tag:blogger.com,1999:blog-66233980654701349212024-03-14T06:23:14.050+09:00東京薬科大学の論文盗用事件東京薬科大学の林正弘 研究室 (薬学部薬物動態制御学教室)の論文(筆頭著者:富田 幹雄、責任著者:瀧沢裕輔)の盗用事件まとめ11jigenhttp://www.blogger.com/profile/03513633746083109180noreply@blogger.comBlogger1125tag:blogger.com,1999:blog-6623398065470134921.post-12177997264008144502014-04-17T01:01:00.001+09:002014-04-17T11:49:37.725+09:00東京薬科大学の論文盗用事件まとめ<span style="font-size: large;">東京薬科大学の林正弘 研究室 (薬学部薬物動態制御学教室)の論文(筆頭著者:富田 幹雄、責任著者:瀧沢裕輔)の文章が盗用であるとして、盗用の被害を受けた雑誌の出版社のリクエストにより撤回されました。AbstractからIntro, Materials and methods, Results, Discussionに至るまで、ほとんどの文章が他者論文からの剽窃です。</span><br />
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<span style="color: red; font-size: large;"><b>富田、瀧沢氏らの不正論文(剽窃・盗用・Plagiarism)</b></span><br />
<b>雑誌名:</b> Int J Pharm.誌<br />
<b>出版日: </b>2012 May 30;428(1-2):33-8. doi: 10.1016/j.ijpharm.2012.02.027. Epub 2012 Feb 25.<br />
<b>論文タイトル: </b>Suppression of efflux transporters in the intestines of endotoxin-treated rats.</div>
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<b>著者: </b>Tomita M (富田幹雄), Takizawa Y (瀧沢裕輔), Kanbayashi A, Murata H, Tanaka A, Nakaike M, Hatanaka M, Kai T, Hayashi M (林正弘).</div>
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<a href="http://www.ncbi.nlm.nih.gov/pubmed/22387888">http://www.ncbi.nlm.nih.gov/pubmed/22387888</a></div>
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(Retraction Watch: <a href="http://retractionwatch.com/2014/04/16/integrity-of-data-undisputed-in-paper-pulled-for-plagiarism/">Integrity of data “undisputed” in paper pulled for plagiarism</a>)<br />
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<b><a href="http://www.sciencedirect.com/science/article/pii/S037851731400180X">撤回告知</a></b></blockquote>
<blockquote class="tr_bq">
"This article has been retracted: please see Elsevier Policy on Article Withdrawal (<a href="http://www.sciencedirect.com/science?_ob=RedirectURL&_method=externObjLink&_locator=url&_cdi=271189&_issn=03785173&_origin=article&_zone=art_page&_plusSign=%2B&_targetURL=http%253A%252F%252Fwww.elsevier.com%252Flocate%252Fwithdrawalpolicy">http://www.elsevier.com/locate/withdrawalpolicy</a>).<br />
This article has been retracted at the request of Publisher Drug Metabolism and Disposition.<br />
The authors have plagiarized text of a paper that had already appeared in Drug MetabDispos32 (2004) 20–27 <a href="http://www.sciencedirect.com/science?_ob=RedirectURL&_method=externObjLink&_locator=url&_cdi=271189&_issn=03785173&_origin=article&_zone=art_page&_plusSign=%2B&_targetURL=http%253A%252F%252Fdx.doi.org%252F10.1124%252Fdmd.32.1.20">http://dx.doi.org/10.1124/dmd.32.1.20</a> The integrity of the research findings in the retracted article is undisputed.<br />
The Publisher apologizes for any inconvenience caused.<br />
Copyright © 2014 Elsevier B.V. All rights reserved."</blockquote>
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<b>林正弘教授</b>は、既に定年退職されています。 </div>
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<a href="http://www.toyaku.ac.jp/event/detail/id/1748/publish/1/">http://www.toyaku.ac.jp/event/detail/id/1748/publish/1/</a> (<a href="http://www.webcitation.org/6OsemGOmJ" target="_blank">写し</a>)</div>
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筆頭著者の<b>富田幹雄氏</b>は現在、東北薬科大学の教授です。 </div>
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<a href="http://www.tohoku-pharm.ac.jp/laboratory/yakudo/index.html">http://www.tohoku-pharm.ac.jp/laboratory/yakudo/index.html</a> (<a href="http://www.webcitation.org/6OsefAGyS">写し</a>)<br />
<a href="http://www.tohoku-pharm.ac.jp/new/sb.cgi?eid=128">http://www.tohoku-pharm.ac.jp/new/sb.cgi?eid=128</a> (<a href="http://www.webcitation.org/6OtKWur4K">写し</a>)</div>
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責任著者の<b>瀧沢裕輔氏</b>は現在、東京薬科大学の助教のようです。 <a href="http://www.ps.toyaku.ac.jp/wp/yakubutsudotai/">http://www.ps.toyaku.ac.jp/wp/yakubutsudotai/</a> (<a href="http://www.webcitation.org/6OseqIaLe" target="_blank">写し</a>)<br />
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<b><span style="color: red; font-size: large;">富田、瀧沢氏らの論文のAbstractの文章</span></b><br />
<b>(黄色でハイライトされた部分が</b><b><a href="http://dmd.aspetjournals.org/content/32/1/20.full">J. Kalitsky-Szirtes氏らの論文</a>との同一文章)</b><br />
<b style="background-color: yellow;">Abstract</b></div>
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<span style="background-color: yellow;">Infection and inflammation</span> suppress<span style="background-color: yellow;"> the expression and activity of several drug transporters</span> <span style="background-color: yellow;">in</span> the <span style="background-color: yellow;">liver. In the intestine, P-glycoprotein (PGP/mdr1) and the multidrug resistance-associated protein 2 (MRP2) are important barriers to the absorption of many clinically important drugs</span>. <span style="background-color: yellow;">The</span> protein<span style="background-color: yellow;"> expression and activity of these transporters were examined </span>during <span style="background-color: yellow;">inflammation </span>induced by lipopolysaccharide (LPS). The<span style="background-color: yellow;"> transport</span> of rhodamine123 (Rho123) and 5-carboxyfluorescein (5-CF) was <span style="background-color: yellow;">determined</span> in <span style="background-color: yellow;">isolated</span> ileal<span style="background-color: yellow;"> segments from endotoxin-treated or control rats</span> in the presence or absence of inhibitors. The<span style="background-color: yellow;"> reverse transcription-polymerase chain reaction was used to measure mRNA levels.</span><span style="background-color: white;"> </span><span style="background-color: yellow;">Compared with</span> the<span style="background-color: yellow;"> controls,</span> the <span style="background-color: yellow;">mRNA</span> <span style="background-color: yellow;">levels of</span> <span style="background-color: yellow;">mdr1a and mrp2 were significantly decreased by approximately 50% in the</span> ilea <span style="background-color: yellow;">of the LPS-treated rats. Corresponding reductions in the basolateral-apical efflux of</span> Rho123 <span style="background-color: yellow;">and 5-CF were observed, resulting in significant increases in the apical-basolateral absorption of these compounds.</span> Neither the<span style="background-color: yellow;"> permeability</span> of fluorescein isothiocyanate labeled dextran 4000 (FD-4), a paracellular marker, nor membrane resistance was <span style="background-color: yellow;">altered. These results indicate that endotoxin-induced inflammation</span> reduces <span style="background-color: yellow;">the intestinal expression and activity of PGP</span> and <span style="background-color: yellow;">MRP2</span> <span style="background-color: yellow;">in rats, which elicit</span>ing<span style="background-color: yellow;"> corresponding changes in the intestinal transport</span> <span style="background-color: yellow;">of their substrates. Hence, infection and inflammatory diseases may</span> induce<span style="background-color: yellow;"> variability in drug bioavailability through alterations in the intestinal expression and activity of drug transporters.</span><b><span style="color: red; font-size: large;"><br /></span></b>
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<b><span style="color: red; font-size: large;">富田、瀧沢氏らの論文のIntroductionの文章</span></b><br />
<b>(黄色でハイライトされた部分が</b><b><a href="http://dmd.aspetjournals.org/content/32/1/20.full">J. Kalitsky-Szirtes氏らの論文</a>との同一文章)</b><br />
<span style="background-color: yellow;">Inflammation is a complex immunological response that is a component of many disease states,</span> and so it is important for the effects of inflammation to be examined<span style="background-color: yellow;"> in </span>the field of <span style="background-color: yellow;">clinical therapeutics. Acute inflammatory reaction</span>s are <span style="background-color: yellow;">initiated by a wide variety of pathological stimuli, including infection, tissue damage, trauma, and cellular stress and result</span> <span style="background-color: yellow;">in the release of proinflammatory cytokine and </span>the <span style="background-color: yellow;">modulation </span>of <span style="background-color: yellow;">the expression of many hapatic proteins. Numerous clinical reports </span>have described <span style="background-color: yellow;">that drug biotransformation </span>reactions are altered <span style="background-color: yellow;">during infection and inflammation due to the downregulation of </span>the expression of several drug efflux transporters, which is <span style="background-color: yellow;">caused by the inflammatory response </span>elicited <span style="background-color: yellow;">(</span>Hartmann et al., 2001, 2002;<span style="background-color: yellow;"> Slaviero et al., 2003).</span> <span style="background-color: yellow;">The concomitant roles (i.e., removing xenobiotics from cells) and close cellular localization of</span> <span style="background-color: yellow;">efflux transporters indicate that these proteins function as a protective mechanism </span>that <span style="background-color: yellow;">limit</span>s the <span style="background-color: yellow;">systemic access of xenobiotics,</span> which probably <span style="background-color: yellow;">contribut</span>es <span style="background-color: yellow;">to the high inter-individual variability that is observed for numerous drugs.</span><br />
<span style="background-color: yellow;">The ATP-dependent drug efflux </span>transporter <span style="background-color: yellow;">P-glycoprotein (PGP),</span> which is <span style="background-color: yellow;">encoded by the multidrug resistance gene (MDR1 in humans; mdr1a, mdr1b in rodents), is responsible for the active excretion of a wide variety of lipophilic cationic drugs from the liver, kidneys, and intestine.</span> <span style="background-color: yellow;">Multidrug resistance-associated proteins 2 (MRP2) is involved in the extrusion of lipophilic anions and their glutathione, glucuronic acid, and sulfate conjugates.</span><br />
<span style="background-color: yellow;">A variety of pharmaceutical and chemical agents, immune mediators, and disease states affect drug disposition </span><span style="background-color: white;">modulation</span><span style="background-color: yellow;"> by</span> <span style="background-color: yellow;">transport</span> mechanisms. The <span style="background-color: yellow;">expression</span> levels of <span style="background-color: yellow;">PGP and MRP2 are reduced in animal livers during infection</span>-induced <span style="background-color: yellow;">inflammation (Piquette-Miller et al., 1998; Tang et al., 2000; Payen et al., 2002). In vivo and in vitro studies </span>have shown <span style="background-color: yellow;">that interleukin-</span>1β <span style="background-color: yellow;">(IL-</span>1β<span style="background-color: yellow;">) and other pro-inflammatory cytokines released during the inflammatory respoinse are primarily involved in mediating this down-regulation (</span>Tomita et al., 2010a,b<span style="background-color: yellow;">). Although the molecular mechanism of this phenomenon has </span>not <span style="background-color: yellow;">yet been elucidated, it is possible that these proteins share common regulatory pathways. Indeed, the pregnane X receptor (PXR) has been shown to regulate a </span>gene <span style="background-color: yellow;">network of drug transporters including MDR1 and MRP2 (Synold et al., 2001) in the liver and intestine.</span><br />
In addtion, a study using <span style="background-color: yellow;">several protein markers </span>showed that<span style="background-color: yellow;"> the intestine exhibited a </span>similar <span style="background-color: yellow;">response</span> to <span style="background-color: yellow;">the liver</span> during the<span style="background-color: yellow;"> acute phase (Molment et al., 1993). However, the</span> effect <span style="background-color: yellow;">of inflammation on transport proteins in the intestine has received little attention, despite the fact that the intestine is the first major barrier to xenobiotic absorption and that the modulation of drug transport affects oral bioavailability. We conducted the present study </span>using <span style="background-color: yellow;">an animal model of inflammation, to ascertain the</span> effects of <span style="background-color: yellow;">PGP</span> and <span style="background-color: yellow;">MRP2</span> protein <span style="background-color: yellow;">expression and activity</span> on drug transport <span style="background-color: yellow;">in</span> the <span style="background-color: yellow;">intestin</span>e.<span style="background-color: yellow;"> Our results indicate that the intestine</span> suppresses<span style="background-color: yellow;"> xenobiotic transport during</span> infection and inflammation in a <span style="background-color: yellow;">similar</span> manner <span style="background-color: yellow;">to the liver.</span><br />
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<b><span style="color: red; font-size: large;">富田、瀧沢氏らの論文のMaterials and methodsの文章</span></b><br />
<b>(黄色でハイライトされた部分が</b><b><a href="http://dmd.aspetjournals.org/content/32/1/20.full">J. Kalitsky-Szirtes氏らの論文</a>との同一文章)</b><br />
<b><i>2.3. <span style="background-color: yellow;">Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis of mRNA.</span> </i></b></div>
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<span style="background-color: yellow;"> Total RNA was extracted from</span> the <span style="background-color: yellow;">intestine segment</span> <span style="background-color: yellow;">mucosal scrapings using the Amersham QuickPrep Total RNA extraction kit (</span>GE Healthcare Life Sciences<span style="background-color: yellow;">), and single-stranded cDNA was synthesized from 3 μg of RNA using the First Strand cDNA Synthesis Kit (</span>TAKARA BIO Inc<span style="background-color: yellow;">) according to manufacturer protocols. Serial dilutions (20- to 16,000-fold) of RT product were used to generate standard curves for PCR, and optimal amounts of template were determined from the linear portions of these curves (data not shown).</span> A <span style="background-color: yellow;">Standard curve</span> <span style="background-color: yellow;">for RT-PCR</span> was produced <span style="background-color: yellow;">for each set of RNA samples analyzed</span>. RT-PCR <span style="background-color: yellow;">standard curves were highly reproducible. Selected RT-PCR results were also confirmed on Northern blots.</span><br />
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The <span style="background-color: yellow;">amounts of RT product (cDNA template) used were</span> as follows<span style="background-color: yellow;">: 25 ng (mdr1a, mrp2),</span> 32 <span style="background-color: yellow;">ng (PXR), 50 ng (IL-</span>1β<span style="background-color: yellow;">), 10 ng (GAPDH). The cDNA templates were amplified in the presence of 1.5 mM MgCl2, 200 μ</span>g <span style="background-color: yellow;">deoxynucleoside-5′-triphosphate, and 50 pmol of</span> the<span style="background-color: yellow;"> forward and reverse primers in a total volume of 100 μ</span>mol <span style="background-color: yellow;">using a GeneAmp 2400 Thermocycler (</span>Scientific Support Inc<span style="background-color: yellow;">). The reaction was initiated by</span> the <span style="background-color: yellow;">addition of 2.5 U of Taq polymerase (MBI Fermentas), and amplification proceeded through 22 cycles for GAPDH, 25 cycles for PXR,</span> and <span style="background-color: yellow;">30 cycles for mdr1a</span> and <span style="background-color: yellow;">mrp2. The PCR products were separated by electrophoresis on 2% agarose gels, stained with SYBR Gold nucleic acid stain (</span>Invitrogen<span style="background-color: yellow;">), and visualized under ultraviolet light.</span> The<span style="background-color: yellow;"> size of DNA bands was confirmed using Gene Ruler 100-bp DNA ladder (Fermentas</span> Life Sciences<span style="background-color: yellow;">). Optical densities were normalized to GAPDH band intensities.</span> The <span style="background-color: yellow;">PCR primers</span> used<span style="background-color: yellow;">,</span> which were <span style="background-color: yellow;">obtained from the DNA Synthesis</span> Center <span style="background-color: yellow;">(</span>SIGN GENOSYS<span style="background-color: yellow;">), are reported in Table 1.</span><br />
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<b><i>2.4. <span style="background-color: yellow;">Ussing Chamber studies</span></i></b></div>
The <span style="background-color: yellow;">procedures for the intestinal Ussing chamber studies were the same as those previously reported </span>(<a href="http://dmd.aspetjournals.org/content/32/1/20.full#ref-2">T</a>omita et al., 2010a,b<span style="background-color: yellow;">). Briefly,</span> The <span style="background-color: yellow;">rat</span> gut <span style="background-color: yellow;">was opened along the mesenteric border, and 1-cm sections of jejunum</span>, ileum, and colon <span style="background-color: yellow;">were excised.</span> The <span style="background-color: yellow;">intestine was visually inspected prior to excision, and Peyer's patches were excluded from sections.</span> The <span style="background-color: yellow;">assembled diffusion chambers were placed in a 38°C heating block, connected to a 95% O2/5% CO2airlift, and filled with 1.0 ml of 38°C Ringer's buffer (141 mM Na</span>Cl<span style="background-color: yellow;">, 5 mM K</span>Cl<span style="background-color: yellow;">, 1.2 mM Ca</span>Cl2<span style="background-color: yellow;">, 1.2 mM Mg</span>Cl2<span style="background-color: yellow;">, 25 mM H</span>2<span style="background-color: yellow;">CO3, 0.4 mM </span>Na<span style="background-color: yellow;">H2PO4, 1.6 mM</span> K2<span style="background-color: yellow;">HPO4</span><span style="background-color: white;">; </span><span style="background-color: yellow;">pH 7.4</span><span style="background-color: yellow;">)</span><span style="background-color: white;"> at 38 °C</span><span style="background-color: yellow;">. Buffers of equal isotonicity containing 10 mM mannitol in the donor compartment and 8 mM glucose with 2 mM mannitol in the receiver compartment were used. The Ussing diffusion chambers and the airlift/heating block were</span> purchased from TOYOBO Inc. (Tokyo, Japan).</div>
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<b><i>2.5. <span style="background-color: yellow;">Determination of PGP-Mediated Transport</span><span style="background-color: white;"> (Iida et al., 2005)</span></i></b><br />
<span style="background-color: yellow;">The PGP transport studies were initiated by the addition of</span> rhodamine123 (Rho123) <span style="background-color: yellow;">to either the apical or basolateral chamber to give a final concentration of</span> 1 μM <span style="background-color: yellow;">in the donor compartment.</span><span style="background-color: white;"> Fluorescein isothiocyanate labeled dextran 4000 (FD-4) </span><span style="background-color: yellow;">was added to the donor side to serve as a </span><span style="background-color: white;">paracellular </span><span style="background-color: yellow;">permeability marker. For inhibition studies,</span> 10 <span style="background-color: yellow;">μM</span> verapamil were <span style="background-color: yellow;">added to the</span> apical <span style="background-color: yellow;">chamber prior to addition of</span> Rho123<span style="background-color: yellow;">. Duplicate samples of 100 μ</span>M <span style="background-color: yellow;">were taken from the receiver chamber at</span> 15, <span style="background-color: yellow;">30, 45, </span>and <span style="background-color: yellow;">60</span> <span style="background-color: yellow;">min</span> and analyzed with a fluorescence spectrophotometer at excitation and emission wavelengths of 485 and 546 nm, respectively, and were<span style="background-color: yellow;"> replaced with fresh buffer. Samples were taken from the donor chamber at the start and end of the transport study.</span><br />
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<b><i>2.6. <span style="background-color: yellow;">Determination of MRP2-Mediated Transport.</span></i></b><br />
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<span style="background-color: yellow;">A previously described</span> <span style="background-color: yellow;">5-carboxyfluorescein </span><span style="background-color: white;">(</span><span style="background-color: yellow;">5-CFDA</span><span style="background-color: white;">)</span> <span style="background-color: yellow;">efflux assay was used to study the functional activity of MRP (Lee and Piquette-Miller, 2001). In this assay, </span><span style="background-color: yellow;">the nonfluorescent 5-CFDA passively and rapidly diffuses into cells, where it is converted to the fluorescent anion 5-CF by intracellular esterases. 5-CF is effluxed from cells by the MRP family of transporters and is not a substrate of PGP or the human organic anion transporter. 5-CFDA was dissolved in ethanol and diluted in Ringer's buffer to a final concentration of 50 μM (in 1% ethanol). </span><span style="background-color: white;">The </span><span style="background-color: yellow;">directional transport of</span> <span style="background-color: yellow;">5-CF was monitored in both B </span>to <span style="background-color: yellow;">A and A </span>to <span style="background-color: yellow;">B directions, with 100-μl samples taken at </span>15, <span style="background-color: yellow;">30, 45,</span> and <span style="background-color: yellow;">60</span> <span style="background-color: yellow;">min, in the presence or absence of the MRP2 inhibitor, MK571 (100 μM). Fresh buffer was</span> added <span style="background-color: yellow;">at each sampling time point. Fluorescence of 5-CF was measured using a SpectraMAX Gemini XS plate reader (Molecular Devices) with excitation and emission wavelengths of 490 and 520 nm, respectively.</span></div>
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<span style="background-color: white; color: #403838; font-family: 'Lucida Sans Unicode', Arial, 'Lucida Grande', Tahoma, Verdana, Helvetica, sans-serif; font-size: 13px; line-height: 19.200000762939453px; text-align: justify;"><br /></span>
<b><span style="color: red; font-size: large;">富田、瀧沢氏らの論文のResultsの文章</span></b><br />
<b>(黄色でハイライトされた部分が</b><b><a href="http://dmd.aspetjournals.org/content/32/1/20.full">J. Kalitsky-Szirtes氏らの論文</a>との同一文章)<br />3. Results</b><br />
<b><i>3.1. <span style="background-color: yellow;">Induction of Acute Inflammation in Rats. </span></i></b><span style="background-color: yellow;"><br />Although rats</span> treated with <span style="background-color: yellow;">LPS displayed pronounced</span> <span style="background-color: yellow;">exudate</span> <span style="background-color: yellow;">formation</span> <span style="background-color: yellow;">around the eyes and nostrils and</span> severe<span style="background-color: yellow;"> diarrhea,</span> <span style="background-color: yellow;">no mortality</span> occurred after the treatment<span style="background-color: yellow;">. IL-</span>1β <span style="background-color: yellow;">mRNA expression, </span>which is<span style="background-color: yellow;"> used as a marker</span> <span style="background-color: yellow;">of inflammation, was induced</span> 3<span style="background-color: yellow;">- to</span> 14<span style="background-color: yellow;">-fold</span> in the whole <span style="background-color: yellow;">intestine </span>at 8h<span style="background-color: yellow;"> after LPS treatment,</span> and was most strongly induced in the colon (Fig.1a). In the jejunum and ileum, the IL-1β mRNA expression level had a tendency to increase (100±25% to 862±388% and 100±33% to 299±101%), although the differences failed reach the 5% level of statistical significane (0.05<p<0.1).<br />
<span style="background-color: yellow;"> Similar cell viability and membrane integrity were seen in</span> ileal <span style="background-color: yellow;">segments obtained from</span> the <span style="background-color: yellow;">LPS-treated and control rats. The directional transport of</span> FD-4<span style="background-color: yellow;"> remained unchanged, and LPS treatment did not </span>increase FD-4<span style="background-color: yellow;"> permeability in</span> the ileal<span style="background-color: yellow;"> segments mounted on Ussing chambers (Fig. </span>1b<span style="background-color: yellow;">). </span>The level of membrane resistance (Rm) did not significantly deccrease over the experimental duration and was similar between the LPS-treatment and control groups (Fig. 1c). <span style="background-color: yellow;">Slight changes in morphological characteristics were seen in</span> the<span style="background-color: yellow;"> hitological sections</span> from<span style="background-color: yellow;"> the LPS-treated animal, including loss of tissue color and enlarged spacing between adjacent villi</span> (data not shown)<span style="background-color: yellow;">. Nevertheless, similar cell viability and integrity were seen in </span>the LPS-<span style="background-color: yellow;">treated and control samples.</span><b><br /></b><br />
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<b><i style="background-color: white;">3.2. </i><i style="background-color: yellow;">Gene expression</i></b><br />
The <span style="background-color: yellow;">levels of mdr1a mRNA were consistently and significantly reduced in</span> both the jejunal and ileal <span style="background-color: yellow;">resions </span>to<span style="background-color: yellow;"> approximately</span> 31±7.3% and 40±3.2<span style="background-color: yellow;">%</span> of the control level, respectively, in the <span style="background-color: yellow;">LPS-treated animals (Fig. </span>2<span style="background-color: yellow;">a).</span> <span style="background-color: yellow;">The levels of mdr1</span>a <span style="background-color: yellow;">mRNA also tended to be lower in the </span>colons<span style="background-color: yellow;"> of LPS-treated animal; however, this change did not reach significance (Fig. </span>2a<span style="background-color: yellow;">).</span> The <span style="background-color: yellow;">expression of </span>mrp2 mRNA<span style="background-color: yellow;"> was also suppressed in the</span> ilea<span style="background-color: yellow;"> of</span> the <span style="background-color: yellow;">LPS-treated animals</span> to 59.8<span style="background-color: yellow;">±</span>8.7<span style="background-color: yellow;">% of</span> the<span style="background-color: yellow;"> control</span> value (Fig. 2b). Likewise, PXR mRNA expression was suppressed to 63.7±3.9% of the control value in response to LPS treatment.</div>
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<i><b>3.3. Rho123 transport by PGP in the ileum</b></i></div>
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In the LPS-treated rats, the efflux clearance of Rho123 in the serosal to mucosal direction <span style="background-color: yellow;">was reduced to</span> 40<span style="background-color: yellow;">% of</span> the <span style="background-color: yellow;">control value</span> <span style="background-color: yellow;">(Fig. </span>3<span style="background-color: yellow;">).</span> The <span style="background-color: yellow;">addition of the PGP-specific inhibitor</span> verapamil <span style="background-color: yellow;">to the </span>mucosal side of<span style="background-color: yellow;"> segments isolated from the control rats reduced transport to </span>60% <span style="background-color: yellow;">of</span> the<span style="background-color: yellow;"> control level, whereas the addition of</span> the <span style="background-color: yellow;">inhibitor to</span> the mucosal side of <span style="background-color: yellow;">segments isolated from the LPS-treated rats did not</span> <span style="background-color: yellow;">inhibit</span> Rho123 <span style="background-color: yellow;">transport</span> <span style="background-color: yellow;">further. The </span>Rho123<span style="background-color: yellow;"> transport</span> to the mucosal to serosal direction, which represents <span style="background-color: yellow;">net absorption</span> <span style="background-color: yellow;">was increased from 100±1</span>8<span style="background-color: yellow;">% in the control</span> <span style="background-color: yellow;">to </span>230<span style="background-color: yellow;">±</span>40<span style="background-color: yellow;">% in</span> the <span style="background-color: yellow;">LPS treated animals</span> (data not shown)<span style="background-color: yellow;">.</span></div>
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<b><i>3.4. <span style="background-color: yellow;">5</span><span style="background-color: yellow;">-Carboxyfluorescein transport by MRP2</span> in the ileum</i></b></div>
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<span style="background-color: yellow;">MRP activity, as</span> shown<span style="background-color: yellow;"> by the </span>efflux clearance of 5-CF in the serosal to mucosal direction<span style="background-color: yellow;">, was reduced to </span>50 <span style="background-color: yellow;">±</span>7<span style="background-color: yellow;">% of</span> the<span style="background-color: yellow;"> control value</span>s (p = 0.08)<span style="background-color: yellow;"> in </span>the<span style="background-color: yellow;"> LPS-treated animals (Fig. </span>4<span style="background-color: yellow;">). Adding the MRP-specific inhibitor MK571 to intestinal segments isolated from </span>the <span style="background-color: yellow;">control rats reduced</span> the efflux to 37<span style="background-color: yellow;">±</span>14<span style="background-color: yellow;">%</span> (Fig. 4)<span style="background-color: yellow;">. In contrast,</span> the<span style="background-color: yellow;"> addition of the inhibitor to </span>the mucosal side of <span style="background-color: yellow;">segments isolated from the LPS-treated rats</span> tended to decrease<span style="background-color: yellow;"> 5-CF</span> efflux, although the differences failed to reach the 5% level of statistical significance (0.05 < p < 0.1) (Fig. 4). 5-CF transport in the mucosal to serosal direction (<span style="background-color: yellow;">net absorption</span>) <span style="background-color: yellow;">was significantly increased from 100%</span> <span style="background-color: yellow;">in</span> the<span style="background-color: yellow;"> controls to </span>190<span style="background-color: yellow;">% in </span>the <span style="background-color: yellow;">LPS-treated rats (p < 0.05)</span> (data not shown)<span style="background-color: yellow;">.</span><br />
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<b><span style="color: red; font-size: large;">富田、瀧沢氏らの論文のDiscussionの文章</span></b><br />
<b>(黄色でハイライトされた部分が</b><b><a href="http://dmd.aspetjournals.org/content/32/1/20.full">J. Kalitsky-Szirtes氏らの論文</a>との同一文章)</b><br />
<b>4. Discussion</b><br />
The suppression of ABC transporters’ expression, the suppression of ABC transporters’ function and unaltered paracellular membrane permeability in the intestine are already recognized at 24, 48 and 72 h after LPS administration (Kalitsky-Szirtes et al., 2004; Moriguchi et al., 2007). On the other hand, we have been focusing on the changes in the expression and function in the early phase after LPS administration (Tomita et al., 2004; 2010). In the present study, we investigated the levels of intestinal P-gp expression and activity of P-gp in the small intestine at 8 h using a rat model induced by LPS.<br />
<span style="background-color: yellow;">Previous studies have demonstrated that inflammation reduces the hepatic expression and activity of the drug efflux transporters mdr1 and mrp2 (Piquette-Miller et al., 1998; Tang et al., 2000). This suppression is</span> <span style="background-color: yellow;">primarily</span> <span style="background-color: yellow;">mediated</span> <span style="background-color: yellow;">by</span> <span style="background-color: yellow;">pro-inflammatory cytokines, particularly IL-</span>1 <span style="background-color: yellow;">(</span>Tomita et al., 2010a,b<span style="background-color: yellow;">). Whether inflammationmediated changes in mdr1 or mrp2 expression occur in epithelial tissues such as the intestine is </span>unknown<span style="background-color: yellow;">. However, there is increasing evidence that the intestinal mucosa responds to endotoxins in a</span> <span style="background-color: yellow;">similar manner</span> <span style="background-color: yellow;">to the well-characterized</span><span style="background-color: white;"> response </span>observed during the <span style="background-color: yellow;">hepatic acute phase response (Molment et al., 1993). Endotoxins </span>are<span style="background-color: yellow;"> known to stimulate the production of IL-</span>1 <span style="background-color: yellow;">in </span>the<span style="background-color: yellow;"> liver (</span>Tomita et al., 2010a,b<span style="background-color: yellow;">). Indeed, we observed an increase in the expression of IL-</span>1 <span style="background-color: yellow;">mRNA in the intestinal segments of LPS-treated rats,</span> which <span style="background-color: yellow;">is consistent with the results reported by </span>Molment<span style="background-color: yellow;"> et al.</span><br />
<span style="background-color: yellow;">(</span>1993<span style="background-color: yellow;">).</span><br />
<span style="background-color: yellow;">We found that LPS-induced inflammation</span> downregulated <span style="background-color: yellow;">the intestinal mRNA expression of mdr1 and mrp2,</span> as was<span style="background-color: yellow;"> previously reported in</span> the<span style="background-color: yellow;"> liver (Piquette-Miller et al., 1998; Tang</span><br />
<span style="background-color: yellow;">et al., 2000; Goralski et al., 2003). Although the levels of mdr1a mRNA were reduced </span>throughout the whole <span style="background-color: yellow;">intestinal regions of </span>the <span style="background-color: yellow;">LPS-treated rats,</span> <span style="background-color: yellow;">mdr1b expression was low and was not significantly altered</span> by LPS-treatment. <span style="background-color: yellow;">Low and often undetectable expression of mdr1b in the intestine has been reported</span> previously <span style="background-color: yellow;">(Salphati and Benet, 1998)</span>, and <span style="background-color: yellow;">investigations in knockout mice have clearly shown that mdr1a is the major determinant of PGP-mediated drug efflux</span> in<span style="background-color: yellow;"> the intestine (Stephens et al., 2002).</span><br />
In accordance<span style="background-color: yellow;"> with the changes in mRNA</span> expression<span style="background-color: yellow;">, we found that LPS treatment</span> induced <span style="background-color: yellow;">significant reductions in PGP- and MRP2-mediated transport in the intestine, whereas the permeability of </span>FD-4 was unchanged.<span style="background-color: yellow;"> Compared with the controls, significant reductions in the</span> serosal to mucosal (S to M) efflux of model substrates of<span style="background-color: yellow;"> PGP and MRP</span> (Rho123 and 5-CF, respectively)<span style="background-color: yellow;"> were</span> observed <span style="background-color: yellow;">in</span> the <span style="background-color: yellow;">intestinal segments isolated from </span>the<span style="background-color: yellow;"> LPS-treated</span> rats<span style="background-color: yellow;">. In these animals, the residual</span> S <span style="background-color: yellow;">to</span> M <span style="background-color: yellow;">transport </span>was<span style="background-color: yellow;"> not</span> reduced<span style="background-color: yellow;"> further</span> <span style="background-color: yellow;">by</span> the <span style="background-color: yellow;">addition of specific inhibitors such as</span> verapamil or <span style="background-color: yellow;">MK571</span>, suggesting that the remaining transport<span style="background-color: yellow;"> reflects passive diffusion. Hence, it is likely that </span>the expression and activity of <span style="background-color: yellow;">PGP and MRP2 were not completely repressed,</span> indicating<span style="background-color: yellow;"> that other post-translational factors may also be involved.</span><br />
<span style="background-color: yellow;">The elevated</span> mucosal to serosal (M to S)<span style="background-color: yellow;"> flux</span> of Rho123 and 5-CF <span style="background-color: yellow;">suggests that a net increase in</span> the<span style="background-color: yellow;"> bioavailability of PGP and MRP2 substrates</span><span style="background-color: white;"> </span><span style="background-color: yellow;">occur</span>s <span style="background-color: yellow;">in vivo during</span> the <span style="background-color: yellow;">inflammatory response.</span><br />
<span style="background-color: yellow;">Our </span>protein <span style="background-color: yellow;">expression and activity data </span>regarding <span style="background-color: yellow;">PGP/mdr1a and MRP2</span> agree with each othe<span style="background-color: yellow;">r, leading us to believe that the reductions in both PGP and MRP activity are the result of a mechanism regulating acute inflammation. Furthermore, these studies indicate that statistically significant alterations in intestinal drug absorption </span>are <span style="background-color: yellow;">predicted</span> by<span style="background-color: yellow;"> altered drug transporter levels.</span> <span style="background-color: yellow;">Since tissue viability (as measured by Rm) and</span> FD-4 <span style="background-color: yellow;">permeability were</span><br />
<span style="background-color: yellow;">not significantly different between the LPS-treated and control animals, it is unlikely that alterations in membrane integrity are responsible for the observed changes in the expression and activity of PGP and MRP2.</span> <span style="background-color: yellow;">It is important to consider the potential contribution of other transporters and metabolic enzymes. Although 5-CF efflux reflects total MRP activity,</span> the <span style="background-color: yellow;">levels of mrp1 and mrp3 were not significantly affected, and</span> the <span style="background-color: yellow;">mRNA expression of other</span><br />
<span style="background-color: yellow;">MRP isoforms was not detectable </span>after LPS treatment<span style="background-color: yellow;">, indicating that changes in 5-CF transport</span> <span style="background-color: yellow;">reflect</span> <span style="background-color: yellow;">changes in MRP2 activity.</span><br />
<span style="background-color: yellow;">From these studies</span>, <span style="background-color: yellow;">significant and concurrent reductions in the expression and activity of PGP/mdr1a and mrp2</span> in the<span style="background-color: yellow;"> intestinal tissue</span> were found <span style="background-color: yellow;">during acute inflammation. Hence, a coordinate system that regulates multiple drug transporters genes</span> is activated <span style="background-color: yellow;">in response to inflammatory stimuli.</span> The <span style="background-color: yellow;">activation of the PXR nuclear receptor has been reported to induce</span> the <span style="background-color: yellow;">expression of MDR1 (Geick et al., 2001) and MRP2 (Kast et al., 2002)</span> <span style="background-color: yellow;">in both the liver and intestine (Staudinger et al., 2001). Furthermore, </span>the<span style="background-color: yellow;"> negative regulation of PXR has been reported to occur via an IL-</span>1 <span style="background-color: yellow;">- mediated mechanism in human hepatocytes (Pascussi et al., 2000). This</span> phenomenon<span style="background-color: yellow;"> has not been examined in vivo or in intestinal tissue. Hence our results, demonstrating a significant reduction in PXR mRNA levels in LPS-treated animals in conjunction with</span> the <span style="background-color: yellow;">suppression of mdr1 and mrp2, suggest </span>the <span style="background-color: yellow;">involvement or coregulation of PXR during inflammation. Further studies examining the involvement of PXR in the basal expression and negative regulation of these genes are currently being performed in PXR knockout and wild-type animal models of inflammation (Jekerle et al., 2003).</span><br />
<span style="background-color: yellow;">Intestinal efflux transporters</span> <span style="background-color: yellow;">contribute to drug clearance, including drug secretion into bile and the direct exsorption of drugs into the intestinal lumen. Hence, inflammatory stimuli are likely to</span> induce <span style="background-color: yellow;">changes in the bioavailability and clearance of numerous drugs that are substrates of PGP and MRP2, thus increasing the possibility of adverse drug reactions or therapeutic failure.</span><br />
<span style="background-color: yellow;"> Our findings indicate that the intestine is</span> changed<span style="background-color: yellow;"> during an inflammatory response. Alterations in</span> <span style="background-color: yellow;">drug transport</span> in <span style="background-color: yellow;">the intestine, as well as the liver, should therefore be considered when predicting drug disposition during inflammation. Increased and variable drug absorption is likely to occur during inflammation, and proper precautions must be taken when determining the appropriate dose</span> <span style="background-color: yellow;">regimen</span> for a particular <span style="background-color: yellow;">drug therapy.</span> The phenomena described in this study are useful for <span style="background-color: yellow;">predicting therapeutic efficacy</span> and understanding <span style="background-color: yellow;">drug–disease interactions.</span><br />
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