro Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: The Effects of Imiquimod on Epithelial Permeability In Vivo Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: The Quantitative Real Time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: Immunohistochemistry for Tight-Junction-Related Proteins Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Info: Title: The Local Activation of Toll-like Receptor 7 (TLR7) Modulates Colonic Epithelial Barrier Function in Rats Author: Javier Estévez and Vicente Martínez Subject: Toll-like receptors (TLRs)-mediated host bacterial interactions participate in the microbial regulation of gastrointestinal functions, including the epithelial barrier function (EBF). We evaluated the effects of TLR7 stimulation on the colonic EBF in rats. TLR7 was stimulated with the selective agonist imiquimod (100/300 g/rat, intracolonic), with or without the intracolonic administration of dimethyl sulfoxide (DMSO). Colonic EBF was assessed in vitro (electrophysiology and permeability to macromolecules, Ussing chamber) and in vivo (passage of macromolecules to blood and urine). Changes in the expression (RT-qPCR) and distribution (immunohistochemistry) of tight junction-related proteins were determined. Expression of proglucagon, precursor of the barrier-enhancer factor glucagon-like peptide 2 (GLP-2) was also assessed (RT-qPCR). Intracolonic imiquimod enhanced the EBF in vitro, reducing the epithelial conductance and the passage of macromolecules, thus indicating a pro-barrier effect of TLR7. However, the combination of TLR7 stimulation and DMSO had a detrimental effect on the EBF, which manifested as an increased passage of macromolecules. DMSO alone had no effect. The modulation of the EBF (imiquimod alone or with DMSO) was not associated with changes in gene expression or the epithelial distribution of the main tight junction-related proteins (occludin, tricellulin, claudin-2, claudin-3, junctional adhesion molecule 1 and Zonula occludens-1). No changes in the proglucagon expression were observed. These results show that TLR7 stimulation leads to the modulation of the colonic EBF, having beneficial or detrimental effects depending upon the state of the epithelium. The underlying mechanisms remain elusive, but seem independent of the modulation of the main tight junction-related proteins or the barrier-enhancer factor GLP-2. Keywords: epithelial barrier function; host bacterial interactions; imiquimod; intestinal permeability; tight junctions; TLR; Toll-like receptors Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Jan 09 04:10:17 CET 2023 ModDate: Mon Jan 09 04:16:22 CET 2023 ID: 0xcd13bc8545d2e2b4a4bdba45dad916df, 0xcd13bc8545d2e2b4a4bdba45dad916df Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: YSPQJI+VnURWPalladioL-Bold FontSubset: true FirstChar: 178 LastChar: 189 FontDescriptor: FontName: YSPQJI+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JLOJZC+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: JLOJZC+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MPUDFL+PazoMath FontSubset: true FirstChar: 68 LastChar: 68 FontDescriptor: FontName: MPUDFL+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: CTLYYG+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 246 FontDescriptor: FontName: CTLYYG+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: NBMIUS+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 243 FontDescriptor: FontName: NBMIUS+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: YFFKLH+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: YFFKLH+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: EDOKCR+URWPalladioL-Ital FontSubset: true FirstChar: 3 LastChar: 121 FontDescriptor: FontName: EDOKCR+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 269, 77, 278 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 180, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 232, 661, 243, 674 Annotation: Subtype: Link Rect: 408, 176, 415, 184 ActionDest: 330 Annotation: Subtype: Link Rect: 418, 176, 425, 184 ActionDest: 330 Annotation: Subtype: Link Rect: 307, 138, 314, 147 ActionDest: 330 Annotation: Subtype: Link Rect: 309, 113, 316, 122 ActionDest: 330 Annotation: Subtype: Link Rect: 319, 113, 326, 122 ActionDest: 330 Annotation: Subtype: Link Rect: 139, 17, 278, 28 Annotation: Subtype: Link Rect: 422, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 354, 87, 373 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 347, 723, 359, 732 ActionDest: 330 Annotation: Subtype: Link Rect: 397, 710, 404, 719 ActionDest: 330 Annotation: Subtype: Link Rect: 405, 710, 412, 719 ActionDest: 330 Annotation: Subtype: Link Rect: 412, 710, 424, 719 ActionDest: 330 Annotation: Subtype: Link Rect: 427, 710, 439, 719 ActionDest: 330 Annotation: Subtype: Link Rect: 427, 673, 439, 682 ActionDest: 330 Annotation: Subtype: Link Rect: 531, 648, 543, 657 ActionDest: 330 Annotation: Subtype: Link Rect: 544, 647, 556, 657 ActionDest: 333 Annotation: Subtype: Link Rect: 275, 622, 287, 631 ActionDest: 333 Annotation: Subtype: Link Rect: 287, 622, 299, 631 ActionDest: 333 Annotation: Subtype: Link Rect: 190, 597, 202, 606 ActionDest: 330 Annotation: Subtype: Link Rect: 529, 585, 541, 594 ActionDest: 333 Annotation: Subtype: Link Rect: 544, 585, 556, 594 ActionDest: 333 Annotation: Subtype: Link Rect: 448, 547, 459, 556 ActionDest: 333 Annotation: Subtype: Link Rect: 460, 547, 472, 556 ActionDest: 333 Annotation: Subtype: Link Rect: 432, 497, 444, 506 ActionDest: 333 Annotation: Subtype: Link Rect: 444, 497, 456, 506 ActionDest: 333 Annotation: Subtype: Link Rect: 400, 472, 412, 481 ActionDest: 333 Annotation: Subtype: Link Rect: 304, 334, 316, 343 ActionDest: 333 Annotation: Subtype: Link Rect: 450, 209, 462, 218 ActionDest: 330 Annotation: Subtype: Link Rect: 463, 209, 475, 218 ActionDest: 333 Annotation: Subtype: Link Rect: 322, 181, 329, 193 ActionDest: 79 Annotation: Subtype: Link Rect: 382, 169, 389, 181 ActionDest: 79 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 337, 86, 343, 98 ActionDest: 133 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 475, 259, 482, 271 ActionDest: 79 Annotation: Subtype: Link Rect: 311, 110, 318, 122 ActionDest: 161 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 479, 138, 486, 150 ActionDest: 133 Annotation: Subtype: Link Rect: 199, 113, 206, 125 ActionDest: 161 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 311, 720, 318, 732 ActionDest: 199 Annotation: Subtype: Link Rect: 340, 695, 347, 707 ActionDest: 199 Annotation: Subtype: Link Rect: 503, 683, 510, 695 ActionDest: 199 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 262, 720, 269, 732 ActionDest: 228 Annotation: Subtype: Link Rect: 453, 670, 460, 682 ActionDest: 228 Annotation: Subtype: Link Rect: 549, 657, 556, 669 ActionDest: 228 Annotation: Subtype: Link Rect: 467, 632, 474, 644 ActionDest: 228 Annotation: Subtype: Link Rect: 494, 632, 501, 644 ActionDest: 274 Annotation: Subtype: Link Rect: 328, 607, 335, 619 ActionDest: 274 Page: Sequence: 8 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 355, 648, 362, 657 ActionDest: 330 Annotation: Subtype: Link Rect: 365, 647, 372, 657 ActionDest: 330 Annotation: Subtype: Link Rect: 267, 560, 274, 569 ActionDest: 330 Annotation: Subtype: Link Rect: 275, 560, 287, 569 ActionDest: 330 Annotation: Subtype: Link Rect: 287, 560, 299, 569 ActionDest: 330 Annotation: Subtype: Link Rect: 300, 560, 312, 569 ActionDest: 333 Annotation: Subtype: Link Rect: 315, 560, 327, 569 ActionDest: 333 Annotation: Subtype: Link Rect: 214, 522, 221, 531 ActionDest: 330 Annotation: Subtype: Link Rect: 221, 522, 233, 531 ActionDest: 333 Annotation: Subtype: Link Rect: 234, 522, 246, 531 ActionDest: 333 Annotation: Subtype: Link Rect: 246, 522, 258, 531 ActionDest: 333 Annotation: Subtype: Link Rect: 359, 434, 371, 443 ActionDest: 333 Annotation: Subtype: Link Rect: 371, 434, 383, 443 ActionDest: 333 Annotation: Subtype: Link Rect: 544, 321, 556, 330 ActionDest: 333 Annotation: Subtype: Link Rect: 264, 259, 276, 267 ActionDest: 333 Annotation: Subtype: Link Rect: 277, 259, 289, 267 ActionDest: 333 Annotation: Subtype: Link Rect: 289, 258, 301, 267 ActionDest: 333 Annotation: Subtype: Link Rect: 433, 208, 445, 217 ActionDest: 333 Annotation: Subtype: Link Rect: 445, 208, 457, 217 ActionDest: 333 Annotation: Subtype: Link Rect: 458, 208, 470, 217 ActionDest: 333 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 338, 622, 350, 631 ActionDest: 336 Annotation: Subtype: Link Rect: 544, 309, 556, 318 ActionDest: 333 Annotation: Subtype: Link Rect: 432, 158, 444, 167 ActionDest: 333 Annotation: Subtype: Link Rect: 430, 133, 442, 142 ActionDest: 330 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 346, 735, 358, 744 ActionDest: 333 Annotation: Subtype: Link Rect: 359, 735, 371, 744 ActionDest: 333 Annotation: Subtype: Link Rect: 397, 660, 409, 669 ActionDest: 336 Annotation: Subtype: Link Rect: 346, 647, 357, 657 ActionDest: 336 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 196, 560, 208, 568 ActionDest: 330 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 212, 459, 224, 468 ActionDest: 336 Annotation: Subtype: Link Rect: 417, 170, 429, 179 ActionDest: 336 Page: Sequence: 14 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 469, 485, 507, 496 Annotation: Subtype: Link Rect: 160, 462, 197, 473 Annotation: Subtype: Link Rect: 203, 462, 239, 473 Annotation: Subtype: Link Rect: 373, 439, 410, 450 Annotation: Subtype: Link Rect: 417, 439, 453, 450 Annotation: Subtype: Link Rect: 490, 416, 526, 427 Annotation: Subtype: Link Rect: 238, 393, 275, 404 Annotation: Subtype: Link Rect: 281, 393, 317, 404 Annotation: Subtype: Link Rect: 59, 371, 96, 381 Annotation: Subtype: Link Rect: 102, 371, 138, 381 Annotation: Subtype: Link Rect: 463, 358, 500, 369 Annotation: Subtype: Link Rect: 508, 335, 546, 346 Annotation: Subtype: Link Rect: 154, 301, 191, 312 Annotation: Subtype: Link Rect: 100, 267, 137, 277 Annotation: Subtype: Link Rect: 59, 232, 96, 243 Annotation: Subtype: Link Rect: 102, 232, 138, 243 Annotation: Subtype: Link Rect: 417, 209, 455, 220 Annotation: Subtype: Link Rect: 59, 175, 96, 185 Annotation: Subtype: Link Rect: 102, 175, 138, 185 Annotation: Subtype: Link Rect: 201, 129, 239, 139 Annotation: Subtype: Link Rect: 245, 129, 281, 139 Annotation: Subtype: Link Rect: 122, 94, 159, 105 Annotation: Subtype: Link Rect: 166, 94, 202, 105 Annotation: Subtype: Link Rect: 519, 82, 557, 93 Annotation: Subtype: Link Rect: 59, 71, 95, 81 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 188, 722, 225, 733 Annotation: Subtype: Link Rect: 416, 710, 454, 721 Annotation: Subtype: Link Rect: 104, 688, 141, 698 Annotation: Subtype: Link Rect: 519, 664, 557, 675 Annotation: Subtype: Link Rect: 59, 653, 95, 664 Annotation: Subtype: Link Rect: 131, 630, 168, 640 Annotation: Subtype: Link Rect: 175, 630, 211, 640 Annotation: Subtype: Link Rect: 217, 595, 254, 606 Annotation: Subtype: Link Rect: 261, 595, 297, 606 Annotation: Subtype: Link Rect: 208, 572, 246, 583 Annotation: Subtype: Link Rect: 164, 549, 202, 560 Annotation: Subtype: Link Rect: 458, 526, 495, 537 Annotation: Subtype: Link Rect: 314, 503, 352, 514 Annotation: Subtype: Link Rect: 59, 469, 96, 479 Annotation: Subtype: Link Rect: 349, 445, 386, 456 Annotation: Subtype: Link Rect: 238, 423, 276, 433 Annotation: Subtype: Link Rect: 237, 388, 274, 399 Annotation: Subtype: Link Rect: 403, 365, 441, 376 Annotation: Subtype: Link Rect: 447, 365, 483, 376 Annotation: Subtype: Link Rect: 478, 342, 515, 353 Annotation: Subtype: Link Rect: 521, 342, 557, 353 Annotation: Subtype: Link Rect: 169, 320, 207, 330 Annotation: Subtype: Link Rect: 86, 297, 123, 307 Annotation: Subtype: Link Rect: 129, 297, 165, 307 Annotation: Subtype: Link Rect: 206, 273, 244, 284 Annotation: Subtype: Link Rect: 378, 250, 415, 261 Annotation: Subtype: Link Rect: 471, 227, 508, 238 Annotation: Subtype: Link Rect: 208, 204, 246, 215 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 424, 169, 461, 180 Annotation: Subtype: Link Rect: 467, 169, 503, 180 Annotation: Subtype: Link Rect: 59, 147, 96, 157 Annotation: Subtype: Link Rect: 102, 147, 138, 157 Annotation: Subtype: Link Rect: 238, 123, 276, 134 Annotation: Subtype: Link Rect: 282, 123, 318, 134 Annotation: Subtype: Link Rect: 59, 90, 96, 100 Annotation: Subtype: Link Rect: 102, 90, 138, 100 Annotation: Subtype: Link Rect: 509, 66, 547, 77 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 417, 733, 454, 744 Annotation: Subtype: Link Rect: 239, 711, 277, 721 Annotation: Subtype: Link Rect: 514, 687, 551, 698 Annotation: Subtype: Link Rect: 224, 665, 261, 675 Checksum: 2142fe49 Type: CRC32 Checksum: cef397ca0dbff2d4a2fba3f3d8a135b0 Type: MD5 Checksum: 08a0e8edc12545d4a5524d74a53a3576df43cc54 Type: SHA-1