e7e98b623976421c0f234accd8e87770 pmc_9913488.pdf 36997f49a9347967d354cafba651e92af4bf0688 pmc_9913488.pdf 0847ff8d3403804aebc2eeae75b7ef1b4d175c2d7f7c42d0d68013d6f4495e3e pmc_9913488.pdf Title: Acute Stress Regulates Sex-Related Molecular Responses in the Human Jejunal Mucosa: Implications for Irritable Bowel Syndrome Subject: Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder linked to intestinal barrier dysfunction and life stress. We have previously reported that female sex per se determines an increased susceptibility to intestinal barrier dysfunction after cold pain stress (CPS). We aimed to identify sex-related molecular differences in response to CPS in healthy subjects to understand the origin of sex bias predominance in IBS. In 13 healthy males and 21 females, two consecutive jejunal biopsies were obtained using Watson’s capsule, at baseline, and ninety minutes after CPS. Total mucosal RNA and protein were isolated from jejunal biopsies. Expression of genes related to epithelial barrier (CLDN1, CLDN2, OCLN, ZO-1, and ZO-3), mast cell (MC) activation (TPSAB1, SERPINA1), and the glucocorticoid receptor (NR3C1) were analyzed using RT-qPCR. NR3C1, ZO-1 and OCLN protein expression were evaluated through immunohistochemistry and western blot, and mucosal inflammation through MC, lymphocyte, and eosinophil numbering. Autonomic, hormonal, and psychological responses to CPS were monitored. We found an increase in jejunal MCs, a reduced CLDN1 and OCLN expression, and an increased CLDN2 and SERPINA1 expression 90 min after CPS. We also found a significant decrease in ZO-1, OCLN, and NR3C1 gene expression, and a decrease in OCLN protein expression only in females, when compared to males. CPS induced a significant increase in blood pressure, plasma cortisol and ACTH, and subjective stress perception in all participants. Specific and independent sex-related molecular responses in epithelial barrier regulation are unraveled by acute stress in the jejunum of healthy subjects and may partially explain female predominance in IBS. Keywords: acute stress; sex; intestinal barrier; human; irritable bowel syndrome Author: Bruno K. Rodiño-Janeiro, Marc Pigrau, Eloísa Salvo-Romero, Adoración Nieto, Elba Expósito, Ana M. González-Castro, Carmen Galán, Inés de Torres, Teodora Pribic, Laura Hernández, Beatriz Lobo, Marina Fortea, Milagros Gallart, Cristina Pardo-Camacho, Danila Guagnozzi, Javier Santos and Carmen Alonso-Cotoner Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Feb 6 07:12:31 2023 CET ModDate: Mon Feb 6 07:31:52 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: 2611236 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- JYPLJJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 GFKJOY+VnURWPalladioL Type 1 Custom yes yes no 15 0 TXPGTR+URWPalladioL-Bold Type 1 Custom yes yes no 19 0 VEQGZO+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 WBUGML+URWPalladioL-Ital Type 1 Custom yes yes no 28 0 PFKOHU+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 46 0 UWJUYN+CMSY10 Type 1 Builtin yes yes no 71 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 78 0 AHDBBC+PalatinoLinotype TrueType WinAnsi yes yes no 88 0 WBVKDJ+URWPalladioL-BoldItal Type 1 Custom yes yes no 102 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 110 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 114 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 118 0 BCDHEE+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 122 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 129 0 BCDJEE+PalatinoLinotype-BoldItalic TrueType WinAnsi yes yes no 136 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 149 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 153 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 157 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 170 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 174 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 178 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 182 0 ArialMT TrueType WinAnsi no no no 189 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 202 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 206 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 210 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 214 0 LWLIVD+EURM10 Type 1 Builtin yes yes no 226 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 234 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 238 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 242 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 246 0 BCDEEE+PalatinoLinotype-Bold TrueType WinAnsi yes yes no 259 0 BCDFEE+PalatinoLinotype-Roman TrueType WinAnsi yes yes no 263 0 BCDGEE+PalatinoLinotype-Italic TrueType WinAnsi yes yes no 267 0 BCDIEE+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 271 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-08 02:27:36 CEST RepresentationInformation: pmc_9913488.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-30 02:16:01 CEST Size: 2611236 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 543 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Participants Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Cold Pain Stress (CPS) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Jejunal Biopsy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Systemic Response to CPS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Hand Pain Perception Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Autonomic Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Psychological Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Hormonal Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Intestinal Response to CPS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Molecular Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Mucosal Inflammation and NR3C1 Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Experimental Design and Procedures (Figure 1) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Demographics, Baseline Stress, and Depression Levels Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Systemic Response to CPS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Children: Item: Title: Autonomic Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Psychological Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Hand Pain Perception Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Hormonal Response Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Intestinal Response to CPS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Children: Item: Title: Mucosal Gene Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Mucosal Inflammation and NR3C1 Protein Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: TJ Protein Expression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Info: Title: Acute Stress Regulates Sex-Related Molecular Responses in the Human Jejunal Mucosa: Implications for Irritable Bowel Syndrome Author: Bruno K. Rodiño-Janeiro, Marc Pigrau, Eloísa Salvo-Romero, Adoración Nieto, Elba Expósito, Ana M. González-Castro, Carmen Galán, Inés de Torres, Teodora Pribic, Laura Hernández, Beatriz Lobo, Marina Fortea, Milagros Gallart, Cristina Pardo-Camacho, Danila Guagnozzi, Javier Santos and Carmen Alonso-Cotoner Subject: Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder linked to intestinal barrier dysfunction and life stress. We have previously reported that female sex per se determines an increased susceptibility to intestinal barrier dysfunction after cold pain stress (CPS). We aimed to identify sex-related molecular differences in response to CPS in healthy subjects to understand the origin of sex bias predominance in IBS. In 13 healthy males and 21 females, two consecutive jejunal biopsies were obtained using Watson’s capsule, at baseline, and ninety minutes after CPS. Total mucosal RNA and protein were isolated from jejunal biopsies. Expression of genes related to epithelial barrier (CLDN1, CLDN2, OCLN, ZO-1, and ZO-3), mast cell (MC) activation (TPSAB1, SERPINA1), and the glucocorticoid receptor (NR3C1) were analyzed using RT-qPCR. NR3C1, ZO-1 and OCLN protein expression were evaluated through immunohistochemistry and western blot, and mucosal inflammation through MC, lymphocyte, and eosinophil numbering. Autonomic, hormonal, and psychological responses to CPS were monitored. We found an increase in jejunal MCs, a reduced CLDN1 and OCLN expression, and an increased CLDN2 and SERPINA1 expression 90 min after CPS. We also found a significant decrease in ZO-1, OCLN, and NR3C1 gene expression, and a decrease in OCLN protein expression only in females, when compared to males. CPS induced a significant increase in blood pressure, plasma cortisol and ACTH, and subjective stress perception in all participants. Specific and independent sex-related molecular responses in epithelial barrier regulation are unraveled by acute stress in the jejunum of healthy subjects and may partially explain female predominance in IBS. Keywords: acute stress; sex; intestinal barrier; human; irritable bowel syndrome Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Feb 06 07:12:31 CET 2023 ModDate: Mon Feb 06 07:31:52 CET 2023 ID: 0x094444fa09bbf494ac2ff76cf0c9ed0b, 0x094444fa09bbf494ac2ff76cf0c9ed0b Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: LWLIVD+EURM10 FontSubset: true FirstChar: 12 LastChar: 12 FontDescriptor: FontName: LWLIVD+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: TXPGTR+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 241 FontDescriptor: FontName: TXPGTR+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WBVKDJ+URWPalladioL-BoldItal FontSubset: true FirstChar: 112 LastChar: 112 FontDescriptor: FontName: WBVKDJ+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: UWJUYN+CMSY10 FontSubset: true FirstChar: 0 LastChar: 21 FontDescriptor: FontName: UWJUYN+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: VEQGZO+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 246 FontDescriptor: FontName: VEQGZO+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: JYPLJJ+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: JYPLJJ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: WBUGML+URWPalladioL-Ital FontSubset: true FirstChar: 40 LastChar: 121 FontDescriptor: FontName: WBUGML+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 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: GFKJOY+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: GFKJOY+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 34, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 271, 78, 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, 121, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 175, 642, 186, 655 Annotation: Subtype: Link Rect: 247, 629, 258, 643 Annotation: Subtype: Link Rect: 423, 629, 434, 643 Annotation: Subtype: Link Rect: 516, 629, 528, 643 Annotation: Subtype: Link Rect: 209, 616, 221, 630 Annotation: Subtype: Link Rect: 409, 604, 420, 617 Annotation: Subtype: Link Rect: 100, 17, 240, 28 Annotation: Subtype: Link Rect: 421, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 402, 87, 421 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 539, 707, 546, 716 ActionDest: 290 Annotation: Subtype: Link Rect: 549, 707, 556, 716 ActionDest: 290 Annotation: Subtype: Link Rect: 217, 644, 224, 654 ActionDest: 290 Annotation: Subtype: Link Rect: 233, 619, 240, 629 ActionDest: 290 Annotation: Subtype: Link Rect: 225, 607, 232, 616 ActionDest: 290 Annotation: Subtype: Link Rect: 235, 607, 242, 616 ActionDest: 290 Annotation: Subtype: Link Rect: 519, 607, 531, 616 ActionDest: 290 Annotation: Subtype: Link Rect: 531, 607, 543, 616 ActionDest: 290 Annotation: Subtype: Link Rect: 348, 569, 359, 578 ActionDest: 290 Annotation: Subtype: Link Rect: 362, 569, 374, 578 ActionDest: 290 Annotation: Subtype: Link Rect: 438, 519, 450, 528 ActionDest: 290 Annotation: Subtype: Link Rect: 450, 519, 462, 528 ActionDest: 290 Annotation: Subtype: Link Rect: 278, 494, 289, 503 ActionDest: 290 Annotation: Subtype: Link Rect: 290, 494, 301, 503 ActionDest: 290 Annotation: Subtype: Link Rect: 302, 494, 313, 503 ActionDest: 290 Annotation: Subtype: Link Rect: 511, 494, 523, 503 ActionDest: 290 Annotation: Subtype: Link Rect: 523, 494, 535, 503 ActionDest: 293 Annotation: Subtype: Link Rect: 188, 469, 200, 478 ActionDest: 293 Annotation: Subtype: Link Rect: 323, 469, 335, 478 ActionDest: 290 Annotation: Subtype: Link Rect: 461, 444, 473, 453 ActionDest: 293 Annotation: Subtype: Link Rect: 325, 419, 337, 428 ActionDest: 293 Annotation: Subtype: Link Rect: 340, 419, 352, 428 ActionDest: 293 Annotation: Subtype: Link Rect: 421, 406, 433, 415 ActionDest: 290 Annotation: Subtype: Link Rect: 434, 406, 446, 415 ActionDest: 293 Annotation: Subtype: Link Rect: 446, 406, 458, 415 ActionDest: 293 Annotation: Subtype: Link Rect: 544, 369, 556, 377 ActionDest: 293 Annotation: Subtype: Link Rect: 244, 343, 256, 352 ActionDest: 293 Annotation: Subtype: Link Rect: 497, 293, 509, 302 ActionDest: 293 Annotation: Subtype: Link Rect: 268, 154, 280, 163 ActionDest: 293 Annotation: Subtype: Link Rect: 475, 154, 487, 163 ActionDest: 293 Annotation: Subtype: Link Rect: 266, 129, 278, 138 ActionDest: 293 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 507, 622, 519, 631 ActionDest: 293 Annotation: Subtype: Link Rect: 279, 243, 291, 253 ActionDest: 293 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 507, 459, 519, 468 ActionDest: 290 Annotation: Subtype: Link Rect: 367, 434, 374, 446 ActionDest: 82 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 200, 215, 208, 227 ActionDest: 82 Annotation: Subtype: Link Rect: 529, 202, 536, 214 ActionDest: 98 Annotation: Subtype: Link Rect: 251, 164, 258, 176 ActionDest: 98 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 288, 473, 295, 484 ActionDest: 98 Annotation: Subtype: Link Rect: 419, 122, 425, 134 ActionDest: 141 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 199, 208, 206, 220 ActionDest: 141 Annotation: Subtype: Link Rect: 199, 107, 206, 119 ActionDest: 141 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 548, 683, 555, 695 ActionDest: 162 Annotation: Subtype: Link Rect: 404, 129, 411, 141 ActionDest: 194 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 246, 297, 253, 309 ActionDest: 194 Annotation: Subtype: Link Rect: 243, 234, 250, 246 ActionDest: 194 Annotation: Subtype: Link Rect: 487, 209, 494, 221 ActionDest: 194 Annotation: Subtype: Link Rect: 283, 58, 290, 70 ActionDest: 222 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 438, 382, 445, 394 ActionDest: 222 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 263, 632, 275, 641 ActionDest: 293 Annotation: Subtype: Link Rect: 275, 632, 287, 641 ActionDest: 293 Annotation: Subtype: Link Rect: 468, 619, 480, 629 ActionDest: 293 Annotation: Subtype: Link Rect: 520, 594, 532, 603 ActionDest: 290 Annotation: Subtype: Link Rect: 532, 594, 544, 603 ActionDest: 293 Annotation: Subtype: Link Rect: 544, 594, 556, 603 ActionDest: 293 Annotation: Subtype: Link Rect: 198, 544, 204, 553 ActionDest: 290 Annotation: Subtype: Link Rect: 205, 544, 217, 553 ActionDest: 293 Annotation: Subtype: Link Rect: 218, 544, 229, 553 ActionDest: 293 Annotation: Subtype: Link Rect: 437, 456, 449, 465 ActionDest: 293 Annotation: Subtype: Link Rect: 449, 456, 461, 465 ActionDest: 293 Annotation: Subtype: Link Rect: 464, 456, 476, 465 ActionDest: 293 Annotation: Subtype: Link Rect: 424, 419, 436, 428 ActionDest: 293 Annotation: Subtype: Link Rect: 322, 381, 334, 390 ActionDest: 293 Annotation: Subtype: Link Rect: 352, 343, 364, 352 ActionDest: 293 Annotation: Subtype: Link Rect: 217, 306, 229, 315 ActionDest: 293 Annotation: Subtype: Link Rect: 408, 256, 420, 264 ActionDest: 293 Annotation: Subtype: Link Rect: 420, 255, 432, 264 ActionDest: 293 Annotation: Subtype: Link Rect: 515, 243, 527, 252 ActionDest: 293 Annotation: Subtype: Link Rect: 222, 218, 234, 227 ActionDest: 296 Annotation: Subtype: Link Rect: 253, 180, 265, 189 ActionDest: 296 Annotation: Subtype: Link Rect: 464, 180, 476, 189 ActionDest: 296 Annotation: Subtype: Link Rect: 307, 167, 319, 177 ActionDest: 296 Annotation: Subtype: Link Rect: 261, 130, 273, 139 ActionDest: 290 Annotation: Subtype: Link Rect: 274, 130, 286, 139 ActionDest: 293 Annotation: Subtype: Link Rect: 286, 130, 298, 139 ActionDest: 293 Annotation: Subtype: Link Rect: 474, 80, 486, 89 ActionDest: 296 Annotation: Subtype: Link Rect: 487, 80, 499, 89 ActionDest: 296 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 380, 660, 391, 669 ActionDest: 296 Annotation: Subtype: Link Rect: 458, 647, 470, 657 ActionDest: 296 Annotation: Subtype: Link Rect: 357, 610, 369, 619 ActionDest: 296 Annotation: Subtype: Link Rect: 486, 522, 498, 531 ActionDest: 293 Annotation: Subtype: Link Rect: 204, 422, 215, 431 ActionDest: 296 Annotation: Subtype: Link Rect: 218, 422, 230, 431 ActionDest: 296 Annotation: Subtype: Link Rect: 328, 422, 339, 431 ActionDest: 290 Annotation: Subtype: Link Rect: 340, 422, 351, 431 ActionDest: 293 Annotation: Subtype: Link Rect: 352, 422, 363, 431 ActionDest: 293 Annotation: Subtype: Link Rect: 468, 409, 479, 418 ActionDest: 293 Annotation: Subtype: Link Rect: 480, 409, 492, 418 ActionDest: 296 Annotation: Subtype: Link Rect: 492, 409, 504, 418 ActionDest: 296 Annotation: Subtype: Link Rect: 506, 409, 518, 418 ActionDest: 296 Annotation: Subtype: Link Rect: 471, 384, 483, 393 ActionDest: 296 Annotation: Subtype: Link Rect: 483, 384, 495, 393 ActionDest: 296 Annotation: Subtype: Link Rect: 495, 384, 507, 393 ActionDest: 296 Annotation: Subtype: Link Rect: 238, 359, 250, 368 ActionDest: 296 Annotation: Subtype: Link Rect: 250, 359, 262, 368 ActionDest: 296 Annotation: Subtype: Link Rect: 411, 359, 423, 368 ActionDest: 296 Annotation: Subtype: Link Rect: 423, 359, 435, 368 ActionDest: 296 Annotation: Subtype: Link Rect: 347, 334, 359, 343 ActionDest: 296 Annotation: Subtype: Link Rect: 346, 309, 358, 318 ActionDest: 296 Annotation: Subtype: Link Rect: 484, 309, 495, 318 ActionDest: 296 Annotation: Subtype: Link Rect: 427, 271, 438, 280 ActionDest: 296 Annotation: Subtype: Link Rect: 406, 246, 417, 255 ActionDest: 299 Annotation: Subtype: Link Rect: 244, 170, 256, 180 ActionDest: 299 Annotation: Subtype: Link Rect: 451, 133, 463, 142 ActionDest: 299 Annotation: Subtype: Link Rect: 257, 95, 269, 104 ActionDest: 299 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 536, 483, 560, 494 Annotation: Subtype: Link Rect: 165, 471, 373, 482 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 95, 613, 133, 623 Annotation: Subtype: Link Rect: 139, 613, 175, 623 Annotation: Subtype: Link Rect: 386, 589, 423, 600 Annotation: Subtype: Link Rect: 430, 589, 466, 600 Annotation: Subtype: Link Rect: 206, 566, 244, 577 Annotation: Subtype: Link Rect: 250, 566, 286, 577 Annotation: Subtype: Link Rect: 402, 531, 439, 542 Annotation: Subtype: Link Rect: 115, 509, 152, 519 Annotation: Subtype: Link Rect: 262, 474, 300, 485 Annotation: Subtype: Link Rect: 113, 452, 150, 462 Annotation: Subtype: Link Rect: 160, 428, 197, 439 Annotation: Subtype: Link Rect: 437, 416, 475, 427 Annotation: Subtype: Link Rect: 59, 383, 96, 393 Annotation: Subtype: Link Rect: 127, 348, 164, 358 Annotation: Subtype: Link Rect: 59, 314, 96, 324 Annotation: Subtype: Link Rect: 479, 278, 516, 289 Annotation: Subtype: Link Rect: 319, 245, 356, 255 Annotation: Subtype: Link Rect: 314, 221, 351, 232 Annotation: Subtype: Link Rect: 509, 186, 547, 197 Annotation: Subtype: Link Rect: 126, 153, 164, 163 Annotation: Subtype: Link Rect: 407, 129, 444, 140 Annotation: Subtype: Link Rect: 271, 106, 309, 117 Annotation: Subtype: Link Rect: 229, 71, 266, 82 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 273, 710, 310, 721 Annotation: Subtype: Link Rect: 317, 710, 353, 721 Annotation: Subtype: Link Rect: 350, 687, 387, 698 Annotation: Subtype: Link Rect: 393, 687, 429, 698 Annotation: Subtype: Link Rect: 122, 665, 159, 675 Annotation: Subtype: Link Rect: 165, 665, 201, 675 Annotation: Subtype: Link Rect: 131, 642, 168, 652 Annotation: Subtype: Link Rect: 174, 642, 210, 652 Annotation: Subtype: Link Rect: 378, 618, 416, 629 Annotation: Subtype: Link Rect: 298, 584, 335, 594 Annotation: Subtype: Link Rect: 330, 549, 368, 560 Annotation: Subtype: Link Rect: 456, 537, 494, 548 Annotation: Subtype: Link Rect: 519, 527, 557, 537 Annotation: Subtype: Link Rect: 104, 504, 141, 514 Annotation: Subtype: Link Rect: 59, 481, 96, 491 Annotation: Subtype: Link Rect: 461, 457, 499, 468 Annotation: Subtype: Link Rect: 210, 422, 248, 433 Annotation: Subtype: Link Rect: 176, 400, 213, 410 Annotation: Subtype: Link Rect: 248, 376, 286, 387 Annotation: Subtype: Link Rect: 409, 353, 446, 364 Annotation: Subtype: Link Rect: 418, 330, 456, 341 Annotation: Subtype: Link Rect: 297, 307, 334, 318 Annotation: Subtype: Link Rect: 310, 273, 348, 284 Annotation: Subtype: Link Rect: 181, 239, 219, 249 Annotation: Subtype: Link Rect: 331, 216, 369, 226 Annotation: Subtype: Link Rect: 375, 216, 411, 226 Annotation: Subtype: Link Rect: 519, 192, 557, 203 Annotation: Subtype: Link Rect: 59, 182, 95, 192 Annotation: Subtype: Link Rect: 326, 158, 363, 169 Annotation: Subtype: Link Rect: 369, 158, 405, 169 Annotation: Subtype: Link Rect: 497, 135, 534, 146 Annotation: Subtype: Link Rect: 512, 112, 549, 123 Annotation: Subtype: Link Rect: 95, 78, 133, 88 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 256, 711, 293, 721 Annotation: Subtype: Link Rect: 135, 676, 173, 687 Annotation: Subtype: Link Rect: 59, 642, 96, 652 Annotation: Subtype: Link Rect: 108, 619, 146, 629 Annotation: Subtype: Link Rect: 164, 595, 202, 606 Annotation: Subtype: Link Rect: 275, 572, 312, 583 Annotation: Subtype: Link Rect: 397, 549, 434, 560 Annotation: Subtype: Link Rect: 197, 527, 234, 537 Annotation: Subtype: Link Rect: 302, 491, 339, 502 Annotation: Subtype: Link Rect: 59, 458, 96, 468 Annotation: Subtype: Link Rect: 104, 435, 142, 445 Annotation: Subtype: Link Rect: 272, 411, 309, 422 Annotation: Subtype: Link Rect: 109, 377, 146, 387 Annotation: Subtype: Link Rect: 371, 353, 408, 364 Annotation: Subtype: Link Rect: 138, 297, 176, 307 Annotation: Subtype: Link Rect: 206, 262, 244, 272 Annotation: Subtype: Link Rect: 250, 262, 286, 272 Annotation: Subtype: Link Rect: 258, 215, 296, 226 Annotation: Subtype: Link Rect: 302, 215, 338, 226 Annotation: Subtype: Link Rect: 470, 204, 507, 215 Annotation: Subtype: Link Rect: 439, 192, 476, 203 Annotation: Subtype: Link Rect: 59, 159, 96, 169 Annotation: Subtype: Link Rect: 437, 135, 474, 146 Annotation: Subtype: Link Rect: 250, 112, 287, 123 Annotation: Subtype: Link Rect: 296, 89, 334, 100 Annotation: Subtype: Link Rect: 95, 67, 132, 77 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 334, 710, 371, 721 Annotation: Subtype: Link Rect: 461, 687, 499, 698 Checksum: e48f96a7 Type: CRC32 Checksum: e7e98b623976421c0f234accd8e87770 Type: MD5 Checksum: 36997f49a9347967d354cafba651e92af4bf0688 Type: SHA-1