f020a66bc43db9403c739e5d053851a7 pmc_33406589.pdf 1faa954a1b6a594b0a9443d35a7b834e768c6153 pmc_33406589.pdf 0736476f69d90257c43659eef6c07f6a1f9a1b09e27b4e7b3c28eb48b7255155 pmc_33406589.pdf Title: Progression of Interstitial Fibrosis and Tubular Atrophyin Low Immunological Risk Renal Transplants Monitoredby Sequential Surveillance Biopsies: The Influence of TAC Exposure and Metabolism Subject: The combination of tacrolimus (TAC) and mycophenolate is the most widely employed maintenance immunosuppression in renal transplants. Different surrogates of tacrolimus exposure or metabolism such as tacrolimus trough levels (TAC-C0), coefficient of variation of tacrolimus (CV-TAC-C0), time in therapeutic range (TTR), and tacrolimus concentration dose ratio (C/D) have been associated with graft outcomes. We explore in a cohort of low immunological risk renal transplants (n = 85) treated with TAC, mycophenolate mofetil (MMF), and steroids and then monitored by paired surveillance biopsies the association between histological lesions and TAC-C0 at the time of biopsy as well as CV-TAC-C0, TTR, and C/D during follow up. Interstitial inflammation (i-Banff score 1) in the first surveillance biopsy was associated with TAC-C0 (odds ratio (OR): 0.69, 95% confidence interval (CI): 0.50–0.96; p = 0.027). In the second surveillance biopsy, inflammation was associated with time below the therapeutic range (OR: 1.05 and 95% CI: 1.01–1.10; p = 0.023). Interstitial inflammation in scarred areas (i-IFTA score 1) was not associated with surrogates of TAC exposure/metabolism. Progression of interstitial fibrosis/tubular atrophy (IF/TA) was observed in 35 cases (41.2%). Multivariate regression logistic analysis showed that mean C/D (OR: 0.48; 95% CI: 0.25–0.92; p = 0.026) and IF/TA in the first biopsy (OR: 0.43, 95% CI: 0.24–0.77, p = 0.005) were associated with IF/TA progression between biopsies. A low C/D ratio is associated with IF/TA progression, suggesting that TAC nephrotoxicity may contribute to fibrosis progression in well immunosuppressed patients. Our data support that TAC exposure is associated with inflammation in healthy kidney areas but not in scarred tissue. Keywords: tacrolimus; renal transplantation; protocol biopsies; concentration dose ratio; time in therapeutic range; coefficient of variation Author: Betty Chamoun, Irina B. Torres, Alejandra Gabaldón, Joana Sellarés, Manel Perelló, Eva Castellá, Xavier Guri, Maite Salcedo, Nestor G. Toapanta, Ignacio Cidraque, Francesc Moreso and Daniel Seron Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jan 8 05:44:18 2021 CET ModDate: Fri Jan 8 05:49:50 2021 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 14 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1093695 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ZZTQUO+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 KYNIJA+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 DEZLIX+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 OTFQEH+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 ZLZLOH+CMSY10 Type 1 Builtin yes yes no 28 0 XPUSBJ+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 36 0 BSDWLC+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 39 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 61 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 65 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 69 0 OIIRUI+EURM10 Type 1 Builtin yes yes no 84 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 96 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 100 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 104 0 ABCDEE+Palatino Linotype CID TrueType Identity-H yes yes yes 108 0 QLBIEB+URWPalladioL-BoldItal Type 1 Custom yes yes no 123 0 ABCDEE+Palatino Linotype,Bold TrueType WinAnsi yes yes no 131 0 ABCDEE+Palatino Linotype TrueType WinAnsi yes yes no 135 0 ABCDEE+Palatino Linotype,Italic TrueType WinAnsi yes yes no 139 0 ABCDEE+Palatino Linotype,BoldItalic TrueType WinAnsi yes yes no 143 0 OGLZWV+VnURWPalladioL Type 1 Custom yes yes no 167 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2021-09-11 03:55:08 CEST RepresentationInformation: pmc_33406589.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2021-01-11 03:39:09 CET Size: 1093695 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 358 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Patients and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Children: Item: Title: Patients Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Biopsies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Immunosuppression Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Therapeutic Drug Monitoring (TDM) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Clinical Variables Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Statistics Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Children: Item: Title: Patients Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Biopsies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Interstitial Inflammation and TDM Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: IF/TA Progression and TDM Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Arteriolar Hyalinosis Progression and TDM Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Progression of Interstitial Fibrosis and Tubular Atrophyin Low Immunological Risk Renal Transplants Monitoredby Sequential Surveillance Biopsies: The Influence of TAC Exposure and Metabolism Author: Betty Chamoun, Irina B. Torres, Alejandra Gabaldón, Joana Sellarés, Manel Perelló, Eva Castellá, Xavier Guri, Maite Salcedo, Nestor G. Toapanta, Ignacio Cidraque, Francesc Moreso and Daniel Seron Subject: The combination of tacrolimus (TAC) and mycophenolate is the most widely employed maintenance immunosuppression in renal transplants. Different surrogates of tacrolimus exposure or metabolism such as tacrolimus trough levels (TAC-C0), coefficient of variation of tacrolimus (CV-TAC-C0), time in therapeutic range (TTR), and tacrolimus concentration dose ratio (C/D) have been associated with graft outcomes. We explore in a cohort of low immunological risk renal transplants (n = 85) treated with TAC, mycophenolate mofetil (MMF), and steroids and then monitored by paired surveillance biopsies the association between histological lesions and TAC-C0 at the time of biopsy as well as CV-TAC-C0, TTR, and C/D during follow up. Interstitial inflammation (i-Banff score 1) in the first surveillance biopsy was associated with TAC-C0 (odds ratio (OR): 0.69, 95% confidence interval (CI): 0.50 0.96; p = 0.027). In the second surveillance biopsy, inflammation was associated with time below the therapeutic range (OR: 1.05 and 95% CI: 1.01 1.10; p = 0.023). Interstitial inflammation in scarred areas (i-IFTA score 1) was not associated with surrogates of TAC exposure/metabolism. Progression of interstitial fibrosis/tubular atrophy (IF/TA) was observed in 35 cases (41.2%). Multivariate regression logistic analysis showed that mean C/D (OR: 0.48; 95% CI: 0.25 0.92; p = 0.026) and IF/TA in the first biopsy (OR: 0.43, 95% CI: 0.24 0.77, p = 0.005) were associated with IF/TA progression between biopsies. A low C/D ratio is associated with IF/TA progression, suggesting that TAC nephrotoxicity may contribute to fibrosis progression in well immunosuppressed patients. Our data support that TAC exposure is associated with inflammation in healthy kidney areas but not in scarred tissue. Keywords: tacrolimus; renal transplantation; protocol biopsies; concentration dose ratio; time in therapeutic range; coefficient of variation Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Fri Jan 08 05:44:18 CET 2021 ModDate: Fri Jan 08 05:49:50 CET 2021 ID: 0xd913b1e80b2bc9e99f563958c7bc3da0, 0xd913b1e80b2bc9e99f563958c7bc3da0 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: DEZLIX+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 134 FontDescriptor: FontName: DEZLIX+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OIIRUI+EURM10 FontSubset: true FirstChar: 20 LastChar: 22 FontDescriptor: FontName: OIIRUI+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: OGLZWV+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: OGLZWV+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: OTFQEH+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: OTFQEH+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZZTQUO+VnURWPalladioL-Bold FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: ZZTQUO+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: QLBIEB+URWPalladioL-BoldItal FontSubset: true FirstChar: 110 LastChar: 112 FontDescriptor: FontName: QLBIEB+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZLZLOH+CMSY10 FontSubset: true FirstChar: 3 LastChar: 21 FontDescriptor: FontName: ZLZLOH+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: KYNIJA+URWPalladioL-Ital FontSubset: true FirstChar: 3 LastChar: 121 FontDescriptor: FontName: KYNIJA+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 176, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 119, 622, 130, 635 Annotation: Subtype: Link Rect: 338, 609, 349, 623 Annotation: Subtype: Link Rect: 130, 599, 141, 610 Annotation: Subtype: Link Rect: 235, 599, 246, 610 Annotation: Subtype: Link Rect: 118, 313, 154, 322 Annotation: Subtype: Link Rect: 35, 301, 155, 310 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 123, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 154, 86 Annotation: Subtype: Link Rect: 35, 66, 49, 74 Annotation: Subtype: Link Rect: 296, 74, 303, 83 ActionDest: 164 Annotation: Subtype: Link Rect: 127, 17, 264, 28 Annotation: Subtype: Link Rect: 422, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 449, 87, 468 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 206, 699, 213, 708 ActionDest: 164 Annotation: Subtype: Link Rect: 222, 636, 229, 645 ActionDest: 164 Annotation: Subtype: Link Rect: 367, 624, 374, 633 ActionDest: 164 Annotation: Subtype: Link Rect: 541, 611, 548, 620 ActionDest: 164 Annotation: Subtype: Link Rect: 549, 611, 556, 620 ActionDest: 164 Annotation: Subtype: Link Rect: 322, 586, 329, 595 ActionDest: 164 Annotation: Subtype: Link Rect: 378, 523, 385, 532 ActionDest: 171 Annotation: Subtype: Link Rect: 386, 523, 393, 532 ActionDest: 171 Annotation: Subtype: Link Rect: 191, 498, 203, 507 ActionDest: 171 Annotation: Subtype: Link Rect: 204, 498, 216, 507 ActionDest: 171 Annotation: Subtype: Link Rect: 239, 486, 251, 495 ActionDest: 171 Annotation: Subtype: Link Rect: 389, 423, 401, 432 ActionDest: 171 Annotation: Subtype: Link Rect: 528, 385, 540, 394 ActionDest: 171 Annotation: Subtype: Link Rect: 541, 385, 553, 394 ActionDest: 171 Annotation: Subtype: Link Rect: 245, 297, 257, 306 ActionDest: 171 Annotation: Subtype: Link Rect: 526, 285, 538, 294 ActionDest: 171 Annotation: Subtype: Link Rect: 460, 272, 472, 281 ActionDest: 171 Annotation: Subtype: Link Rect: 201, 247, 213, 256 ActionDest: 171 Annotation: Subtype: Link Rect: 213, 247, 225, 256 ActionDest: 171 Annotation: Subtype: Link Rect: 208, 222, 219, 231 ActionDest: 171 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 513, 371, 525, 380 ActionDest: 171 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 246, 493, 253, 505 ActionDest: 88 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 219, 722, 231, 731 ActionDest: 171 Annotation: Subtype: Link Rect: 549, 694, 556, 706 ActionDest: 88 Annotation: Subtype: Link Rect: 392, 446, 404, 455 ActionDest: 171 Annotation: Subtype: Link Rect: 448, 140, 455, 152 ActionDest: 119 Annotation: Subtype: Link Rect: 481, 128, 488, 140 ActionDest: 119 Annotation: Subtype: Link Rect: 484, 115, 491, 127 ActionDest: 148 Page: Sequence: 6 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 233, 204, 240, 216 ActionDest: 151 Annotation: Subtype: Link Rect: 257, 129, 264, 141 ActionDest: 151 Annotation: Subtype: Link Rect: 329, 66, 336, 78 ActionDest: 154 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 253, 130, 260, 142 ActionDest: 154 Annotation: Subtype: Link Rect: 280, 130, 287, 142 ActionDest: 157 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 230, 412, 242, 421 ActionDest: 171 Annotation: Subtype: Link Rect: 347, 412, 359, 421 ActionDest: 171 Annotation: Subtype: Link Rect: 359, 412, 371, 421 ActionDest: 171 Annotation: Subtype: Link Rect: 448, 412, 460, 421 ActionDest: 171 Annotation: Subtype: Link Rect: 472, 324, 484, 333 ActionDest: 171 Annotation: Subtype: Link Rect: 198, 274, 210, 283 ActionDest: 171 Annotation: Subtype: Link Rect: 503, 249, 515, 258 ActionDest: 171 Annotation: Subtype: Link Rect: 516, 249, 527, 258 ActionDest: 171 Annotation: Subtype: Link Rect: 311, 236, 323, 245 ActionDest: 171 Annotation: Subtype: Link Rect: 451, 224, 463, 233 ActionDest: 171 Annotation: Subtype: Link Rect: 463, 224, 475, 233 ActionDest: 171 Annotation: Subtype: Link Rect: 345, 211, 357, 220 ActionDest: 174 Annotation: Subtype: Link Rect: 496, 211, 508, 220 ActionDest: 174 Annotation: Subtype: Link Rect: 387, 148, 399, 157 ActionDest: 174 Annotation: Subtype: Link Rect: 399, 148, 411, 157 ActionDest: 174 Annotation: Subtype: Link Rect: 371, 73, 378, 82 ActionDest: 164 Annotation: Subtype: Link Rect: 378, 73, 390, 82 ActionDest: 171 Annotation: Subtype: Link Rect: 391, 73, 403, 82 ActionDest: 174 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 222, 710, 234, 719 ActionDest: 174 Annotation: Subtype: Link Rect: 237, 710, 249, 719 ActionDest: 174 Annotation: Subtype: Link Rect: 459, 597, 470, 606 ActionDest: 174 Annotation: Subtype: Link Rect: 473, 572, 485, 581 ActionDest: 174 Annotation: Subtype: Link Rect: 429, 496, 441, 505 ActionDest: 174 Annotation: Subtype: Link Rect: 444, 496, 456, 505 ActionDest: 174 Annotation: Subtype: Link Rect: 441, 446, 453, 455 ActionDest: 174 Annotation: Subtype: Link Rect: 454, 446, 466, 455 ActionDest: 174 Annotation: Subtype: Link Rect: 484, 433, 496, 443 ActionDest: 174 Annotation: Subtype: Link Rect: 496, 433, 508, 443 ActionDest: 174 Annotation: Subtype: Link Rect: 209, 308, 221, 317 ActionDest: 171 Annotation: Subtype: Link Rect: 221, 308, 233, 317 ActionDest: 174 Annotation: Subtype: Link Rect: 236, 308, 248, 317 ActionDest: 174 Annotation: Subtype: Link Rect: 251, 283, 263, 292 ActionDest: 174 Annotation: Subtype: Link Rect: 264, 283, 276, 292 ActionDest: 174 Annotation: Subtype: Link Rect: 392, 132, 404, 141 ActionDest: 174 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 477, 630, 560, 641 Annotation: Subtype: Link Rect: 166, 618, 289, 629 Annotation: Subtype: Link Rect: 144, 266, 182, 276 Annotation: Subtype: Link Rect: 188, 266, 224, 276 Annotation: Subtype: Link Rect: 131, 231, 169, 241 Annotation: Subtype: Link Rect: 175, 231, 211, 241 Annotation: Subtype: Link Rect: 59, 185, 97, 195 Annotation: Subtype: Link Rect: 103, 185, 139, 195 Annotation: Subtype: Link Rect: 426, 173, 560, 184 Annotation: Subtype: Link Rect: 56, 161, 288, 172 Annotation: Subtype: Link Rect: 392, 138, 429, 149 Annotation: Subtype: Link Rect: 435, 138, 472, 149 Annotation: Subtype: Link Rect: 59, 116, 97, 126 Annotation: Subtype: Link Rect: 122, 82, 160, 92 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 204, 709, 242, 720 Annotation: Subtype: Link Rect: 224, 686, 262, 697 Annotation: Subtype: Link Rect: 392, 663, 430, 674 Annotation: Subtype: Link Rect: 82, 630, 119, 640 Annotation: Subtype: Link Rect: 192, 594, 229, 605 Annotation: Subtype: Link Rect: 236, 594, 272, 605 Annotation: Subtype: Link Rect: 303, 560, 340, 571 Annotation: Subtype: Link Rect: 347, 560, 383, 571 Annotation: Subtype: Link Rect: 519, 537, 557, 548 Annotation: Subtype: Link Rect: 59, 526, 95, 536 Annotation: Subtype: Link Rect: 59, 492, 97, 502 Annotation: Subtype: Link Rect: 95, 469, 133, 479 Annotation: Subtype: Link Rect: 306, 433, 343, 444 Annotation: Subtype: Link Rect: 415, 410, 452, 421 Annotation: Subtype: Link Rect: 59, 377, 97, 387 Annotation: Subtype: Link Rect: 113, 342, 151, 352 Annotation: Subtype: Link Rect: 287, 307, 324, 318 Annotation: Subtype: Link Rect: 225, 272, 262, 283 Annotation: Subtype: Link Rect: 268, 272, 304, 283 Annotation: Subtype: Link Rect: 298, 249, 335, 260 Annotation: Subtype: Link Rect: 341, 249, 377, 260 Annotation: Subtype: Link Rect: 59, 215, 97, 226 Annotation: Subtype: Link Rect: 103, 215, 139, 226 Annotation: Subtype: Link Rect: 486, 192, 524, 203 Annotation: Subtype: Link Rect: 300, 169, 337, 180 Annotation: Subtype: Link Rect: 518, 134, 555, 145 Annotation: Subtype: Link Rect: 489, 111, 527, 122 Annotation: Subtype: Link Rect: 300, 77, 337, 88 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 356, 721, 394, 732 Annotation: Subtype: Link Rect: 136, 687, 173, 697 Annotation: Subtype: Link Rect: 479, 663, 516, 674 Annotation: Subtype: Link Rect: 521, 663, 557, 674 Annotation: Subtype: Link Rect: 276, 629, 314, 640 Annotation: Subtype: Link Rect: 320, 629, 356, 640 Annotation: Subtype: Link Rect: 118, 595, 155, 605 Annotation: Subtype: Link Rect: 161, 595, 197, 605 Annotation: Subtype: Link Rect: 59, 561, 97, 571 Annotation: Subtype: Link Rect: 103, 561, 139, 571 Annotation: Subtype: Link Rect: 296, 537, 334, 548 Annotation: Subtype: Link Rect: 340, 537, 376, 548 Annotation: Subtype: Link Rect: 95, 503, 133, 513 Annotation: Subtype: Link Rect: 108, 469, 146, 479 Annotation: Subtype: Link Rect: 483, 445, 520, 456 Annotation: Subtype: Link Rect: 261, 423, 298, 433 Annotation: Subtype: Link Rect: 519, 410, 557, 421 Annotation: Subtype: Link Rect: 140, 377, 178, 387 Annotation: Subtype: Link Rect: 184, 377, 220, 387 Annotation: Subtype: Link Rect: 457, 353, 494, 364 Annotation: Subtype: Link Rect: 500, 353, 536, 364 Annotation: Subtype: Link Rect: 488, 330, 525, 341 Annotation: Subtype: Link Rect: 178, 296, 215, 306 Annotation: Subtype: Link Rect: 222, 296, 258, 306 Annotation: Subtype: Link Rect: 393, 272, 430, 283 Annotation: Subtype: Link Rect: 436, 272, 472, 283 Annotation: Subtype: Link Rect: 300, 238, 338, 249 Annotation: Subtype: Link Rect: 344, 238, 380, 249 Annotation: Subtype: Link Rect: 202, 203, 239, 214 Annotation: Subtype: Link Rect: 339, 181, 377, 191 Annotation: Subtype: Link Rect: 207, 146, 244, 157 Annotation: Subtype: Link Rect: 398, 111, 436, 122 Annotation: Subtype: Link Rect: 291, 88, 328, 99 Annotation: Subtype: Link Rect: 335, 88, 371, 99 Checksum: 6a881dcd Type: CRC32 Checksum: f020a66bc43db9403c739e5d053851a7 Type: MD5 Checksum: 1faa954a1b6a594b0a9443d35a7b834e768c6153 Type: SHA-1