Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Cell Prevents Silicosis Accepted: November 21, 2013 Administration of Stem 1421-9778/13/0326-1681$38.00/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.

Original Paper

Repeated Administration of Bone Marrow– Derived Cells Prevents Disease Progression in Experimental Silicosis Miquéias Lopes-Pachecoa,b,c Debora G. Xistoa,b Felipe M. Ornellasa Mariana A. Antunesb Soraia C. Abreub Patricia R. M. Roccob Christina M. Takiyac Marcelo M. Moralesa Laboratory of Cellular and Molecular Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, bLaboratory of Pulmonary Investigation, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, cLaboratory of Cellular Pathology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil a

Key Words &HOOWKHUDS\‡,QÁDPPDWLRQ‡)LEURVLV‡(ODVWLFÀEHU‡6LOLFRVLV Abstract Background/Aims: Bone marrow–derived cells (BMDCs) reduced mechanical and histologic FKDQJHV LQ WKH OXQJ LQ D PXULQH PRGHO RI VLOLFRVLV EXW WKHVH EHQHÀFLDO HIIHFWV GLG QRW persist in the course of lung injury. We hypothesized that repeated administration of BMDCs PD\ GHFUHDVH OXQJ LQÁDPPDWLRQ DQG UHPRGHOLQJ WKXV SUHYHQWLQJ GLVHDVH SURJUHVVLRQ Methods: One hundred and two C57BL/6 mice were randomly divided into SIL (silica, 20 mg intratracheally [IT]) and control (C) groups (saline, IT). C and SIL groups were further randomized to receive BMDCs (2×106 cells) or saline IT 15 and 30 days after the start of the protocol. Results: By day 60, BMDCs had decreased the fractional area of granuloma and the number of polymorphonuclear cells, macrophages (total and M1 phenotype), apoptotic cells, the level of transforming growth factor (TGF)-DŽDQGW\SHV,DQG,,,FROODJHQÀEHUFRQWHQW in the granuloma. In the alveolar septa, BMDCs reduced the amount of collagen and elastic ÀEHUV 7*)DŽ, and the number of M1 and apoptotic cells. Furthermore, interleukin (IL)-1DŽ, IL-1R1, caspase-3 mRNA levels decreased and the level of IL-1RN mRNA increased. Lung mechanics improved after BMDC therapy. The presence of male donor cells in lung tissue was not observed using detection of Y chromosome DNA. Conclusion: repeated administration RI %0'&V UHGXFHG LQÁDPPDWLRQ ÀEURJHQHVLV DQG HODVWRJHQHVLV WKXV LPSURYLQJ OXQJ mechanics through the release of paracrine factors. Copyright © 2013 S. Karger AG, Basel Marcelo Marcos Morales, MD, PhD

Universidade Federal do Rio de Janeiro, Instituto de Biofísica Carlos Chagas Filho C.C.S., Laboratório de Fisiologia Celular e Molecular, Ilha do Fundão, 21941-902 - Rio de Janeiro - RJ (Brazil) Tel. +55 21 2562 6572, Fax +55 21 2280 8193. E-Mail [email protected]

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Introduction

Š”‘‹… ‹ŠƒŽƒ–‹‘ ‘ˆ •‹Ž‹…ƒ ’ƒ”–‹…Ž‡• Ž‡ƒ†• –‘ ƒ ’‡”•‹•–‡– ‹ϐŽƒƒ–‘”› ’”‘…‡••ǡ ˆ‘”ƒ–‹‘‘ˆ‰”ƒ—Ž‘ƒ–‘—•‘†—Ž‡•ǡƒ†Ž—‰ϐ‹„”‘•‹•Ǥ‹Ž‹…‘•‹•‹•ƒ‘……—’ƒ–‹‘ƒŽ†‹•‡ƒ•‡ –Šƒ–…ƒ—•‡•”‡•’‹”ƒ–‘”›ˆƒ‹Ž—”‡†—‡–‘”‡†—…–‹‘‹–Š‡ƒ”‡ƒ‘ˆ‰ƒ•‡š…Šƒ‰‡ƒ††‡–‡”‹‘”ƒ–‹‘ ‹ Ž—‰ ˆ—…–‹‘ ȏͳȂ͵ȐǤ ‘ †ƒ–‡ǡ –Š‡”‡ Šƒ• „‡‡ ‘ ‡ˆˆ‡…–‹˜‡ –”‡ƒ–‡– –‘ ‹‹‹œ‡ –Š‡ †‡˜‡Ž‘’‡–‘ˆ–Š‹•†‹•‡ƒ•‡ǤŽ‹‹…ƒŽ–”‡ƒ–‡–‹•„ƒ•‡†‘…‘–”‘ŽŽ‹‰–Š‡•›’–‘•ƒ† ’”‡˜‡–‹‰…‘’Ž‹…ƒ–‹‘•ȏͶȐǤ ‡˜‡”ƒŽ•–—†‹‡•Šƒ˜‡”‡’‘”–‡†‘–Š‡„‡‡ϐ‹…‹ƒŽ‡ˆˆ‡…–•‘ˆ„‘‡ƒ””‘™…‡ŽŽ–Š‡”ƒ’›‹ †‹ˆˆ‡”‡–‘†‡Ž•‘ˆŽ—‰‹Œ—”›ȏͷȂͳͲȐǤ ‡š’‡”‹‡–ƒŽ‘†‡Ž•‘ˆŽ—‰ϐ‹„”‘•‹•ǡ„‘‡ƒ””‘™Ȃ †‡”‹˜‡†…‡ŽŽ•ȋ•Ȍ™‡”‡ƒ„Ž‡–‘”‡’ƒ‹”†ƒƒ‰‡†–‹••—‡„›‘†—Žƒ–‹‰–Š‡‹ϐŽƒƒ–‘”› ’”‘…‡••ƒ†”‡’Žƒ…‹‰†ƒƒ‰‡†…‡ŽŽ•ȏ͵ǡͷȂ͹ȐǤ ƒ’”‡˜‹‘—••–—†›ǡ™‡‘„•‡”˜‡†–Šƒ–‹‡š’‡”‹‡–ƒŽ•‹Ž‹…‘•‹•ǡ–Š‡‡ƒ”Ž›„‡‡ϐ‹…‹ƒŽ‡ˆˆ‡…–• ‘ˆ–Š‡”ƒ’›‘Ž—‰‹ϐŽƒƒ–‹‘ƒ†‡…Šƒ‹…•™‡”‡–”ƒ•‹‡–ȏͳͳȐǤŠ‡”‡ˆ‘”‡ǡ™‡ –‡•–‡†–Š‡Š›’‘–Š‡•‹•–Šƒ–ƒ’”‘–‘…‘Ž™‹–Š”‡’‡ƒ–‡†‹–”ƒ–”ƒ…Š‡ƒŽƒ†‹‹•–”ƒ–‹‘‘ˆ• …‘—Ž† ”‡†—…‡ –Š‡ ‘‰‘‹‰ ‹ϐŽƒƒ–‘”› ƒ† ϐ‹„”‘‡Žƒ•–‘‰‡‹… ’”‘…‡••‡•ǡ –Š—• ‹’”‘˜‹‰ Ž—‰‡…Šƒ‹…•Ǥ‡‡š’Ž‘”‡†–Š‡‡ˆˆ‡…–•‘ˆ”‡’‡ƒ–‡†‹–”ƒ–”ƒ…Š‡ƒŽƒ†‹‹•–”ƒ–‹‘‘ˆ• ‘ †ƒ›• ͳͷ ƒ† ͵Ͳ ‹ ƒ ™‡ŽŽǦ‡•–ƒ„Ž‹•Š‡† ‘†‡Ž ‘ˆ •‹Ž‹…‘•‹•Ǥ —‰ ‡…Šƒ‹…•ǡ Š‹•–‘Ž‘‰›ǡ ƒ’‘’–‘•‹•ǡ ‘”’Š‘‡–”›ǡ ƒ† Š—‘”ƒŽ ƒ† …‡ŽŽ—Žƒ” ƒ••‡••‡–• ‘ˆ ‹ϐŽƒƒ–‹‘ ™‡”‡ ƒƒŽ›œ‡†Ǥ‡‘†‡Ž‹‰™ƒ•‡˜ƒŽ—ƒ–‡†„›ƒ••‡••‡–‘ˆ…‘ŽŽƒ‰‡ƒ†‡Žƒ•–‹…ϐ‹„‡”†‡’‘•‹–‹‘ǡ –”ƒ•ˆ‘”‹‰‰”‘™–Šˆƒ…–‘”ȋ ȌǦEǡƒ†…Šƒ‰‡•‹ƒ…”‘’Šƒ‰‡’Š‡‘–›’‡Ǥ Materials and Methods Š‹••–—†›™ƒ•ƒ’’”‘˜‡†„›–Š‡‡ƒŽ–Š…‹‡…‡•‡–‡”–Š‹…•‘‹––‡‡ƒ––Š‡ ‡†‡”ƒŽ‹˜‡”•‹–› ‘ˆ‹‘†‡ ƒ‡‹”‘ȋǦǦͲͳͻȌǤŽŽƒ‹ƒŽ•”‡…‡‹˜‡†Š—ƒ‡…ƒ”‡‹…‘’Ž‹ƒ…‡™‹–Š–Š‡”‹…‹’Ž‡•‘ˆ ƒ„‘”ƒ–‘”›‹ƒŽƒ”‡ˆ‘”—Žƒ–‡†„›–Š‡ƒ–‹‘ƒŽ‘…‹‡–›ˆ‘”‡†‹…ƒŽ‡•‡ƒ”…Šƒ†–Š‡ —‹†‡ˆ‘”–Š‡ƒ”‡ ƒ†•‡‘ˆƒ„‘”ƒ–‘”›‹ƒŽ•’”‡’ƒ”‡†„›–Š‡ƒ–‹‘ƒŽ…ƒ†‡›‘ˆ…‹‡…‡•ǡǤ Animal preparation and experimental protocol –‘–ƒŽ‘ˆͳͲʹ‹…‡ȋͺȂͳʹ™‡‡•‘ˆƒ‰‡Ȍ™‡”‡—•‡†‹–Š‹••–—†›Ǥ‡˜‡–›ƒŽ‡‹…‡™‡”‡—•‡†ƒ•„‘‡ ƒ””‘™†‘‘”•ǤŠ‹”–›Ǧ–™‘ˆ‡ƒŽ‡‹…‡™‡”‡”ƒ†‘Ž›†‹˜‹†‡†‹–‘–™‘‰”‘—’•ǣ‰”‘—’ǡ‹•–‹ŽŽ‡†™‹–Š •–‡”‹Ž‡•ƒŽ‹‡ȋͷͲP‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȏ ȐȌƒ† ‰”‘—’ǡ‹•–‹ŽŽ‡†™‹–Šƒ•‹Ž‹…ƒ’ƒ”–‹…Ž‡•—•’‡•‹‘ȋʹͲ‰ȀͷͲ P‘ˆ•ƒŽ‹‡Ȍȋ’ƒ”–‹…Ž‡•‹œ‡ǣͺͲΨ„‡–™‡‡ͳƒ†ͷPǢ‹‰ƒǦŽ†”‹…Šǡ–Ǥ‘—‹•ǡǡȌǤŠ‡•‡‰”‘—’•™‡”‡ –Š‡•—„†‹˜‹†‡†‹–‘–™‘‰”‘—’•ǣ‹…‡–Šƒ–”‡…‡‹˜‡†•ƒŽ‹‡ȋƒŽȌ‘”•ȋ‡ŽŽȌǤ‹ƒŽ•™‡”‡–”‡ƒ–‡†™‹–Š •ȋʹέͳͲ6 …‡ŽŽ•ȀͷͲP‘ˆ•ƒŽ‹‡ Ȍͳͷƒ†͵Ͳ†ƒ›•ƒˆ–‡”–Š‡•–ƒ”–‘ˆ–Š‡’”‘–‘…‘ŽȋǦ‡ŽŽƒ† Ǧ‡ŽŽ ‰”‘—’•Ȍȋ ‹‰ǤͳȌǤ Extraction and culture of BMDCs •™‡”‡‘„–ƒ‹‡†ˆ”‘–Š‡ˆ‡—”ƒ†–‹„‹ƒ‘ˆƒŽ‡‹…‡ƒ•’”‡˜‹‘—•Ž›†‡•…”‹„‡†ȏͻǡͳͳȐǤˆ–‡”–Š‡ ‡’‹’Š›•‡•™‡”‡…—–ǡ–Š‡„‘‡•™‡”‡…‡–”‹ˆ—‰‡†ȋͶͲͲέgˆ‘”ͳͲ‹Ȍƒ†–Š‡„‘‡ƒ””‘™™ƒ•…‘ŽŽ‡…–‡†Ǥ Š‡ …‡ŽŽ• ™‡”‡ ”‡•—•’‡†‡† ‹ —Ž„‡……‘ǯ• ‘†‹ϐ‹‡† ƒ‰Ž‡ǯ• ‡†‹— ȋǢ ‹ˆ‡ ‡…Š‘Ž‘‰‹‡•ǡ ”ƒ† •Žƒ†ǡǡȌ•—’’Ž‡‡–‡†™‹–ŠʹͲΨˆ‡–ƒŽ„‘˜‹‡•‡”—ȋ ‹„…‘ȌǡʹđǦ‰Ž—–ƒ‹‡ȋ‹‰ƒǦŽ†”‹…ŠȌǡ ƒ†ƒ–‹„‹‘–‹…•ȋ’‡‹…‹ŽŽ‹Ȁ•–”‡’–‘›…‹Ȍƒ†’Žƒ–‡†‹…—Ž–—”‡ϐŽƒ••ȋͳͲ6…‡ŽŽ•Ȁ…ʹȌǤŠ‡ˆ‘ŽŽ‘™‹‰†ƒ›ǡ–Š‡ •—’‡”ƒ–ƒ–™ƒ•†‹•…ƒ”†‡†ƒ†–Š‡ƒ†Š‡”‡†…‡ŽŽ•™‡”‡™ƒ•Š‡†™‹–Š’Š‘•’Šƒ–‡Ǧ„—ˆˆ‡”‡†•ƒŽ‹‡Ǥ‡ŽŽ•™‡”‡ ”‡Ǧˆ‡†™‹–Šǡ™Š‹…Š™ƒ•…Šƒ‰‡†‡˜‡”›ʹ†ƒ›•Ǥ†ƒ›͹ǡƒ†Š‡”‡–…‡ŽŽ•™‡”‡–”›’•‹‹œ‡†ƒ†ƒˆ–‡” ”›’ƒŽ—‡•–ƒ‹‹‰ǡ–Š‡›™‡”‡…‘—–‡†‹ƒ‡—„ƒ—‡”…Šƒ„‡”ˆ‘”‡˜ƒŽ—ƒ–‹‘‘ˆ˜‹ƒ„‹Ž‹–›Ǥ Lung mechanics ‹š–› †ƒ›• ƒˆ–‡” ‹•–‹ŽŽƒ–‹‘ ‘ˆ •ƒŽ‹‡ ‘” •‹Ž‹…ƒǡ –Š‡ ƒ‹ƒŽ• ™‡”‡ •‡†ƒ–‡† ȋ†‹ƒœ‡’ƒ ͳ ‰Ȁ‰ ‹–”ƒ’‡”‹–‘‡ƒŽŽ›Ȍǡƒ‡•–Š‡–‹œ‡†ȋ–Š‹‘’‡–ƒŽ•‘†‹—ʹͲ‰Ȁ‰‹–”ƒ’‡”‹–‘‡ƒŽŽ›Ȍǡ–”ƒ…Š‡‘–‘‹œ‡†ǡ’ƒ”ƒŽ›œ‡† ȋ˜‡…—”‘‹—„”‘‹†‡ͲǤͲͲͷ‰Ȁ‰‹–”ƒ˜‡‘—•Ž›Ȍǡƒ†˜‡–‹Žƒ–‡†™‹–Šƒ…‘•–ƒ–ϐŽ‘™˜‡–‹Žƒ–‘”ȋƒƒ› ͳͷǢ‹˜‡”•‹†ƒ††‡Žƒ‡’—„Ž‹…ƒǡ‘–‡˜‹†‡‘ǡ”—‰—ƒ›Ȍ—•‹‰–Š‡ˆ‘ŽŽ‘™‹‰’ƒ”ƒ‡–‡”•ǣˆ”‡“—‡…›ǡͳͲͲ

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

„”‡ƒ–Š•Ȁ‹Ǣ –‹†ƒŽ ˜‘Ž—‡ ȋVȌǡ ͲǤʹ Ǣ ˆ”ƒ…–‹‘ ‘ˆ ‹•’‹”‡† ‘š›‰‡ǡ ͲǤʹͳǤ Š‡ …Š‡•– ™ƒŽŽ ™ƒ• •—”‰‹…ƒŽŽ› ”‡‘˜‡†ƒ†ƒ’‘•‹–‹˜‡‡†Ǧ‡š’‹”ƒ–‘”›’”‡••—”‡‘ˆʹǤͲ…ʹ™ƒ•ƒ††‡†Ǥˆ–‡”ƒͳͲǦ‹˜‡–‹Žƒ–‹‘’‡”‹‘†ǡ Ž—‰‡…Šƒ‹…•™‡”‡…‘’—–‡†ƒ†ƒ––Š‡‡†‘ˆ–Š‡‡š’‡”‹‡–•ȋʹͲȂʹͷ‹ȌǡŽ—‰•™‡”‡’”‡’ƒ”‡†ˆ‘” Š‹•–‘Ž‘‰›ƒ†‘Ž‡…—Žƒ”ƒƒŽ›•‹•Ǥ —‰ •–ƒ–‹… ‡Žƒ•–ƒ…‡ ȋE•–ǡđȌǡ ”‡•‹•–‹˜‡ ’”‡••—”‡ ȋ'PͳǡđȌǡ ƒ† ˜‹•…‘‡Žƒ•–‹…Ȁ‹Š‘‘‰‡‡‘—• ’”‡••—”‡ ȋ'PʹǡđȌ ™‡”‡ ‡ƒ•—”‡† —•‹‰ –Š‡ ‡†Ǧ‹ϐŽƒ–‹‘ ‘……Ž—•‹‘ ‡–Š‘† ȏͳʹȐǤ ŽŽ †ƒ–ƒ ™‡”‡ ƒƒŽ›œ‡† —•‹‰ †ƒ–ƒƒƒŽ›•‹••‘ˆ–™ƒ”‡ȋǦ ˆ‘ƒ–ƒǡ …Ǥǡ‘–”‡ƒŽǡ—‡„‡…ǡƒƒ†ƒȌǤ Histology ‡†‹ƒ–‡Ž›ƒˆ–‡”Ž—‰ ‡…Šƒ‹…•™‡”‡ƒ••‡••‡†ǡŠ‡’ƒ”‹ȋͳͲͲͲ Ȍ™ƒ•‹–”ƒ˜‡‘—•Ž›‹–”‘†—…‡† ‹–‘ –Š‡ ˜‡ƒ …ƒ˜ƒ ƒ† ƒ Žƒ’ƒ”‘–‘› ™ƒ• ’‡”ˆ‘”‡†Ǥ Š‡ –”ƒ…Š‡ƒ ™ƒ• …Žƒ’‡† ƒ– ‡† ‡š’‹”ƒ–‹‘ǡ ƒ† –Š‡ ƒ„†‘‹ƒŽ ƒ‘”–ƒ ƒ† ˜‡ƒ …ƒ˜ƒ ™‡”‡ •‡…–‹‘‡†ǡ Ž‡ƒ†‹‰ –‘ ƒ ƒ••‹˜‡ Š‡‘””Šƒ‰‡ –Šƒ– “—‹…Ž› ‹ŽŽ‡†–Š‡ƒ‹ƒŽ•ǤŠ‡Ž‡ˆ–Ž—‰™ƒ•‹•‘Žƒ–‡†ǡˆ”‘œ‡“—‹…Ž›„›‹‡”•‹‘‹Ž‹“—‹†‹–”‘‰‡ǡϐ‹š‡†™‹–Š ƒ”‘›ǯ••‘Ž—–‹‘ǡƒ†‡„‡††‡†‹’ƒ”ƒˆϐ‹ǤŠ”‡‡ͶǦPǦ–Š‹…•Ž‹…‡•’‡”Ž—‰™‡”‡…—–ƒ†•–ƒ‹‡†™‹–Š Š‡ƒ–‘š›Ž‹Ȃ‡‘•‹ˆ‘”Ž—‰‘”’Š‘‡–”‹…ƒƒŽ›•‹•ǡ‘”‡‹‰‡”–ǯ•”‡•‘”…‹Ǧˆ—…Š•‹‡–Š‘†™‹–Š‘š‹†ƒ–‹‘ ˆ‘”“—ƒ–‹ϐ‹…ƒ–‹‘‘ˆ‡Žƒ•–‹…ϐ‹„‡”•‹ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†•‹Ž‹…‘–‹…‘†—Ž‡•Ǥ ‘” Ž—‰ ‘”’Š‘‡–”‹… ƒƒŽ›•‹•ǡ ƒ reticle …‘’‘•‡† ‘ˆ ͳͲͲ ’‘‹–• ƒ† ͷͲ Ž‹‡• ȋ‘™ Ž‡‰–ŠȌ …‘—’Ž‡† –‘ –Š‡ ”‹‰Š– ‡›‡’‹‡…‡ ‘ˆ ƒ Ž‹‰Š– ‹…”‘•…‘’‡ ™ƒ• —•‡†Ǥ Š‡ ˆ”ƒ…–‹‘ƒŽ ƒ”‡ƒ ‘ˆ ‰”ƒ—Ž‘ƒ ƒ† –Š‡ –‘–ƒŽƒ††‹ˆˆ‡”‡–‹ƒŽ…‡ŽŽ—Žƒ”‹–›‘ˆ‰”ƒ—Ž‘ƒ™‡”‡†‡–‡”‹‡†„›–Š‡’‘‹–Ǧ…‘—–‹‰–‡…Š‹“—‡ȏͳ͵Ȑ‹ʹͲ ”ƒ†‘‘Ǧ…‘‹…‹†‡–‹…”‘•…‘’‹…ϐ‹‡Ž†•ǤʹͲέ‘„Œ‡…–‹˜‡Ž‡•™ƒ•—•‡†–‘†‡–‡”‹‡–Š‡ˆ”ƒ…–‹‘ƒŽƒ”‡ƒ‘ˆ ‰”ƒ—Ž‘ƒǡƒ†ƒͳͲͲέ‘„Œ‡…–‹˜‡Ž‡•™ƒ•—•‡†–‘ƒ••‡••–Š‡–‘–ƒŽƒ††‹ˆˆ‡”‡–‹ƒŽ…‡ŽŽ—Žƒ”‹–›‘ˆ‰”ƒ—Ž‘ƒǤ ‘–ƒŽ—„‡”‘ˆƒ…”‘’Šƒ‰‡•ȋ]Ȍƒ†•—„’‘’—Žƒ–‹‘•ȋͳƒ†ʹȌǡŽ‡˜‡Ž‘ˆ ǦEǡƒ†–›’‡ ƒ† –›’‡ …‘ŽŽƒ‰‡ϐ‹„‡”…‘–‡–™‡”‡“—ƒ–‹ϐ‹‡†•‡’ƒ”ƒ–‡Ž›‹ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†‰”ƒ—Ž‘ƒƒˆ–‡”Žƒ„‡Ž‹‰„› ‹—‘Š‹•–‘…Š‡‹•–”›Ǥ‘‘…Ž‘ƒŽƒ–‹„‘†› ͶȀͺͲ”ƒ–ƒ–‹Ǧ‘—•‡ȋ…ƒ–ƒŽ‘‰‘ǤͶͻ͹ǡ„‡”‘–‡…Ȍ™ƒ• —•‡†–‘Žƒ„‡Žƒ…”‘’Šƒ‰‡•]Ǥƒ…”‘’Šƒ‰‡•ͳƒ†ʹ™‡”‡Žƒ„‡Ž‡†—•‹‰’‘Ž›…Ž‘ƒŽƒ–‹„‘†›‹†—…‹„Ž‡ ‹–”‹… ‘š‹†‡ •›–Šƒ•‡ ȋ‹Ȍ ”ƒ„„‹– ƒ–‹Ǧ‘—•‡ ȋ…ƒ–ƒŽ‘‰ ‘Ǥ „ǦͻʹͶʹǡ Š‡”‘ …‹‡–‹ϐ‹…Ȍ ƒ† ’‘Ž›…Ž‘ƒŽ ƒ–‹„‘†› ƒ”‰‹ƒ•‡Ǧͳ ”ƒ„„‹– ƒ–‹Ǧ‘—•‡ ȋ…ƒ–ƒŽ‘‰ ‘Ǥ •…ǦʹͲͳͷͲǡ ƒ–ƒ ”—œ ‹‘–‡…Š‘Ž‘‰›Ȍǡ ”‡•’‡…–‹˜‡Ž›Ǥ  ǦE™ƒ•†‡–‡…–‡†—•‹‰ƒ”ƒ„„‹–’‘Ž›…Ž‘ƒŽƒ–‹Ǧ’ƒǦ ǦEȋ…ƒ–ƒŽ‘‰‘ǤǦͳͲͲǡƬ›•–‡•ǡȌǤ Š‡‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹š…‘’‘‡–•™‡”‡Žƒ„‡Ž‡†™‹–Š”ƒ„„‹–ƒ–‹Ǧ‘—•‡’‘Ž›…Ž‘ƒŽƒ–‹„‘†‹‡•–‘–›’‡  ƒ†–›’‡ …‘ŽŽƒ‰‡•ȋ…ƒ–ƒŽ‘‰‘•ǤʹͲͳͶͳƒ†ʹͲ͵Ͷͳǡ”‡•’‡…–‹˜‡Ž›Ǣ‘˜‘–‡…ǡ ”ƒ…‡ȌǤ –‹„‘†‹‡• ™‡”‡ †‡–‡…–‡† —•‹‰ ƒ •‡…‘†ƒ”› ƒ–‹„‘†› Žƒ„‡Ž‡† ™‹–Š ’‡”‘š‹†ƒ•‡ ˆ”‘ ‹…Š‹”‡‹ ‹‘•…‹‡…‡•ȋ‘›‘ǡ ƒ’ƒȌȋ‹•–‘ϐ‹‡‘—•‡ƒ–‹Ǧ”ƒ–ƒ†ƒ–‹Ǧ”ƒ„„‹–Ȍˆ‘ŽŽ‘™‡†„›–Š‡…Š”‘‘‰‡ •—„•–”ƒ–‡ǡ †‹ƒ‹‘„‡œ‹†‹‡ ȋŽ‹“—‹† Ǣ …ƒ–ƒŽ‘‰ ‘Ǥ ͵Ͷ͸ͺǡ ƒ‘…›–‘ƒ–‹‘ǡ ȌǤ Š‹”–› ‹…”‘•…‘’‹… ϐ‹‡Ž†• ™‡”‡ ”ƒ†‘Ž› •‡Ž‡…–‡†ǡ ƒ˜‘‹†‹‰ ˜‡••‡Ž• ƒ† „”‘…Š‹ǡ —•‹‰ ƒ †‹‰‹–ƒŽ …ƒ‡”ƒ ȋ˜‘Ž—–‹‘ǡ ‡†‹ƒ ›„‡”‡–‹…•ǡ‹Ž˜‡”’”‹‰•ǡǡȌ…‘—’Ž‡†–‘ƒŽ‹‰Š–‹…”‘•…‘’‡ȋ…Ž‹’•‡ͶͲͲǢ‹‘ǡ‘›‘ǡ ƒ’ƒȌǡƒ† ƒ…‘’—–‡”™‹–Š‰”ƒ’Š‹…ƒŽ‹–‡”ˆƒ…‡•‘ˆ–™ƒ”‡ȋǦƒ’–—”‡ʹǤͻͷǤͲǡ˜‡”•‹‘ʹǤͲǤͷǢ‹Ž‹…‘ ”ƒ’Š‹…• …ǡȌǤ ‹‰ŠǦ“—ƒŽ‹–›‹ƒ‰‡•ȋʹͲͶͺέͳͷ͵͸’‹š‡Ž„—ˆˆ‡”Ȍ™‡”‡…ƒ’–—”‡†ƒˆ–‡”•‡––‹‰—’ƒ†…ƒŽ‹„”ƒ–‹‰–Š‡’”‘‰”ƒǡ —•‹‰–Š‡ͶͲέ‘„Œ‡…–‹˜‡Ž‡•ǤŠ‡—„‡”‘ˆŽƒ„‡Ž‡†ƒ…”‘’Šƒ‰‡•™ƒ•†‹˜‹†‡†„›–Š‡–‘–ƒŽ—„‡”‘ˆ…‡ŽŽ• ’‡” ϐ‹‡Ž†Ǥ Š‡ ƒ”‡ƒ ‘……—’‹‡† „›  ǦȾ ƒ† …‘ŽŽƒ‰‡ ™ƒ• †‹˜‹†‡† „› –Š‡ –‘–ƒŽ –‹••—‡ ƒ”‡ƒǡ ƒ† –Š‡ ”‡•—Ž–• ™‡”‡‡š’”‡••‡†ƒ••—”ˆƒ…‡†‡•‹–›ǤŠ‡‹ƒ‰‡•™‡”‡ƒƒŽ›œ‡†—•‹‰ ƒ‰‡Ǧ”‘Ž—•ͶǤͷǤͳ•‘ˆ–™ƒ”‡ȋ‡†‹ƒ ›„‡”‡–‹…•ȌǤ TUNEL assay ˆ”ƒ‰‡–ƒ–‹‘ƒ••‘…‹ƒ–‡†™‹–Šƒ’‘’–‘•‹•™ƒ•†‡–‡…–‡†„›–‡”‹ƒŽ†‡‘š›—…Ž‡‘–‹†›Ž–”ƒ•ˆ‡”ƒ•‡ ȋ†ȌǦ‡†‹ƒ–‡††‹…Ǧ‡†Žƒ„‡Ž‹‰ȋȌǡ—•‹‰ƒ’‘’ƒ‰’‡”‘š‹†ƒ•‡‹•‹–—ƒ’‘’–‘•‹•†‡–‡…–‹‘ ‹–ȋ…ƒ–ƒŽ‘‰‘Ǥ͹ͳͲͳǡ‹ŽŽ‹’‘”‡ǡ‹ŽŽ‡”‹…ƒǡǡȌǡƒ……‘”†‹‰–‘–Š‡ƒ—ˆƒ…–—”‡”ǯ•‹•–”—…–‹‘•Ǥ ‘” “—ƒ–‹ϐ‹…ƒ–‹‘ǡͷͲ‹…”‘•…‘’‹…ϐ‹‡Ž†•™‡”‡”ƒ†‘Ž›…ƒ’–—”‡†ȋʹͷ‹‰”ƒ—Ž‘ƒƒ†ʹͷ‹ƒŽ˜‡‘Žƒ”•‡’–ƒȌ —•‹‰ƒͶͲέ‘„Œ‡…–‹˜‡Ž‡•Ǥ‘•‹–‹˜‡—…Ž‡‹”‡•—Ž–•ƒ”‡‡š’”‡••‡†ƒ•–Š‡’‡”…‡–ƒ‰‡‘ˆƒ’‘’–‘–‹……‡ŽŽ•Ǥ Levels of interleukin (IL)-1D, IL-1E, IL-1 receptor type 1, IL-1 receptor antagonist, IL-10, metalloproteinase-9, TGF-E and caspase-3 mRNA ‡–”ƒŽ •Ž‹…‡• ‘ˆ ”‹‰Š– Ž—‰ ™‡”‡ …—–ǡ …‘ŽŽ‡…–‡† ‹ …”›‘–—„‡•ǡ “—‹… ˆ”‘œ‡ „› ‹‡”•‹‘ ‹ Ž‹“—‹† ‹–”‘‰‡ǡƒ†Š‡Ž†ƒ–ȂͺͲιǤ“—ƒ–‹–ƒ–‹˜‡”‡ƒŽǦ–‹‡”‡˜‡”•‡–”ƒ•…”‹’–‹‘ȋȌ’‘Ž›‡”ƒ•‡…Šƒ‹”‡ƒ…–‹‘ ȋȌ ™ƒ• ’‡”ˆ‘”‡† –‘ ‡ƒ•—”‡ –Š‡ ”‡Žƒ–‹˜‡ Ž‡˜‡Ž• ‘ˆ  ‘ˆ ǦͳDǡ ǦͳEǡ Ǧͳ ”‡…‡’–‘” –›’‡ ͳ ȋ Ǧ

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

ͳͳȌǡ Ǧͳ ”‡…‡’–‘” ƒ–ƒ‰‘‹•– ȋ ǦͳȌǡ ǦͳͲǡ ‡–ƒŽŽ‘’”‘–‡‹ƒ•‡ ȋȌǦͻǡ  ǦE ƒ† …ƒ•’ƒ•‡Ǧ͵Ǥ ‘–ƒŽ ™ƒ•‡š–”ƒ…–‡†ˆ”‘–Š‡ˆ”‘œ‡–‹••—‡•—•‹‰ƒ‡ƒ•›‹‹‹–ȋ‹ƒ‰‡ǡƒŽ‡…‹ƒǡǡȌƒ……‘”†‹‰ –‘ –Š‡ ƒ—ˆƒ…–—”‡”ǯ• ”‡…‘‡†ƒ–‹‘•Ǥ Š‡  …‘…‡–”ƒ–‹‘ ™ƒ• ‡ƒ•—”‡† „› •’‡…–”‘’Š‘–‘‡–”› ‹ ƒ‘†”‘’ ǦͳͲͲͲǤ ‹”•–Ǧ•–”ƒ† … ™ƒ• •›–Š‡•‹œ‡† ˆ”‘ –‘–ƒŽ  —•‹‰ ƒ Ǧ ‡˜‡”•‡ ”ƒ•…”‹’–ƒ•‡‹–ȋ ˜‹–”‘‰‡ȌǤ‡Žƒ–‹˜‡Ž‡˜‡Ž•™‡”‡‡ƒ•—”‡†™‹–Šƒ ”‡‡†‡–‡…–‹‘•›•–‡ —•‹‰ ͹ͷͲͲ”‡ƒŽǦ–‹‡ȋ’’Ž‹‡†‹‘•›•–‡•ǡ ‘•–‡”‹–›ǡȌǤŽŽ•ƒ’Ž‡•™‡”‡‡ƒ•—”‡†‹†—’Ž‹…ƒ–‡Ǥ Š‡ ”‡Žƒ–‹˜‡ Ž‡˜‡Ž ‘ˆ ‡ƒ…Š ‰‡‡ ™ƒ• …ƒŽ…—Žƒ–‡† ƒ• –Š‡ ”ƒ–‹‘ ‘ˆ –Š‡ •–—†› ‰‡‡ –‘ –Š‡ …‘–”‘Ž ‰‡‡ ȋƒ…‹†‹… ”‹„‘•‘ƒŽ’Š‘•’Š‘’”‘–‡‹Ͳȏ͵͸EͶȐȌƒ†‰‹˜‡ƒ•–Š‡ˆ‘Ž†…Šƒ‰‡”‡Žƒ–‹˜‡–‘ ǦƒŽǤŠ‡ˆ‘ŽŽ‘™‹‰ ’”‹‡”•™‡”‡—•‡†ǣ ǦͳDˆ‘”™ƒ”†ͷcǦ

 

Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ  

 Ǧ͵cͳͲʹ„’Ǣ ǦͳEˆ‘”™ƒ”†ͷcǦ  

 Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ

      Ǧ͵cͻ͵„’Ǣ Ǧͳͳˆ‘”™ƒ”†ͷcǦ      Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ 

   Ǧ͵c͸͸„’Ǣ Ǧͳˆ‘”™ƒ”†ͷcǦ 

Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ     Ǧ͵cͳͷͲ„’Ǣ ǦͳͲˆ‘”™ƒ”†ͷcǦ     Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ  

Ǧ͵cǢǦͻˆ‘”™ƒ”†ͷcǦ    Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ 

Ǧ͵cǢ…ƒ•’ƒ•‡Ǧ͵ˆ‘”™ƒ”†ͷcǦ





 Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ   

   Ǧ͵cͳͲͶ„’Ǣ ǦEˆ‘”™ƒ”†ͷcǦ   

 Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ    ͵c͹͹„’Ǣ͵͸EͶˆ‘”™ƒ”†ͷcǦ 

 Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ      Ǧ͵cͳͷͲ„’Ǥ Detection of Y chromosome DNA —ƒ–‹ϐ‹…ƒ–‹‘‘ˆ—”‹‡…Š”‘‘•‘‡‹Ž—‰–‹••—‡™ƒ•ƒ…Š‹‡˜‡†—•‹‰“—ƒ–‹–ƒ–‹˜‡”‡ƒŽǦ–‹‡ ƒ–†ƒ›͸Ͳƒ•’”‡˜‹‘—•Ž›†‡•…”‹„‡†ȏ͵ȐǤŠ‡ˆ‘ŽŽ‘™‹‰’”‹‡”•™‡”‡—•‡†ǣˆ‘”™ƒ”†ͷcǦ   

  Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ   

Ǧ͵cǢ ˆ‘”™ƒ”†ͷcǦ   Ǧ͵cƒ†”‡˜‡”•‡ͷcǦ  

Ǧ͵cǤ Statistical analysis Š‡‘”ƒŽ‹–›‘ˆ–Š‡†ƒ–ƒȋ‘Ž‘‰‘”‘˜Ȃ‹”‘˜–‡•–™‹–Š‹ŽŽ‹‡ˆ‘”•ǯ…‘””‡…–‹‘Ȍƒ†–Š‡Š‘‘‰‡‡‹–› ‘ˆ˜ƒ”‹ƒ…‡•ȋ‡˜‡‡‡†‹ƒ–‡•–Ȍ™‡”‡–‡•–‡†Ǥ ˆ„‘–Š…‘†‹–‹‘•™‡”‡•ƒ–‹•ˆƒ…–‘”›ǡ†‹ˆˆ‡”‡…‡•„‡–™‡‡–Š‡ ‰”‘—’•™‡”‡ƒ••‡••‡†„›–™‘Ǧ™ƒ›ƒƒŽ›•‹•‘ˆ˜ƒ”‹ƒ…‡ˆ‘ŽŽ‘™‡†„›–Š‡—‡›–‡•–ǤŠ‡…‘’ƒ”‹•‘ „‡–™‡‡–Š‡ ǦƒŽƒ† Ǧ‡ŽŽ‰”‘—’•™ƒ•’‡”ˆ‘”‡†—•‹‰–Š‡—’ƒ‹”‡†t–‡•–‘”–Š‡ƒȂŠ‹–‡›U –‡•–ˆ‘”’ƒ”ƒ‡–”‹…ƒ†‘Ǧ’ƒ”ƒ‡–”‹…†ƒ–ƒǡ”‡•’‡…–‹˜‡Ž›Ǥƒ–ƒƒ”‡’”‡•‡–‡†ƒ•–Š‡‡ƒά‘”‡†‹ƒ ȋʹͷ–ŠȂ͹ͷ–Š ’‡”…‡–‹Ž‡•Ȍ ™Š‡ ƒ’’”‘’”‹ƒ–‡Ǥ  ƒŽŽ –‡•–•ǡ –Š‡ •‹‰‹ϐ‹…ƒ…‡ Ž‡˜‡Ž ™ƒ• •‡– ƒ– ͷΨǤ –ƒ–‹•–‹…ƒŽ ƒƒŽ›•‡•™‡”‡’‡”ˆ‘”‡†—•‹‰‹‰ƒŽ‘–ͳͳǤͲȋ›•–ƒ–‘ˆ–™ƒ”‡ǡŠ‹…ƒ‰‘ǡ ǡȌǤ

Results

Lung mechanics and engraftment ‘•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡•™‡”‡‘„•‡”˜‡†‹–‹†ƒŽ˜‘Ž—‡ƒ†ϐŽ‘™„‡–™‡‡–Š‡‰”‘—’•Ǥ Š‡ ǦƒŽ ƒ† Ǧ‡ŽŽ ‰”‘—’• •Š‘™‡† •‹‹Žƒ” ˜ƒŽ—‡• ˆ‘” Est,đǡ 'Pͳ,đǡ ƒ† 'Pʹ,đ ’”‡••—”‡•Ǥ ‹‰Š‡”˜ƒŽ—‡•ˆ‘”Est,đǡ'Pͳ,đǡƒ†'Pʹ,𙇔‡ˆ‘—†ˆ‘”–Š‡ ǦƒŽ‰”‘—’–Šƒ–Š‡ǦƒŽ‰”‘—’ ȋͳ͵ͶΨǡ ͳ͹ͲΨǡ ƒ† ͳͶͺΨǡ ”‡•’‡…–‹˜‡Ž›ȌǤ  –Š‡  Ǧ‡ŽŽ ‰”‘—’ǡ ƒŽŽ ‡…Šƒ‹…ƒŽ ’ƒ”ƒ‡–‡”• ™‡”‡”‡†—…‡†…‘’ƒ”‡†™‹–Š–Š‡ ǦƒŽ‰”‘—’„—–'Pͳ,đ”‡ƒ‹‡†Š‹‰Š‡”–Šƒˆ‘”–Š‡Ǧ‡ŽŽ ‰”‘—’ȋ ‹‰ǤʹǡȌǤŠ‡”‡™ƒ•‘†‡–‡…–ƒ„Ž‡ …Š”‘‘•‘‡ ‘†ƒ›͸Ͳ‹–Š‡Ǧ‡ŽŽƒ†  Ǧ‡ŽŽ‰”‘—’•ȋ†ƒ–ƒ‘–•Š‘™ȌǤ Lung inϔlammation  –Š‡  ǦƒŽ ‰”‘—’ǡ ™‡ ‘„•‡”˜‡† ‰”ƒ—Ž‘ƒ–‘—• ‘†—Ž‡• ™‹–Š ‹ϐ‹Ž–”ƒ–‹‘ ‘ˆ ’‘Ž›‘”’Š‘—…Ž‡ƒ”ƒ†‘‘—…Ž‡ƒ”…‡ŽŽ•ǡƒ‹Ž›ƒ…”‘’Šƒ‰‡•ǡ‹š‡†™‹–Š’”‘–‡‹ƒ…‡‘—• ƒ–‡”‹ƒŽǤ‘”‡‘˜‡”ǡ–Š‡”‡™‡”‡‹…”‡ƒ•‡†ƒ”‡ƒ•‘ˆƒŽ˜‡‘Žƒ”…‘ŽŽƒ’•‡ǡ”‡†—…‡†ƒ‹”™ƒ›Ž—‡ǡ ƒ† …‡ŽŽ ‹ϐ‹Ž–”ƒ–‹‘ ‹ –Š‡ ƒŽ˜‡‘Žƒ” •‡’–ƒ ȋ ‹‰Ǥ ͵ȌǤ Š‡ ˆ”ƒ…–‹‘ƒŽ ƒ”‡ƒ ‘ˆ ‰”ƒ—Ž‘ƒ ƒ† –Š‡–‘–ƒŽƒ††‹ˆˆ‡”‡–‹ƒŽ…‡ŽŽ…‘—–™‡”‡”‡†—…‡†ƒˆ–‡””‡’‡ƒ–‡†‹–”ƒ–”ƒ…Š‡ƒŽ–Š‡”ƒ’›™‹–Š •ȋ ‹‰Ǥ͵ǡȌǤ ƒ…”‘’Šƒ‰‡•ƒ”‡–Š‡ƒ‹…‡ŽŽ•‹˜‘Ž˜‡†‹–Š‡’ƒ–Š‘’Š›•‹‘Ž‘‰›‘ˆ•‹Ž‹…‘•‹•ǤŠ‡•‡…‡ŽŽ• …ƒ„‡ƒ…–‹˜ƒ–‡†„›ƒ”ƒ‰‡‘ˆ‡š–”ƒ…‡ŽŽ—Žƒ”•‹‰ƒŽ•–‘’‘Žƒ”‹œ‡‹ͳƒ…”‘’Šƒ‰‡•ȋ”‡•’‘•‹„Ž‡

1684

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 1. ȋȌ …Š‡ƒ–‹… ϐŽ‘™ …Šƒ”– ƒ† ȋȌ –‹‡Ž‹‡ ‘ˆ –Š‡ •–—†› †‡•‹‰Ǥ ǡ …‘–”‘ŽǢ  ǡ •‹Ž‹…‘•‹•ǤƒŽ‹‡ȋƒŽȌƒ† „‘‡ ƒ””‘™Ȃ†‡”‹˜‡† …‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡ ‹–”ƒ–”ƒ…Š‡ƒŽŽ› ȋ Ȍ ƒ†‹‹•–‡”‡† ‘ †ƒ›• ͳͷƒ†͵ͲǤ‹ƒŽ•™‡”‡ •ƒ…”‹ϐ‹…‡†‘†ƒ›͸ͲǤ

A

B

Fig. 2. —‰ ‡…Šƒ‹…•Ǥ ȋȌ —‰ •–ƒ–‹… ‡Žƒ•–ƒ…‡ (E•–ǡđȌ ƒ† ȋȌ ”‡•‹•–‹˜‡ ’”‡••—”‡ ȋ'PͳǡđȌ ƒ† ˜‹•…‘‡Žƒ•–‹…Ȁ‹Š‘‘‰‡‡‘—•’”‡••—”‡('PʹǡđȌǤǡ…‘–”‘ŽǢ ǡ•‹Ž‹…‘•‹•ǤƒŽ‹‡ȋƒŽȌƒ†„‘‡ƒ””‘™Ȃ †‡”‹˜‡†…‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȋ Ȍƒ†‹‹•–‡”‡†‘†ƒ›•ͳͷƒ†͵ͲǤƒ–ƒƒ”‡’”‡•‡–‡† ƒ• ‡ƒ•άǢ nαͺ ƒ‹ƒŽ• ’‡” ‰”‘—’Ǥ ȗ‹‰‹ϐ‹…ƒ–Ž› †‹ˆˆ‡”‡– ˆ”‘ ǦƒŽ ƒ† Ǧ‡ŽŽǤ Ț‹‰‹ϐ‹…ƒ–Ž› †‹ˆˆ‡”‡–ˆ”‘ ǦƒŽǤ

ˆ‘” –Š‡ ƒ–‹‹…”‘„‹ƒŽ ”‡•’‘•‡ ƒ† ‹ϐŽƒƒ–‹‘Ȍ ‘” ʹ ƒ…”‘’Šƒ‰‡• ȋ”‡•’‘•‹„Ž‡ ˆ‘” ”‡•‘Ž˜‹‰–Š‡‹ϐŽƒƒ–‹‘ƒ†™‘—†”‡’ƒ‹”ȌǤŠ‡–‘–ƒŽ—„‡”‘ˆƒ…”‘’Šƒ‰‡•ȋ]Ȍƒ† •—„’‘’—Žƒ–‹‘•ȋͳƒ†ʹȌ™‡”‡“—ƒ–‹ϐ‹‡†ȋ ‹‰ǤͶȂȌǤ –Š‡ ǦƒŽ‰”‘—’ǡ–Š‡—„‡” ‘ˆƒ…”‘’Šƒ‰‡•™ƒ•Š‹‰Š‡”‹‰”ƒ—Ž‘ƒƒ†ƒŽ˜‡‘Žƒ”•‡’–ƒ…‘’ƒ”‡†™‹–Š–Š‡ǦƒŽ‰”‘—’Ǥ  Ǧ‡ŽŽƒ‹ƒŽ•ǡ–Š‡–‘–ƒŽ—„‡”‘ˆƒ…”‘’Šƒ‰‡•ƒ†–Š‡—„‡”‘ˆͳƒ…”‘’Šƒ‰‡•‹ ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†‰”ƒ—Ž‘ƒ™‡”‡Ž‘™‡”–Šƒ‹–Š‡ ǦƒŽ‰”‘—’ȋ ‹‰ǤͶȂ ȌǤ‘•‹‰‹ϐ‹…ƒ– †‹ˆˆ‡”‡…‡™ƒ•‘„•‡”˜‡†‹–Š‡—„‡”‘ˆʹƒ…”‘’Šƒ‰‡•„‡–™‡‡–Š‡ ǦƒŽƒ† Ǧ‡ŽŽ ‰”‘—’•ȋ ‹‰ǤͶǡ ȌǤ Expression of IL-1D, IL-1E, IL-1R1, IL-1RN, IL-10, and MMP-9 Š‡Ž‡˜‡Ž‘ˆ ǦͳEǡ Ǧͳͳǡ Ǧͳǡ ǦͳDǡ ǦͳͲǡƒ†Ǧͻ™ƒ•Š‹‰Š‡”‹–Š‡  ǦƒŽ‰”‘—’…‘’ƒ”‡†™‹–Š–Š‡ǦƒŽ‰”‘—’ǤŠ‡‡š’”‡••‹‘‘ˆ ǦͳEƒ† Ǧͳͳ”‡†—…‡† ƒˆ–‡”  –Š‡”ƒ’› ȋ ‹‰Ǥ ͷǡȌǡ ™Š‡”‡ƒ• Ǧͳ Ž‡˜‡Ž ‹…”‡ƒ•‡† ȋ ‹‰Ǥ ͷȌǤ ‘ •‹‰‹ϐ‹…ƒ– †‹ˆˆ‡”‡…‡•™‡”‡‘„•‡”˜‡†‹–Š‡Ž‡˜‡Ž‘ˆ ǦͳDǡ ǦͳͲǡƒ†Ǧͻ„‡–™‡‡–Š‡ Ǧ ƒŽƒ† Ǧ‡ŽŽ‰”‘—’•ȋ ‹‰ǤͷȂ ȌǤ Lung remodeling Š‡ƒ‘—–‘ˆ ǦEƒ†–›’‡ ƒ† …‘ŽŽƒ‰‡ϐ‹„‡”•™ƒ•‹…”‡ƒ•‡†‹ƒŽ˜‡‘Žƒ”•‡’–ƒ ƒ†‰”ƒ—Ž‘ƒ‹–Š‡ ǦƒŽ‰”‘—’…‘’ƒ”‡†™‹–Š–Š‡ǦƒŽ‰”‘—’Ǥ•”‡†—…‡†

1685

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 3. ȋȌ ‡’”‡•‡–ƒǦ –‹˜‡ ’Š‘–‘‹…”‘‰”ƒ’Š• ‘ˆ Ž—‰ ’ƒ”‡…Š›ƒ •–ƒ‹‡† ™‹–Š Š‡ƒ–‘š›Ǧ Ž‹Ȃ‡‘•‹ ȋȌǤ ‘–‡ –Š‡ ’”‡•‡…‡ ‘ˆ ‰”ƒ—Ž‘ƒ• ‹ –Š‡   ‰”‘—’• ȋ*ȌǤ ȋȌ—ƒ–‹ϐ‹…ƒ–‹‘‘ˆ–Š‡ ˆ”ƒ…–‹‘ƒŽƒ”‡ƒ‘ˆ‰”ƒ—Ǧ Ž‘ƒǤƒ–ƒƒ”‡’”‡•‡–‡† ƒ• ‡†‹ƒ ȋʹͷ–ŠȂ͹ͷ–Š ’‡”…‡–‹Ž‡•ȌǤ ȋȌ ‘–ƒŽ ƒ† †‹ˆˆ‡”‡–‹ƒŽ –‹••—‡ …‡ŽŽ—Žƒ”‹–› ‹ –Š‡ ‰”ƒ—Ǧ Ž‘ƒǤ ƒ–ƒ ƒ”‡ ’”‡•‡Ǧ –‡† ƒ• ‡ƒ•άǤ ǡ …‘–”‘ŽǢ ǡ ’‘Ž›‘”Ǧ ’Š‘—…Ž‡ƒ” …‡ŽŽ•Ǣ ǡ ‘‘—…Ž‡ƒ” …‡ŽŽ•Ǣ  ǡ •‹Ž‹…‘•‹•ǤƒŽ‹‡ȋƒŽȌƒ† „‘‡ ƒ””‘™Ȃ†‡”‹˜‡† …‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡ ‹–”ƒ–”ƒ…Š‡ƒŽŽ› ȋ Ȍ ƒ†Ǧ ‹‹•–‡”‡† ‘ †ƒ›• ͳͷ ƒ†͵ͲǢnαͺƒ‹ƒŽ•’‡” ‰”‘—’ǤȚ‹‰‹ϐ‹…ƒ–Ž›†‹ˆǦ ˆ‡”‡–ˆ”‘ ǦƒŽǤ

‡š’”‡••‹‘ ‘ˆ  ǦE ȋ ‹‰Ǥ ͸Ȍ ƒ† …‘ŽŽƒ‰‡ †‡’‘•‹–‹‘ ȋ–›’‡•  ƒ† Ȍ ‹ ‰”ƒ—Ž‘ƒ ƒ† ƒŽ˜‡‘Žƒ” •‡’–ƒ ȋ ‹‰Ǥ ͹ȌǤ Žƒ•–‹… ϐ‹„‡” …‘–‡– ™ƒ• ƒŽ•‘ ‹…”‡ƒ•‡† ‹•‹†‡ –Š‡ ‰”ƒ—Ž‘ƒ ƒ† ƒŽ˜‡‘Žƒ” •‡’–ƒ ‹ –Š‡  ǦƒŽ ‰”‘—’ „—– ”‡†—…‡† ƒˆ–‡”  –Š‡”ƒ’› ‹ –Š‡ ƒŽ˜‡‘Žƒ” •‡’–ƒ ȋ ‹‰ǤͺȌǤ Apoptosis Š‡—„‡”‘ˆ…‡ŽŽ•—†‡”‰‘‹‰ƒ’‘’–‘•‹•™ƒ•”ƒ”‡‹–Š‡ǦƒŽƒ†Ǧ‡ŽŽ‰”‘—’•ȋ ‹‰Ǥ ͻȌǤŠ‡—„‡”‘ˆƒ’‘’–‘–‹……‡ŽŽ•ƒ†–Š‡Ž‡˜‡Ž‘ˆ…ƒ•’ƒ•‡Ǧ͵™‡”‡Š‹‰Š‡”‹–Š‡ Ǧ ƒŽ‰”‘—’…‘’ƒ”‡†™‹–Š–Š‡ǦƒŽ‰”‘—’ȋ ‹‰ǤͻȂȌǤ‡’‡ƒ–‡†ƒ†‹‹•–”ƒ–‹‘‘ˆ•‹ •‹Ž‹…‘–‹…‹…‡”‡†—…‡†ƒ’‘’–‘•‹•‹–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†‰”ƒ—Ž‘ƒǡƒ•™‡ŽŽƒ•…ƒ•’ƒ•‡Ǧ͵ Ž‡˜‡ŽǤ Discussion

 ƒ ’”‡˜‹‘—• •–—†›ǡ ƒ••ƒ…‡ ‡– ƒŽǤ ȏͳͳȐ ‘„•‡”˜‡† –Šƒ– –Š‡ ‡ƒ”Ž› „‡‡ϐ‹…‹ƒŽ ‡ˆˆ‡…–• ‘ˆ –Š‡”ƒ’› ™‹–Š • ™‡”‡ ‡’Š‡‡”ƒŽ ͸Ͳ †ƒ›• ƒˆ–‡” ‹–”ƒ–”ƒ…Š‡ƒŽ ‹•–‹ŽŽƒ–‹‘ ‘ˆ •‹Ž‹…ƒ ’ƒ”–‹…Ž‡•‹—”‹‡•‹Ž‹…‘•‹•Ǥ –Š‡…—””‡–•–—†›ǡ™‡‘„•‡”˜‡†–Šƒ––™‘‹ˆ—•‹‘•‘ˆ• Ž‡†–‘ƒ”‡†—…–‹‘‹…‘ŽŽƒ‰‡ƒ†‡Žƒ•–‹…ϐ‹„‡”†‡’‘•‹–‹‘ǡ–Š‡–‘–ƒŽ—„‡”‘ˆƒ…”‘’Šƒ‰‡• ƒ†•—„’‘’—Žƒ–‹‘ͳȋ‹ϐŽƒƒ–‘”›ƒ†ƒ–‹‹…”‘„‹ƒŽȌǡƒ•™‡ŽŽƒ•–Š‡ƒ‘—–‘ˆƒ’‘’–‘–‹… …‡ŽŽ• ƒ† –Š‡ ˆ”ƒ…–‹‘ƒŽ ƒ”‡ƒ ‘ˆ ‰”ƒ—Ž‘ƒǡ ”‡•—Ž–‹‰ ‹ ‹’”‘˜‡‡– ‹ Est,đǡ 'Pͳ,đǡ ƒ† 'Pʹ,đ ’”‡••—”‡• ȋ ‹‰Ǥ ʹȌǤ —”–Š‡”‘”‡ǡ –Š‡•‡ „‡‡ϐ‹…‹ƒŽ ‡ˆˆ‡…–• ™‡”‡ ‹†‡’‡†‡– ‘ˆ –Š‡ ’‡”ƒ‡…‡‘ˆ•‹Ž—‰–‹••—‡ǡ•—‰‰‡•–‹‰ƒ’ƒ”ƒ…”‹‡‡ˆˆ‡…–Ǥ •‹‰Ž‡‡š’‘•—”‡–‘…”›•–ƒŽŽ‹‡•‹Ž‹…ƒ…ƒŽ‡ƒ†–‘‘”’Š‘ˆ—…–‹‘ƒŽ…Šƒ‰‡•ƒˆ–‡”ͳͷ†ƒ›• ‹ƒ—”‹‡‘†‡ŽǤŠ‡ˆ—…–‹‘ƒŽ…Šƒ‰‡•ƒ”‡•‹‹Žƒ”–‘–Š‘•‡‘„•‡”˜‡†‹–Š‡…Ž‹‹…ƒŽ•‡––‹‰ ȏ͵ǡͳͶȐǤ‹Ž‹…‘–‹…‹…‡•Š‘™‡†‹…”‡ƒ•‡†˜ƒŽ—‡•ˆ‘”Est,đǡ'Pͳ,đǡƒ†'Pʹ,đǤŠ‡‘†‹ϐ‹…ƒ–‹‘•

1686

Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 4. ‡’”‡•‡–ƒ–‹˜‡ ’Š‘–‘‹…”‘‰”ƒ’Š• ‘ˆ Ž—‰ ’ƒ”‡…Š›ƒǤ —‘Š‹•–‘…Š‡‹•–”› ˆ‘”ǣ ȋȌ ͶȀͺͲǡ ȋȌ‹ǡƒ†ȋȌƒ”‰‹ƒ•‡Ǧͳƒ–‹„‘†‹‡•Ǥ‘–‡–Š‡’”‡•‡…‡‘ˆƒ…”‘’Šƒ‰‡•ȋarrowsȌ‹–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒ ƒ†‹–Š‡‰”ƒ—Ž‘ƒǤ—ƒ–‹ϐ‹…ƒ–‹‘‘ˆƒ…”‘’Šƒ‰‡•]‹ȋȌ–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†ȋȌ–Š‡‰”ƒ—Ž‘ƒǤ —ƒ–‹ϐ‹…ƒ–‹‘ ‘ˆ ƒ…”‘’Šƒ‰‡• ͳ ‹ ȋ Ȍ –Š‡ ƒŽ˜‡‘Žƒ” •‡’–ƒ ƒ† ȋ Ȍ –Š‡ ‰”ƒ—Ž‘ƒǤ —ƒ–‹ϐ‹…ƒ–‹‘ ‘ˆ ƒ…”‘’Šƒ‰‡•ʹ‹ȋȌ–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†ȋ Ȍ–Š‡‰”ƒ—Ž‘ƒǤǡ…‘–”‘ŽǢ ǡ•‹Ž‹…‘•‹•ǤƒŽ‹‡ȋƒŽȌƒ† „‘‡ƒ””‘™Ȃ†‡”‹˜‡†…‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȋ Ȍƒ†‹‹•–‡”‡†‘†ƒ›•ͳͷƒ†͵ͲǤƒ–ƒ ƒ”‡ ’”‡•‡–‡† ƒ• ‡ƒ•άǢ nαͺ ƒ‹ƒŽ• ’‡” ‰”‘—’Ǥ ȗ‹‰‹ϐ‹…ƒ–Ž› †‹ˆˆ‡”‡– ˆ”‘ ǦƒŽǤ Ț‹‰‹ϐ‹…ƒ–Ž› †‹ˆˆ‡”‡–ˆ”‘ ǦƒŽǤ

‘„•‡”˜‡†‹Est,đ and 'Pʹ,đ…‘—Ž†„‡ƒ••‘…‹ƒ–‡†™‹–ŠƒŽ˜‡‘Žƒ”…‘ŽŽƒ’•‡ǡ‹–‡”•–‹–‹ƒŽ‡†‡ƒǡ –Š‹…‡‹‰‘ˆƒŽ˜‡‘Žƒ”•‡’–ƒǡ‹…”‡ƒ•‡†—„‡”‘ˆ…‡ŽŽ•ǡƒ†–Š‡’”‡•‡…‡‘ˆ•‹Ž‹…‘–‹…‘†—Ž‡•Ǥ Š‡ …Šƒ‰‡• ‹ 'Pͳ,đ ƒ› „‡ ƒ––”‹„—–‡† –‘ ‹–”ƒ„”‘…Š‹ƒŽ …‡ŽŽ—Žƒ” ‹ϐ‹Ž–”ƒ–‹‘ Ž‡ƒ†‹‰ –‘ ‘„•–”—…–‹‘ ‘ˆ –Š‡ Ž—‡ ȋ ‹‰•Ǥ ʹ ƒ† ͵Ȍ ‹ ƒ……‘”†ƒ…‡ ™‹–Š ’”‡˜‹‘—• •–—†‹‡• ‘ •‹Ž‹…‘–‹… ‹…‡ȏ͵ǡͳͶȂͳ͸ȐǤ‹…”‡ƒ•‡‹–Š‡—„‡”‘ˆƒ…”‘’Šƒ‰‡•ǡ„‘–Š‹ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†‹•‹†‡

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 5. ‡ƒŽǦ–‹‡”‡˜‡”•‡–”ƒ•…”‹’–‹‘ƒƒŽ›•‹•‘ˆȋȌ ǦͳEǡȋȌ ǦͳͳǡȋȌ ǦͳǡȋȌ ǦͳDǡȋȌ Ǧ ͳͲǡƒ†ȋ ȌǦͻǤǡ…‘–”‘ŽǢ ǡ•‹Ž‹…‘•‹•ǤƒŽ‹‡ȋƒŽȌƒ†„‘‡ƒ””‘™Ȃ†‡”‹˜‡†…‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡ ‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȋ Ȍƒ†‹‹•–‡”‡†‘†ƒ›•ͳͷƒ†͵ͲǤƒ–ƒƒ”‡’”‡•‡–‡†ƒ•‡ƒ•άǢnα͸ƒ‹ƒŽ•’‡” ‰”‘—’Ǥȗ‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡–ˆ”‘ǦƒŽǤȚ‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡–ˆ”‘ ǦƒŽǤ

‰”ƒ—Ž‘ƒǡ ™ƒ• ˆ‘—† ‹ –Š‡  ǦƒŽ ‰”‘—’ ȋ ‹‰Ǥ ͶȌǤ Š‹• …‡ŽŽ ’‘’—Žƒ–‹‘ ‹• ‘‡ ‘ˆ –Š‡ ‡› –ƒ”‰‡–•‹•‹Ž‹…‘•‹•ȏʹȐǤŠ‡‹–‡”ƒ…–‹‘„‡–™‡‡•‹Ž‹…ƒ’ƒ”–‹…Ž‡•ƒ†ƒŽ˜‡‘Žƒ”ƒ…”‘’Šƒ‰‡•…ƒ Ž‡ƒ†–‘‹–‡”ƒŽ‹œƒ–‹‘‘ˆ•‹Ž‹…ƒ’ƒ”–‹…Ž‡•„›–Š‡ƒŽ˜‡‘Žƒ”ƒ…”‘’Šƒ‰‡•–‘…Ž‡ƒ”–Š‡’ƒ”–‹…Ž‡•‘—– ‘ˆ–Š‡Ž—‰•ǡ…‡ŽŽ†‡ƒ–Š„›ƒ’‘’–‘•‹•‘”ƒ…–‹˜ƒ–‹‘‘ˆ…‡ŽŽ•™‹–Š–Š‡”‡Ž‡ƒ•‡‘ˆ’”‘‹ϐŽƒƒ–‘”› …›–‘‹‡••—…Šƒ• ǦͳȏͳȐǡƒ†–Š‡”‡…”—‹–‡–‘ˆ‹ϐŽƒƒ–‘”›…‡ŽŽ•‹–‘–Š‡Ž—‰–‹••—‡ȏͳ͹ǡ ͳͺȐǤ ǦͳDǡ ͳǦEǡƒ† Ǧͳƒ”‡‡„‡”•‘ˆ–Š‡ Ǧͳˆƒ‹Ž›Ǥ ǦͳȽƒ† ǦͳȾƒ”‡ƒ‰‘‹•–• ƒ† Ǧͳ‹•ƒƒ–—”ƒŽŽ›‘……—””‹‰”‡…‡’–‘”ƒ–ƒ‰‘‹•––Šƒ–‹•’”‘†—…‡†Ž‘…ƒŽŽ›‹˜ƒ”‹‘—• –‹••—‡• ‹ ”‡•’‘•‡ –‘ ‹ˆ‡…–‹‘ ‘” ‹ϐŽƒƒ–‹‘ǡ ƒ• ƒ ‹–”ƒ…‡ŽŽ—Žƒ” ’”‘–‡‹ ‘” ƒ •‡…”‡–‡† ˆ‘”ǡ„‘–Š‘ˆ™Š‹…Šƒ”‡…ƒ’ƒ„Ž‡‘ˆ…‘’‡–‹–‹˜‡Ž›‹Š‹„‹–‹‰–Š‡Ž‘…ƒŽ‹ϐŽƒƒ–‘”›‡ˆˆ‡…–• ‘ˆ ǦͳȏͳͺǡͳͻȐǤ –Š‹•…‘–‡š–ǡ–Š‡‹…”‡ƒ•‡‹–Š‡Ž‡˜‡Ž‘ˆ Ǧͳ‹Ž—‰–‹••—‡™ƒ• …‘””‡Žƒ–‡†™‹–Š–Š‡†‡˜‡Ž‘’‡–‘ˆ’—Ž‘ƒ”›‹ϐŽƒƒ–‹‘ƒ†ϐ‹„”‘•‹•Ǥ‘™‡˜‡”ǡ –Š‡”ƒ’› ‹‹‹œ‡† –Š‡ Ž‡˜‡Ž• ‘ˆ ǦͳE ƒ† Ǧͳͳ  ȋ ‹‰Ǥ ͷǡȌ ‹ …‘…‘”†ƒ…‡ ™‹–Š ”–‹œ‡–ƒŽǤȏͻȐƒ†‡˜‹•‡–ƒŽǤȏʹͲȐǡ™Š‘‘„•‡”˜‡†ƒ†‘™”‡‰—Žƒ–‹‘‘ˆ„‘–Šƒˆ–‡”‹ˆ—•‹‘‘ˆ

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 6. ‡’”‡•‡–ƒ–‹˜‡’Š‘–‘‹…”‘‰”ƒ’Š•‘ˆŽ—‰’ƒ”‡…Š›ƒǤ —‘Š‹•–‘…Š‡‹•–”›ˆ‘” ǦEƒ–‹„‘†› ȋȌǤ —ƒ–‹ϐ‹…ƒ–‹‘ ‘ˆ  ǦE  ‹ ȋȌ –Š‡ ƒŽ˜‡‘Žƒ” •‡’–ƒ ƒ† ȋȌ –Š‡ ‰”ƒ—Ž‘ƒǤ ƒ–ƒ ƒ”‡ ’”‡•‡–‡† ƒ• ‡ƒ•άǢnαͺƒ‹ƒŽ•’‡”‰”‘—’Ǥ‡ƒŽǦ–‹‡”‡˜‡”•‡–”ƒ•…”‹’–‹‘ƒƒŽ›•‹•‘ˆ ǦEȋȌǤƒ–ƒƒ”‡ ’”‡•‡–‡†ƒ•‡ƒ•άǢnα͸ƒ‹ƒŽ•’‡”‰”‘—’Ǥǡ…‘–”‘ŽǢ ǡ•‹Ž‹…‘•‹•ǤƒŽ‹‡ȋƒŽȌƒ†„‘‡ƒ””‘™Ȃ †‡”‹˜‡†…‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȋ Ȍƒ†‹‹•–‡”‡†‘†ƒ›•ͳͷƒ†͵ͲǤȗ‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡– ˆ”‘ǦƒŽǤȚ‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡–ˆ”‘ ǦƒŽǤ

•‹‡š’‡”‹‡–ƒŽŽ—‰ϐ‹„”‘•‹•ǤŠ‡Š‹‰ŠŽ‡˜‡Ž•‘ˆ Ǧͳ‹†—…‡†ƒˆ–‡”–Š‡”ƒ’› ȋ ‹‰ǤͷȌƒ›Šƒ˜‡ƒ…”—…‹ƒŽ”‘Ž‡‹’”‘‘–‹‰ƒ–‹‹ϐŽƒƒ–‘”›ƒ†ƒ–‹ϐ‹„”‘–‹…”‡•’‘•‡• •‹‹Žƒ”–‘–Š‘•‡•Š‘™„›”–‹œ‡–ƒŽǤȏͻȐƒˆ–‡”‹ˆ—•‹‘‘ˆ‡•‡…Š›ƒŽ…‡ŽŽ•‹ƒ„Ž‡‘›…‹ ‘†‡ŽǤ”‡˜‹‘—••–—†‹‡•Šƒ˜‡•Š‘™–Šƒ– ǦͳͲƒ›„‡‹˜‘Ž˜‡†‹–Š‡’”‘…‡••‘ˆϐ‹„”‘•‹• ‹’—Ž‘ƒ”›•‹Ž‹…‘•‹•ȏʹͳǡʹʹȐǤ —”–Š‡”‘”‡ǡƒ‹…”‡ƒ•‡‹Ǧͻƒ…–‹˜‹–›‹•”‡’‘”–‡†‹ •‹Ž‹…ƒǦ‹†—…‡†Ž—‰‹Œ—”›ȏʹ͵ǡʹͶȐǤ™‘‹ˆ—•‹‘•‘ˆ™‡”‡‘–ƒ„Ž‡–‘ƒ…–‘–Š‡Ž‡˜‡Ž• ‘ˆ ǦͳDǡ ǦͳͲǡƒ†Ǧͻȋ ‹‰ǤͷȂ ȌǤ—”ϐ‹†‹‰••—’’‘”––Š‡Š›’‘–Š‡•‹•–Šƒ––Š‡ ‹…”‡ƒ•‡‹ ǦͳŠƒ†ƒ†‡…‹•‹˜‡”‘Ž‡‹–Š‡„‡‡ϐ‹…‹ƒŽ‡ˆˆ‡…–‘ˆ–”‡ƒ–‡–™‹–Š‹ƒ —”‹‡‘†‡Ž‘ˆ•‹Ž‹…‘•‹•Ǥ ‘…‘‹–ƒ–™‹–Š–Š‡‹ϐŽƒƒ–‘”›’”‘…‡••ǡ–Š‡’”‡•‡…‡‘ˆ•‹Ž‹…ƒ‹Ž—‰–‹••—‡ƒŽ•‘ –”‹‰‰‡”‡†ƒ’‘’–‘•‹•ȏͳ͸ǡʹͷȐǡ™‹–Š‹…”‡ƒ•‡†‡š’”‡••‹‘‘ˆ…ƒ•’ƒ•‡Ǧ͵ȏͳ͹ǡʹ͸ȐǤŠ‡‹…”‡ƒ•‡ ‹ƒ’‘’–‘–‹……‡ŽŽ•ƒ›„‡ƒ••‘…‹ƒ–‡†™‹–Š”‡Ž‡ƒ•‡‘ˆ‡†‹ƒ–‘”•–‘”‡…”—‹–…‡ŽŽ•ƒ†ƒ‹–ƒ‹ –Š‡‹ϐŽƒƒ–‘”›’”‘…‡••Ǥ –Š‹•…‘–‡š–ǡ”‡’‡ƒ–‡†‹ˆ—•‹‘‘ˆ•”‡†—…‡†–Š‡—„‡” ‘ˆƒ’‘’–‘–‹……‡ŽŽ•ƒ†–Š‡Ž‡˜‡Ž‘ˆ…ƒ•’ƒ•‡Ǧ͵ȋ ‹‰ǤͻȌƒ†–Š‡Ž‡˜‡Ž‘ˆ‹ϐŽƒƒ–‹‘ǡ†‡…”‡ƒ•‹‰ –Š‡—„‡”‘ˆƒ…”‘’Šƒ‰‡•ȋ ‹‰ǤͶȌǡ™Š‹…Š’Žƒ›•ƒ…‡–”ƒŽ”‘Ž‡‹–Š‡’ƒ–Š‘’Š›•‹‘Ž‘‰›‘ˆ •‹Ž‹…‘•‹•Ǥ†‡…”‡ƒ•‡‹–Š‡—„‡”‘ˆƒ’‘’–‘–‹……‡ŽŽ•‹–Š‡ Ǧ‡ŽŽ‰”‘—’‹••‡‡‹ƒ•‡”‹‡• ‘ˆ†‹•‡ƒ•‡‘†‡Ž•ƒˆ–‡”‹ˆ—•‹‘‘ˆ•ȋ‘‘—…Ž‡ƒ”ǡŠ‡ƒ–‘’‘‹‡–‹…ǡ‘”‡•‡…Š›ƒŽ …‡ŽŽ•Ȍȏʹ͹Ȃ͵ʹȐǤ Š‡Ž‘™‡”—„‡”‘ˆƒ…”‘’Šƒ‰‡•‹ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†•‹Ž‹…‘–‹…‘†—Ž‡•ƒˆ–‡” –Š‡”ƒ’› ƒ› „‡ ƒ••‘…‹ƒ–‡† ™‹–Š –Š‡ †‡…”‡ƒ•‡ ‹ –Š‡ Ž‡˜‡Ž ‘ˆ ǦͳE  ȋ ‹‰Ǥ ͷȌǤ Š‹• …›–‘‹‡ ’Žƒ›• ƒ ‡› ”‘Ž‡ ‹ –Š‡ †‡˜‡Ž‘’‡– ‘ˆ •‹Ž‹…‘•‹• „› ”‡‰—Žƒ–‹‰ –Š‡ ‡†‹ƒ–‘”• –Šƒ– ƒ”‡”‡•’‘•‹„Ž‡ˆ‘”–Š‡’‡”•‹•–‡–‹ϐŽƒƒ–‹‘ƒ††‡˜‡Ž‘’‡–‘ˆϐ‹„”‘•‹•Ǥ —”–Š‡”‘”‡ǡ ǦͳEŠƒ•„‡‡‹’Ž‹…ƒ–‡†‹ϐ‹„”‘„Žƒ•–ƒ…–‹˜ƒ–‹‘ƒ†…‘ŽŽƒ‰‡†‡’‘•‹–‹‘ȏ͵͵ȐǤ•ƒŽ”‡ƒ†› †‡‘•–”ƒ–‡† ‹ ‘–Š‡” ‘†‡Ž• ‘ˆ Ž—‰ ‹ϐŽƒƒ–‹‘ ƒ† ϐ‹„”‘•‹• ȏͳͲȐǡ • ™‡”‡ ƒ„Ž‡ –‘’”‡˜‡–ƒ‹…”‡ƒ•‡‹–Š‡ƒ‘—–‘ˆ ǦE‹Ž—‰–‹••—‡ƒ•™‡ŽŽƒ• ǦEƒˆ–‡” ‹•–‹ŽŽƒ–‹‘‘ˆ•‹Ž‹…ƒȏ͵ȐǤ ǦE”‡‰—Žƒ–‡•ƒ™‹†‡˜ƒ”‹‡–›‘ˆ…‡ŽŽ—Žƒ”’”‘…‡••‡•‹…Ž—†‹‰…‡ŽŽ ‰”‘™–Šǡƒ’‘’–‘•‹•ǡ†‹ˆˆ‡”‡–‹ƒ–‹‘ǡ‹‰”ƒ–‹‘ǡƒ†‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹š’”‘†—…–‹‘ȏ͵ͶȐǤ ‘—” ™‘”ǡ–Š‡ƒ‘—–‘ˆ ǦE‹ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†‰”ƒ—Ž‘ƒƒ† ǦE™ƒ•”‡†—…‡†

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 7. ‡’”‡•‡–ƒ–‹˜‡’Š‘–‘‹…”‘‰”ƒ’Š•‘ˆŽ—‰’ƒ”‡…Š›ƒǤ —‘Š‹•–‘…Š‡‹•–”›ˆ‘”–›’‡ ȋȌƒ† –›’‡ ȋȌ…‘ŽŽƒ‰‡ϐ‹„‡”Ǥ—ƒ–‹ϐ‹…ƒ–‹‘‘ˆ…‘ŽŽƒ‰‡–›’‡ ‹ȋȌ–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†ȋȌ–Š‡‰”ƒ—Ž‘ƒǤ —ƒ–‹ϐ‹…ƒ–‹‘‘ˆ…‘ŽŽƒ‰‡–›’‡ ‹ȋȌ–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†ȋ Ȍ–Š‡‰”ƒ—Ž‘ƒǤǡ…‘–”‘ŽǢ ǡ•‹Ž‹…‘•‹•Ǥ ƒŽ‹‡ȋƒŽȌƒ†„‘‡ƒ””‘™Ȃ†‡”‹˜‡†…‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȋ Ȍƒ†‹‹•–‡”‡†‘†ƒ›• ͳͷƒ†͵ͲǤƒ–ƒƒ”‡’”‡•‡–‡†ƒ•‡ƒ•άǢnαͺƒ‹ƒŽ•’‡”‰”‘—’Ǥȗ‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡–ˆ”‘ǦƒŽǤ Ț‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡–ˆ”‘ ǦƒŽǤ

‹•‹Ž‹…‘–‹…‹…‡–”‡ƒ–‡†™‹–Š–™‘‹ˆ—•‹‘•‘ˆ•ȋ ‹‰Ǥ͸ȌǤŠ‡‹…”‡ƒ•‡†‡š’”‡••‹‘‘ˆ  ǦEƒ›„‡ƒ••‘…‹ƒ–‡†™‹–Šƒ’‘’–‘•‹•ƒ†…‘ŽŽƒ‰‡†‡’‘•‹–‹‘‹–Š‡ ǦƒŽ‰”‘—’ǡ™Š‹…Š …‘—Ž†‹ϐŽ—‡…‡ϐ‹„”‘„Žƒ•–…‡ŽŽ‹‰”ƒ–‹‘ǡ’”‘Ž‹ˆ‡”ƒ–‹‘ǡƒ†–Š‡”‡‘†‡Ž‹‰’”‘…‡••Ǥ –Š‹• …‘–‡š–ǡ –Š‡  Ǧ‡ŽŽ ‰”‘—’ •Š‘™‡† ƒ †‡…”‡ƒ•‡ ‹ –Š‡ †‡’‘•‹–‹‘ ‘ˆ –›’‡  ƒ†  …‘ŽŽƒ‰‡ ϐ‹„‡”•‹ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†‰”ƒ—Ž‘ƒȋ ‹‰Ǥ͹ȌǤ‡•‹†‡•–Š‡‹…”‡ƒ•‡††‡’‘•‹–‹‘‘ˆ…‘ŽŽƒ‰‡ ‹–Š‡‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹šǡ–Š‡…Šƒ‰‡‹–Š‡ƒ‘—–‘ˆ‡Žƒ•–‹…ϐ‹„‡”•‹•ƒ‹’‘”–ƒ–ˆƒ…–‘” ‹˜‘Ž˜‡†‹Ž—‰”‡‘†‡Ž‹‰‹•‹Ž‹…ƒǦ‹†—…‡†Ž—‰‹Œ—”›ȏͳͶǡ͵ͷȐǤŠ‡‹ˆ—•‹‘‘ˆ• ‹ –Š‡   ‰”‘—’ ™ƒ• ƒ„Ž‡ –‘ ”‡†—…‡ ‡Žƒ•–‹… ϐ‹„‡” †‡’‘•‹–‹‘ ‹ ƒŽ˜‡‘Žƒ” •‡’–ƒ ȋ ‹‰Ǥ ͺȌǤŠ‡ †‡…”‡ƒ•‡‹–Š‡‹ϐŽƒƒ–‘”›’”‘…‡••ƒ††‡’‘•‹–‹‘‘ˆ‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹š‡Ž‡‡–•Ž‡† –‘ ƒ ‹’”‘˜‡‡– ‹ ”‡•’‹”ƒ–‘”› ˆ—…–‹‘ „‡…ƒ—•‡ –Š‡› ƒŽŽ‘™‡† ”‡‘†‡Ž‹‰ ‘ˆ –Š‡ Ž—‰ ’ƒ”‡…Š›ƒˆƒ˜‘”‹‰‡š’ƒ•‹‘‘ˆƒŽ˜‡‘Ž‹Ǥ ‡•’‹–‡ –Š‡ ’‘•‹–‹˜‡ ‡ˆˆ‡…–• ‘„•‡”˜‡†ǡ ‘—” •–—†› Šƒ† •‘‡ Ž‹‹–ƒ–‹‘•ǣ ȋͳȌ –Š‡ •–—†› ™ƒ•†‘‡—•‹‰ƒ‘†‡Ž‘ˆ•‹Ž‹…‘•‹•Ǣ™‡†‘‘–‘™–Š‡‡ˆˆ‡…–•‘ˆ–Š‹•–Š‡”ƒ’›‘†‹ˆˆ‡”‡– †‡‰”‡‡•‘ˆŽ—‰‹Œ—”›‘”‘–Š‡”•‘†‡Ž•‘ˆϐ‹„”‘•‹•ǤȋʹȌ‡…Š‘•‡ƒ‡†’‘‹–‘ˆ͸Ͳ†ƒ›•ƒˆ–‡”

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Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 8. ‡’”‡•‡–ƒ–‹˜‡ ’Š‘–‘‹…”‘‰”ƒ’Š• ‘ˆ Ž—‰ ’ƒ”‡…Š›ƒ •–ƒ‹‡† ™‹–Š ‡‹‰‡”–ǯ• ”‡•‘”…‹Ǧˆ—…Š•‹ ‡–Š‘†™‹–Š‘š‹†ƒ–‹‘ȋȌǤ—ƒ–‹ϐ‹…ƒ–‹‘‘ˆ‡Žƒ•–‹…ϐ‹„‡”•‹–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒȋȌƒ†–Š‡‰”ƒ—Ž‘ƒȋȌǤ ǡ…‘–”‘ŽǢ ǡ•‹Ž‹…‘•‹•ǤƒŽ‹‡ȋƒŽȌƒ†„‘‡ƒ””‘™Ȃ†‡”‹˜‡†…‡ŽŽ•ȋ•ǡ‡ŽŽȌ™‡”‡‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȋ Ȍ ƒ†‹‹•–‡”‡†‘†ƒ›•ͳͷƒ†͵ͲǤƒ–ƒƒ”‡’”‡•‡–‡†ƒ•‡ƒ•άǢnαͺƒ‹ƒŽ•’‡”‰”‘—’Ǥȗ‹‰‹ϐ‹…ƒ–Ž› †‹ˆˆ‡”‡–ˆ”‘ǦƒŽǤȚ‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡–ˆ”‘ ǦƒŽǤ

ƒ†‹‹•–”ƒ–‹‘‘ˆ•‹Ž‹…ƒ’ƒ”–‹…Ž‡•–‘…‘’ƒ”‡–Š‡”‡•—Ž–•™‹–Š’”‡˜‹‘—•™‘”ȏͳͳȐƒ†„‡––‡” ‡˜ƒŽ—ƒ–‡–Š‡”ƒ’›™‹–Š‘Ž—‰”‡‘†‡Ž‹‰Ǥ‘™‡˜‡”ǡ‹–™‘—Ž†„‡‹–‡”‡•–‹‰–‘•–—†› Žƒ–‡”–‹‡’‘‹–•„‡…ƒ—•‡•‹Ž‹…‘•‹•‹•ƒ…Š”‘‹…‹””‡˜‡”•‹„Ž‡†‹•‡ƒ•‡Ǥȋ͵Ȍ‘‡”‡’‘”–•ȏ͵͸ǡ͵͹Ȑ Šƒ˜‡•—‰‰‡•–‡†ƒ•–ƒ†ƒ”††‡ϐ‹‹–‹‘‘ˆ„‘‡ƒ””‘™‡•‡…Š›ƒŽ…‡ŽŽ•ǣȋƒȌ…ƒƒ†Š‡”‡–‘ ’Žƒ•–‹…‹…—Ž–—”‡…‘†‹–‹‘•ǡȋ„Ȍ…ƒ„‡†‹ˆˆ‡”‡–‹ƒ–‡†in vitro‹ƒ†‹’‘…›–‡•ǡ‘•–‡‘…›–‡•ǡƒ† …Š‘†”‘…›–‡•Ǣƒ†ȋ…Ȍ•—”ˆƒ…‡ƒ”‡”•ͳͳ„‘”ͳͶǡͳͻ‘”͹ͻƒǡ͵ͶǡͶͷǡƒ† Ǧƒ”‡ƒ„•‡–Ǣ͹͵ǡͻͲǡƒ†ͳͲͷƒ”‡’”‡•‡–Ǥ Conclusion

Š‡‡ˆˆ‡…–•’”‘‘–‡†„›–™‘†‘•‡•‘ˆ•‹•–‹ŽŽ‡†‹–”ƒ–”ƒ…Š‡ƒŽŽ›•‡‡–‘‡Šƒ…‡ –Š‡ –‡’‘”ƒ”› ‡…Šƒ‹•• ‘ˆ –‹••—‡ ’”‘–‡…–‹‘ ƒŽ”‡ƒ†› •‡‡ ƒˆ–‡” ‘‡ †‘•‡ǡ ƒ† ’”‘˜‹†‡ ˆ—”–Š‡”‹ˆ‘”ƒ–‹‘ƒ„‘—––Š‡…Ž‹‹…ƒŽƒ’’Ž‹…ƒ–‹‘•‘ˆ…‡ŽŽ–Š‡”ƒ’›ǤŠ‡•—•–ƒ‹‡†„‡‡ϐ‹…‹ƒŽ

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Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Fig. 9.‡’”‡•‡–ƒ–‹˜‡’Š‘–‘‹…”‘‰”ƒ’Š•‘ˆŽ—‰’ƒ”‡…Š›ƒ•–ƒ‹‡†™‹–ŠȋȌǤ‘–‡–Š‡’”‡•‡…‡ ‘ˆƒ’‘’–‘–‹……‡ŽŽ•ȋarrowsȌ‹–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†‹–Š‡‰”ƒ—Ž‘ƒǤ—ƒ–‹ϐ‹…ƒ–‹‘‘ˆƒ’‘’–‘–‹……‡ŽŽ•‹ ȋȌ–Š‡ƒŽ˜‡‘Žƒ”•‡’–ƒƒ†ȋȌ–Š‡‰”ƒ—Ž‘ƒǤƒ–ƒƒ”‡’”‡•‡–‡†ƒ•‡ƒ•άǢnαͺƒ‹ƒŽ•’‡”‰”‘—’Ǥ ‡ƒŽǦ–‹‡ ”‡˜‡”•‡ –”ƒ•…”‹’–‹‘  ƒƒŽ›•‹• ‘ˆ …ƒ•’ƒ•‡Ǧ͵ ȋȌǤ ƒ–ƒ ƒ”‡ ’”‡•‡–‡† ƒ• ‡ƒ•άǢ n=6 ƒ‹ƒŽ• ’‡” ‰”‘—’Ǥ ǡ …‘–”‘ŽǢ  ǡ •‹Ž‹…‘•‹•Ǥ ƒŽ‹‡ ȋƒŽȌ ƒ† „‘‡ ƒ””‘™Ȃ†‡”‹˜‡† …‡ŽŽ• ȋ•ǡ ‡ŽŽȌ ™‡”‡‹–”ƒ–”ƒ…Š‡ƒŽŽ›ȋ Ȍƒ†‹‹•–‡”‡†‘†ƒ›•ͳͷƒ†͵ͲǤȗ‹‰‹ϐ‹…ƒ–Ž›†‹ˆˆ‡”‡–ˆ”‘ǦƒŽǤȚ‹‰‹ϐ‹…ƒ–Ž› †‹ˆˆ‡”‡–ˆ”‘ ǦƒŽǤ

‡ˆˆ‡…–• ‘ˆ …‡ŽŽ –Š‡”ƒ’› –Š”‘—‰Š ”‡’‡ƒ–‡† ƒ†‹‹•–”ƒ–‹‘ ‘ˆ • ƒ› „‡ ‹†‡’‡†‡– ‘ˆ ‡‰”ƒˆ–‡–‘ˆ„‘‡ƒ””‘™…‡ŽŽ•‹–‘–Š‡Ž—‰ǡ„—–ƒ›„‡‡†‹ƒ–‡†„›ƒ‹…”‡ƒ•‡‹–Š‡ Ž‡˜‡Ž ‘ˆ ͳǦ ’”‘‘–‹‰ †‘™”‡‰—Žƒ–‹‘ ‘ˆ ‹ϐŽƒƒ–‘”› ƒ† ϐ‹„”‘‰‡‹… ”‡•’‘•‡• –‘ •‹Ž‹…ƒǦ‹†—…‡†Ž—‰‹Œ—”›Ǥ Acknowledgments

Š‡ƒ—–Š‘”•™‘—Ž†Ž‹‡–‘‡š’”‡••–Š‡‹”‰”ƒ–‹–—†‡–‘”•‘”ƒǯ”‹‡ˆ‘”Š‡”ƒ••‹•–ƒ…‡ ‹‡†‹–‹‰–Š‡ƒ—•…”‹’–ǡ”†”‡‡‡†‹–‘†ƒ‹Ž˜ƒˆ‘”ƒ‹ƒŽ…ƒ”‡ǡ”•ƒ—…‹ƒ‡˜‡• †ƒ‹Ž˜ƒˆ‘”Š‡”Š‡Ž’™‹–Š‹…”‘•…‘’›ǤŠ‹••–—†›™ƒ••—’’‘”–‡†„›–Š‡‡–‡”•‘ˆš…‡ŽŽ‡…‡ ”‘‰”ƒȋǦ  Ȍǡ”ƒœ‹Ž‹ƒ‘—…‹Žˆ‘”…‹‡–‹ϐ‹…ƒ†‡…Š‘Ž‘‰‹…ƒŽ‡˜‡Ž‘’‡– ȋ“Ȍǡ‹‘†‡ ƒ‡‹”‘–ƒ–‡‡•‡ƒ”…Š—’’‘”–‹‰ ‘—†ƒ–‹‘ȋ  Ȍǡƒ–‹‘ƒŽ •–‹–—–‡ ‘ˆ…‹‡…‡ƒ†‡…Š‘Ž‘‰›‘ˆ”—‰•ƒ†‡†‹…‹‡ȋ Ǧ  Ȍǡƒ†‘‘”†‹ƒ–‹‘ˆ‘”–Š‡ ’”‘˜‡‡–‘ˆ‹‰Š‡”‡˜‡Ž‡”•‘‡ŽȋȌǤ References 1

2 3

”‹˜ƒ•–ƒ˜ƒǡ‘ǡ ƒ‰‹”†ƒ” ǡ‹‡ǡ ‘”†‘ǡ…Š‘—Ǧ‘‰ǣ”—…‹ƒŽ”‘Ž‡‘ˆ‹–‡”Ž‡—‹Ǧͳ„‡–ƒ ƒ†‹–”‹…‘š‹†‡•›–Šƒ•‡‹•‹Ž‹…ƒǦ‹†—…‡†‹ϐŽƒƒ–‹‘ƒ†ƒ’‘’–‘•‹•‹‹…‡Ǥ ‡•’‹””‹–ƒ”‡‡† ʹͲͲʹǢͳ͸ͷǣͷʹ͹Ȃͷ͵͵Ǥ —ƒ—š ǣ‡™†‡˜‡Ž‘’‡–•‹–Š‡—†‡”•–ƒ†‹‰‘ˆ‹—‘Ž‘‰›‹•‹Ž‹…‘•‹•Ǥ—””’‹ŽŽ‡”›Ž‹ —‘ŽʹͲͲ͹Ǣ͹ǣͳ͸ͺȂͳ͹͵Ǥ ƒ”‘Ǧ —–‹‡””‡œǡƒ•–‹‰Ž‹‘‡ǡ‹•–‘ ǡŽ‹˜‡‹”ƒ ǡ”—œ ǡ‡­ƒŠƒǡƒ””‡‹”ƒǦ —‹‘”ǡ”‡ŽŽƒ• ǡ‘”ƒ‡•ǡƒ‹›ƒǡ‘……‘ǡ‘”ƒŽ‡•ǣ‘‡ƒ””‘™Ǧ†‡”‹˜‡†‘‘—…Ž‡ƒ”…‡ŽŽ–Š‡”ƒ’› ƒ––‡—ƒ–‡••‹Ž‹…ƒǦ‹†—…‡†Ž—‰ϐ‹„”‘•‹•Ǥ—”‡•’‹” ʹͲͳͳǢ͵͹ǣͳʹͳ͹ȂͳʹʹͷǤ

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Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

‡—‰ǡ— ǡŠ‡ǣ‹Ž‹…‘•‹•Ǥƒ…‡–ʹͲͳʹǢ͵͹ͻǣʹͲͲͺȂʹͲͳͺǤ „”‡—ǡ–—‡•ǡ‡Ž‘•‹ǡ‘”ƒŽ‡•ǡ‘……‘ǣ‡…Šƒ‹••‘ˆ…‡ŽŽ—Žƒ”–Š‡”ƒ’›‹”‡•’‹”ƒ–‘”› diseases. –‡•‹˜‡ƒ”‡‡†ʹͲͳͳǢ͵͹ǣͳͶʹͳȂͳͶ͵ͳǤ ‘Œƒ•ǡ— ǡ‘‘†•ǡ‘”ƒǡ’‡ƒ”•ǡ‘ƒ ǡ”‹‰Šƒǣ‘‡ƒ””‘™Ǧ†‡”‹˜‡†‡•‡…Š›ƒŽ •–‡…‡ŽŽ•‹”‡’ƒ‹”‘ˆ–Š‡‹Œ—”‡†Ž—‰Ǥ ‡•’‹”‡ŽŽ‘Ž‹‘ŽʹͲͲͷǢ͵͵ǣͳͶͷȂͳͷʹǤ ’‡‡• ǡ‘…‹ƒ•ǡ—ŽŽ‹˜ƒǡŠ‹–‡›ǡƒ•› ǡ”‘…‘’ ǡ”‘†›ǣ‰”ƒˆ–‡–‘ˆ„‘‡ ƒ””‘™’”‘‰‡‹–‘”…‡ŽŽ•‹ƒ”ƒ–‘†‡Ž‘ˆƒ•„‡•–‘•Ǧ‹†—…‡†’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ ‡•’‹””‹–ƒ”‡‡† ʹͲͲ͹Ǣͳ͹͸ǣ͵ͺͷȂ͵ͻͶǤ ‘––‘ǡƒǡƒ”†‘•‘ǡƒ†‡”•‘ǡ—‡”ǡ‹ŽŽ‹ƒ•ǡ ‹‡ǣ ‘‡ƒ””‘™Ǧ†‡”‹˜‡† …‡ŽŽ•ƒ•’”‘‰‡‹–‘”•‘ˆŽ—‰ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹—Ǥ‡˜‡Ž‘’‡–ʹͲͲͳǢʹͺǣͷͳͺͳȂͷͳͺͺǤ ”–‹œǡ—–”‡‹Žǡ ƒ––ƒǡƒ†‡›ǡ‘””‡• ǡ ‘ǡŠ‹‡› ǣ –‡”Ž‡—‹ͳ”‡…‡’–‘”ƒ–ƒ‰‘‹•– ‡†‹ƒ–‡•–Š‡ƒ–‹Ǧ‹ϐŽƒƒ–‘”›ƒ†ƒ–‹ϐ‹„”‘–‹…‡ˆˆ‡…–‘ˆ‡•‡…Š›ƒŽ•–‡…‡ŽŽ•†—”‹‰Ž—‰‹Œ—”›Ǥ”‘… ƒ–Ž…ƒ†…‹ʹͲͲ͹ǢͳͲͶǣͳͳͲͲʹȂͳͳͲͲ͹Ǥ ”—œ ǡ–—‡•ǡ„”‡—ǡ —Œ‹•ƒ‹ǡ‹Ž˜ƒ ǡ‹•–‘ ǡƒ”‘Ǧ —–‹‡””‡œǡ”‡ŽŽƒ•ǡžǡ‘…Šƒ ǡƒ’‡Ž‘œœ‹ǡ‘”ƒŽ‡•ǡ‘……‘ǣ”‘–‡…–‹˜‡‡ˆˆ‡…–•‘ˆ„‘‡ƒ””‘™‘‘—…Ž‡ƒ”…‡ŽŽ–Š‡”ƒ’›‘ Ž—‰ƒ†Š‡ƒ”–‹ƒ‡Žƒ•–ƒ•‡Ǧ‹†—…‡†‡’Š›•‡ƒ‘†‡ŽǤ‡•’‹”Š›•‹‘Ž‡—”‘„‹‘ŽʹͲͳʹǢͳͺʹǣʹ͸Ȃ͵͸Ǥ ƒ••ƒ…‡ǡ”‘–ƒ ǡƒ”‘Ǧ —–‹‡””‡œǡ ƒ”…‹ƒǡ„”‡—ǡž••ƒ”‘ǡ‹•–‘ ǡƒ•–‹‰Ž‹‘‡ǡ ƒ””‡‹”ƒ ”ǡ”‡ŽŽƒ•ǡƒ–ƒƒǡ‘—œƒǡ —Žϐ‹ŽŽ‡ǡ ‘•‡…ƒǡ‘……‘ǡ‘”ƒŽ‡•ǣ –”ƒ–”ƒ…Š‡ƒŽ‹•–‹ŽŽƒ–‹‘‘ˆ„‘‡ƒ””‘™Ǧ†‡”‹˜‡†…‡ŽŽ‹ƒ‡š’‡”‹‡–ƒŽ‘†‡Ž‘ˆ•‹Ž‹…‘•‹•Ǥ‡•’‹”Š›•‹‘Ž ‡—”‘„‹‘ŽʹͲͲͻǢͳ͸ͻǣʹʹ͹Ȃʹ͵͵Ǥ ƒ–‡• ǡ‡…”ƒ‡”ǡŠƒ”–”ƒ†ǡ‹ǡ‘††‡‡”ǡ‹Ž‹…Ǧ‹Ž‹ ǣ‘Ž—‡Ǧ–‹‡’”‘ϐ‹Ž‡†—”‹‰”‡Žƒš‡† ‡š’‹”ƒ–‹‘‹–Š‡‘”ƒŽ†‘‰Ǥ ’’ŽŠ›•‹‘ŽͳͻͺͷǢͷͻǣ͹͵ʹȂ͹͵͹Ǥ ‡‹„‡Žǣ‘”’Š‘‡–”›ǣ•–‡”‡‘Ž‘‰‹…ƒŽ–Š‡‘”›ƒ†’”ƒ…–‹…ƒŽ‡–Š‘†•Ǣ‹ ‹Ž ȋ‡†Ȍǣ‘†‡Ž•‘ˆ—‰‹•‡ƒ•‡ ‹…”‘•…‘’›ƒ†–”—…–—”ƒŽ‡–Š‘†•Ǥ‡™‘”ǡƒ”…‡Ž‡‡”ǡͳͻͻͲǡ’’ͳͻͻȂʹͶ͹Ǥ ƒˆˆ‡ǡ‹Ž˜ƒ ǡ—”–œǡ‡‰”‹ǡƒ’‡Ž‘œœ‹ǡ‘……‘ǡ‹ǣ—‰–‹••—‡‡…Šƒ‹…•ƒ† ‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹š…‘’‘•‹–‹‘‹ƒ—”‹‡‘†‡Ž‘ˆ•‹Ž‹…‘•‹•Ǥ ’’ŽŠ›•‹‘ŽʹͲͲͳǢͻͲǣͳͶͲͲȂͳͶͲ͸Ǥ ‘”‰‡•ǡ ƒŽ… ‘ǡ‡‹–‡Ǧ ‘” ǡŽ˜‹ǡ‡‹š‡‹”ƒ ǡ—••‘ǡׄ”‡‰ƒ ǡ‘’‡• ǡ‘……‘ǡ ƒ˜‹†•‘ ǡ‹†‡ǡƒ‰‹–ƒǡ‹ǡ‘•‡‹• ǣ ƒ•Ž‹‰ƒ†–”‹‰‰‡”•’—Ž‘ƒ”›•‹Ž‹…‘•‹•Ǥ š’‡† ʹͲͲͳǢͳͻͶǣͳͷͷȂͳ͸ͶǤ ‘”‰‡•ǡ‘’‡• ǡ ƒŽ… ‘ǡ‡‹–‡Ǧ ‘”ǡ‘……‘ǡƒ˜‹†•‘ ǡ‹†‡ǡ‹ǡ‘•‡‹•

ǣ’‘’–‘•‹•—†‡”Ž‹‡•‹—‘’ƒ–Š‘‰‡‹…‡…Šƒ‹••‹ƒ…—–‡•‹Ž‹…‘•‹•Ǥ ‡•’‹”‡ŽŽ‘Ž‹‘Ž ʹͲͲʹǢʹ͹ǣ͹ͺȂͺͶǤ ‹ƒŽǡ ”‡‡„‡”‰ǡ‘ǣƒ•‹…’ƒ–Š‘‰‡‡–‹…‡…Šƒ‹••‘•‹Ž‹…‘•‹•ǣ…—””‡–—†‡”•–ƒ†‹‰Ǥ—”” ’‹—Ž‡†ʹͲͲͷǢͳͳǣͳ͸ͻȂͳ͹͵Ǥ

”‡‡„‡”‰ ǡƒ•ƒ ǡ—”–‹• ǣ‹Ž‹…‘•‹•ǣƒ”‡˜‹‡™Ǥ‹•‘ʹͲͲ͹Ǣͷ͵ǣ͵ͻͶȂͶͳ͸Ǥ ”‡†ǡ ƒ„ƒ›ǣŠ›•‹‘Ž‘‰‹…”‘Ž‡‘ˆ‹–‡”Ž‡—‹Ǧͳ”‡…‡’–‘”ƒ–ƒ‰‘‹•–Ǥ”–Š”‹–‹•‡•ʹͲͲͲǢʹǣʹͶͷȂʹͶͺǤ ‡˜‹• ǡ‘‹ǡ—–‘” ǡƒ…‡ǡ–‡‡Ž‡ǡ‘••ƒǡ‡‹•• ǣ‡…”‡ƒ•‡†ƒ•„‡•–‘•Ǧ‹†—…‡†Ž—‰ ‹ϐŽƒƒ–‹‘ƒ†ϐ‹„”‘•‹•ƒˆ–‡””ƒ†‹ƒ–‹‘ƒ†„‘‡ƒ””‘™–”ƒ•’Žƒ–Ǥ ‡•’‹”‡ŽŽ‘Ž‹‘Ž ʹͲͲͺǢ͵ͺǣͳ͸ȂʹͷǤ —ƒ—š ǡ‘—ƒŠ‡† ǡ—†•’‹–Šǡ‡”‡†‹–Šǡ‡Ž‘•ǡ‡ƒ—Ž† ǡ‹•‘ǣ‘Ž‡‘ˆ‹–‡”Ž‡—‹ǦͳͲ‹–Š‡Ž—‰ ”‡•’‘•‡–‘•‹Ž‹…ƒ‹‹…‡Ǥ ‡•’‹”‘Ž‹‘ŽͳͻͻͺǢͳͺǣͷͳȂͷͻǤ ƒ”„ƒ”‹ǡ””ƒ•ǡ‹••‘ǡ‡Ž‘•ǡ… ƒ””›ǡŠƒǡ‹•‘ǡ—ƒ—š ǣŠƒ”ƒ…–‡”‹œƒ–‹‘‘ˆ–Š‡ ‡ˆˆ‡…–‘ˆ‹–‡”Ž‡—‹ǦͳͲ‘•‹Ž‹…ƒǦ‹†—…‡†Ž—‰ϐ‹„”‘•‹•‹‹…‡Ǥ ‡•’‹”‡ŽŽ‘Ž‹‘ŽʹͲͲͶǢ͵ͳǣ͹ͺȂͺͷǤ •Š‹Šƒ”ƒǡ‹•Š‹ƒ™ƒǡ ‹Œ‹ƒǡƒ–•—ƒ‰ƒǣš’”‡••‹‘‘ˆƒ–”‹š‡–ƒŽŽ‘’”‘–‡‹ƒ•‡ǡ–‹••—‡‹Š‹„‹–‘” ‘ˆ‡–ƒŽŽ‘’”‘–‡‹ƒ•‡ƒ†ƒ†Š‡•‹‘‘Ž‡…—Ž‡•‹•‹Ž‹…‘–‹…‹…‡™‹–Š–—‘”‡–ƒ•–ƒ•‹•Ǥ‘š‹…‘Ž‡–– ʹͲͲ͵ǢͳͶʹǣ͹ͳȂ͹ͷǤ …ƒ„‹ŽŽ‘‹ ǡƒ‰ǡ–‘‹‹ ǡ‘„‡”–• ǡƒ•–”ƒ‘˜ƒǡ‡”…‡”ǣƒ–”‹š‡–ƒŽŽ‘’”‘–‡‹ƒ•‡‹†—…–‹‘ ‹ϐ‹„”‘•‹•ƒ†ϐ‹„”‘–‹…‘†—Ž‡•ˆ‘”ƒ–‹‘†—‡–‘•‹Ž‹…ƒ‹ŠƒŽƒ–‹‘Ǥ Š›•‹‘Ž—‰‡ŽŽ‘ŽŠ›•‹‘Ž ʹͲͲͷǢʹͺͺǣ͹ͲͻȂ͹ͳ͹Ǥ ƒŽƒƒ…Š ǡ‹‘–ǡ ‘†ƒ–ǡ‡…ƒ‹ŽŽ‡ ǡ‡”˜±Ǧ ”±’‹‡–ǣ›•–‡‹‡…ƒ–Š‡’•‹•ƒ†…ƒ•’ƒ•‡•‹•‹Ž‹…‘•‹•Ǥ ‹‘ŽŠ‡ʹͲͲ͸Ǣ͵ͺ͹ǣͺ͸͹Ȃͺ͹ͲǤ Š‡ǡŠƒ‰ǡŠƒ‰ ǡ‰ǣ‡ƒ…–‹˜‡‘š›‰‡•’‡…‹‡•ƒ†…ƒ•’ƒ•‡ƒ…–‹˜ƒ–‹‘‡†‹ƒ–‡•‹Ž‹…ƒǦ ‹†—…‡†ƒ’‘’–‘•‹•‹ƒŽ˜‡‘Žƒ”ƒ…”‘’Šƒ‰‡•Ǥ Š›•‹‘Ž—‰‡ŽŽ‘ŽŠ›•‹‘ŽʹͲͲͳǢʹͺͲǣͳͲȂͳ͹Ǥ

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Cell Physiol Biochem 2013;32:1681-1694 DOI: 10.1159/000356603 Published online: December 13, 2013

© 2013 S. Karger AG, Basel www.karger.com/cpb

Lopes-Pacheco et al.: Repeated Administration of Stem Cell Prevents Silicosis

Š‡‰ǡŠ‡ǡ‹

ǡ‹— ǡŠ‡ǣˆˆ‡…–•‘ˆƒ—–‘Ž‘‰‘—•„‘‡ƒ””‘™‘‘—…Ž‡ƒ”…‡ŽŽ• ‹’Žƒ–ƒ–‹‘‘…Š‘Žƒ‰‹‘…›–‡•ƒ’‘’–‘•‹•‹‘†‡Ž‘ˆ‹–”ƒŠ‡’ƒ–‹…‹•…Š‡‹…–›’‡„‹Ž‹ƒ”›Ž‡•‹‘‹”ƒ„„‹–•Ǥ ”ƒ•’Žƒ–”‘…ʹͲͳʹǢͶͶǣͳͶ͵ͷȂͳͶ͵ͺǤ ‘ ǡŠ‘‰ǡŠ‘— ǡ‡‰ǡŠƒ‰ǣ‘‡ƒ””‘™Ǧ†‡”‹˜‡†‡•‡…Š›ƒŽ•–‡…‡ŽŽ•’”‡˜‡––Š‡ ƒ’‘’–‘•‹•‘ˆ‡—”‘ǦŽ‹‡ͳʹ…‡ŽŽ•˜‹ƒ‡”›–Š”‘’‘‹‡–‹‡š’”‡••‹‘Ǥ‡—”‘•…‹‡––ʹͲͳʹǢͷʹʹǣͻʹȂͻ͹Ǥ ‘•‡„‡”‰ǡŠƒ‰ǡŠƒ‰ ǣ †‡ϐ‹…‹‡…›‹’ƒ‹”•‡ƒ”Ž›Š‡ƒ–‘’‘‹‡•‹•‹–Š‡„‘‡ƒ””‘™Ǥ  —‘ŽʹͲͳʹǢͳͺ͸ǣʹͲ͵Ȃʹͳ͵Ǥ ƒƒœƒ™ƒǡ —Œ‹‘–‘ǡ‡”ƒ–ƒ‹ǡ ™ƒ•ƒ‹ ǡƒ•ƒŠƒ”ƒǡ‡‰‹•Š‹ǡ•—”—›ƒƒǡ‡‘–‘ǡ‘„ƒ›ƒ•Š‹ ǣ‘‡ƒ””‘™Ǧ†‡”‹˜‡†‡•‡…Š›ƒŽ•–‡…‡ŽŽ•ƒ‡Ž‹‘”ƒ–‡Š‡’ƒ–‹…‹•…Š‡‹ƒ”‡’‡”ˆ—•‹‘‹Œ—”›‹ƒ”ƒ– ‘†‡ŽǤ‘‡ʹͲͳͳǢ͸ǣ‡ͳͻͳͻͷǤ ‘˜‡ŽŽ ǡƒ•‹ǡ‡‡ǡŠ‡—‰ǡŠ‹–ƒ‹ǡ‡—‰ǡƒƒŠƒ•Š‹ǡƒ’‘‘”ǡƒ‰‘‘„ǡ—œ—‹ ǡ›–Š‰‘‡ ǡƒ”–‹ ǡ—”‘‡ǡŠ‹‡‡”ƒǡƒ–Š—”ǣ‘‡ƒ””‘™‘‘—…Ž‡ƒ”…‡ŽŽ•”‡†—…‡ ›‘…ƒ”†‹ƒŽ”‡’‡”ˆ—•‹‘‹Œ—”›„›ƒ…–‹˜ƒ–‹‰–Š‡ ͵Ȁ–•—”˜‹˜ƒŽ’ƒ–Š™ƒ›Ǥ–Š‡”‘•…Ž‡”‘•‹•ʹͲͳͲǢʹͳ͵ǣ͸͹Ȃ ͹͸Ǥ ƒ””‡‹”ƒǡƒ‹›ƒǡƒ•–‹‰Ž‹‘‡ǡƒ”‘Ǧ —–‹‡””‡œǡƒ”„‘•ƒǡ”‡ŽŽƒ•ǡ‡”†‘‘”ǡ ƒ•…ƒ”‡ŽŽ‹ǡ‘”‘Œ‡˜‹…ǡ‹‹…‡”Ǧƒƒ•ǡ‡‹–‡ ”ǡ‘”ƒŽ‡•ǡ‹‡›”ƒǣ‘‡ƒ””‘™‘‘—…Ž‡ƒ” …‡ŽŽ•ƒ––‡—ƒ–‡‹–‡”•–‹–‹ƒŽϐ‹„”‘•‹•ƒ†•–‹—Žƒ–‡–Š‡”‡’ƒ‹”‘ˆ–—„—Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ƒˆ–‡”—‹Žƒ–‡”ƒŽ —”‡–‡”ƒŽ‘„•–”—…–‹‘Ǥ‡ŽŽŠ›•‹‘Ž‹‘…Š‡ ʹͲͲͻǢʹͶǣͷͺͷȂͷͻͶǤ ƒ”‹ƒ‹ ǡ‘„› ǡ‡…Šƒǡƒ”•ǡ”‘—…Šǡ‹‡”…‡ǣ‘…ƒŽ‹œƒ–‹‘‘ˆ–›’‡ ’”‘…‘ŽŽƒ‰‡‰‡‡ ‡š’”‡••‹‘‹•‹Ž‹…ƒǦ‹†—…‡†‰”ƒ—Ž‘ƒ–‘—•Ž—‰†‹•‡ƒ•‡ƒ†‹’Ž‹…ƒ–‹‘‘ˆ–”ƒ•ˆ‘”‹‰‰”‘™–Šˆƒ…–‘”Ǧ „‡–ƒƒ•ƒ‡†‹ƒ–‘”‘ˆϐ‹„”‘•‹•Ǥ ƒ–Š‘Žͳͻͻ͸ǢͳͶͺǣͳͷͳȂͳ͸ͶǤ ƒŠ‹‹ǡ‡‘ˆǣ Ǧ„‡–ƒ•‹‰ƒŽ‹‰ǣƒ–ƒŽ‡‘ˆ–™‘”‡•’‘•‡•Ǥ ‡ŽŽ‹‘…Š‡ ʹͲͲ͹ǢͳͲʹǣͷͻͳȂ͸ͲͺǤ ƒ”‹ƒ‹ ǡ”‘—…Šǡ‘„› ǡ–ƒ”…Š‡”ǡ‹‡”…‡ǣ …”‡ƒ•‡†‡Žƒ•–‹’”‘†—…–‹‘‹‡š’‡”‹‡–ƒŽ ‰”ƒ—Ž‘ƒ–‘—•Ž—‰†‹•‡ƒ•‡Ǥ ƒ–Š‘ŽͳͻͻͷǢͳͶ͹ǣͻͺͺǦͳͲͲͲǤ ‘‹‹…‹ǡ‡Žƒ…ǡ—‡ŽŽ‡” ǡŽƒ’‡”Ǧ‘”–‡„ƒ…Š ǡƒ”‹‹ ǡ”ƒ—•‡ǡ‡ƒ•ǡ‡ƒ–‹‰ǡ”‘…‘’ ǡ ‘”™‹–œǣ‹‹ƒŽ…”‹–‡”‹ƒˆ‘”†‡ϐ‹‹‰—Ž–‹’‘–‡–‡•‡…Š›ƒŽ•–”‘ƒŽ…‡ŽŽ•ǤŠ‡ –‡”ƒ–‹‘ƒŽ‘…‹‡–› ˆ‘”‡ŽŽ—Žƒ”Š‡”ƒ’›’‘•‹–‹‘•–ƒ–‡‡–Ǥ›–‘–Š‡”ƒ’›ʹͲͲ͸Ǣͺǣ͵ͳͷǦ͵ͳ͹Ǥ ƒ‹Ž˜ƒ‡‹”‡Ž‡•ǡƒ’Žƒ ǡƒ”†‹ǣ •‡ƒ”…Š‘ˆ–Š‡‹˜‹˜‘‹†‡–‹ˆ›‘ˆ‡•‡…Š›ƒŽ•–‡…‡ŽŽ•Ǥ–‡ …‡ŽŽ•ʹͲͲͺǢʹ͸ǣͳ͵Ͳ͹Ǧͳ͵ͳͶǤ

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Copyright: S. Karger AG, Basel 2013. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.