Biz sırasıyla, PA-824 (pretomanid), AZD5847, radezolid ve tedizolid Bedaquiline sağlamak için Johnson &amp; Johnson, TB Alliance, Astra Zeneca, Kaburga-X ve Trius Therapeutics teşekkür etmek istiyorum. Brendan Prideaux Matthew Zimmerman, Stephen Juzwin Emma Rey-Jurado Nancy Ruel, Leyan Li ve Danielle Weiner MALDI analizi, biyoanalitik yöntemler, caseum vekil hazırlanması, kimyasal sentez ve tavşan caseum izolasyonu ile destek sağlamıştır. Bu çalışma V. Dartois NIH Paylaşılan Enstrümantasyon Grant S10OD018072 yanı sıra Mükemmellik Bir Merkezi için Bill ve Melinda Gates Vakfı ve Wellcome Trust ortak finansmanı için Bill ve Melinda Gates Vakfı tarafından finanse ödül # OPP1044966 ve OPP1024050 ile yürütülmüştür P. VVyatt&#39;a Gelişen Dünya Hastalıklar Kurşun Optimizasyonu için.

Tüm hayvan çalışmaları NIAID Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (NIH), Bethesda, MD onayı ile Ulusal Sağlık Enstitüleri Laboratuvar Hayvanları Bakım ve Kullanımı Kılavuzu uyarınca gerçekleştirilmiştir. M. tuberculosis içeren tüm çalışmalarda biyo-güvenlik tutucu seviye 3 (BSL-3) ile birlikte bir laboratuarda gerçekleştirilmiştir. 1. Tavşan Enfeksiyon Modeli ve Caseum Koleksiyonu <ol><li> Daha önce tarif edildiği gibi bir sadece burundan bir aerosol uygulama sistemi kullanılarak Yeni Zelanda M. tuberculosis ile beyaz tavşan Infect. 12, 13 enfeksiyon 12-16 hafta boyunca ilerlemeye izin verin. Oturaklı 35 mg / kg ketamin ve 5 mg / kg ksilazin kas içinden, birlikte tavşan 0.22 ml / kg pentobarbital sodyum ve fenitoin sodyum ile tavşan damar ötenazi ve nekropsilerden devam edin. </li><li> cımbız ve bir neşter kullanılarak, göğüs ca akciğerleri kaldırmakvity. Her akciğer lobundan, bir neşter yardımıyla bireysel boşlukları ve geniş nekrotik granülomları inceleyin. Kaseyi dikkatli bir şekilde boşluk ve granülom duvarlarından kazıyın. 2 mL vidalı kaptaki tüpleri tartın, kaydedin ve depolayın -20 ° C&#39;de kullanıma hazır oluncaya kadar saklayın. </li><li> Kuru buz üzerinde 3 MegaRad&#39;de bulaşıcı kazein örneklerini gamma radyasyona maruz bırakarak onları bir bulaşıcı olmayan ve güvenli bir BSL-2 laboratuarında kullanın. </li></ol> 2. THP-1 Hücrelerinden Kaseum Vekilinin in vitro Üretimi <ol><li> T175 hücre kültürü şişeleri (80 mL / şişe) içinde RPMI 1640 ortamında (2 mM L-glutamin ve% 10 sığır fetüsü serumu) THP-1 monositleri büyütün. Şişeleri 37 ° C&#39;de% 5 CO 2 atmosferinde 3-4 gün inkübe edin. </li><li> Kültürü, 150 xg&#39;de iki 50 mL konik tüp içinde T175 bir şişeden 5 dakika boyunca santrifüjleyin. Süpernatantı atın ve 10 mL RPMI 1640 ortamda pelet askıya alınız. </li><li> 45 uL t içeren 1.5 mL tüp içine bu kültürden 5 μL pipetleMavi rippan. Pipetleme ile iyice karıştırın. 10 μL&#39;lik bir hemositometreye aktarın ve canlı bir THP-1 monosit sayısını (renklendirilmemiş) bir ışık mikroskopu (10X büyütme) kullanarak sayın. Kültür mL başına canlı hücrelerin sayısını hesaplayın. RPMI ortamı ile 1.25 x 106 hücre / mL nihai yoğunluğa kadar seyreltin. </li><li> Büyük bir hücre kültürü plakasına (50 x 10 6 hücre / tabağa) 40 mL kültür yükleyin. 40 uL 100 uM PMA (etanol içinde hazırlanmış forbol 12-miristat 13-asetat) ilave edin ve hücrelerin inkübatöre gece boyunca yapışmasına izin verin. NOT: PMA&#39;nın nihai konsantrasyonu 100 nM&#39;dir. </li><li> Saf oleik asit (OA) (0.89 g / mL), 0.1 M konsantrasyonunda etanol içinde ( yani , 968.3 uL etanolde 31.7 μL OA) seyreltin. Bu çözeltiyi taze önceden ısıtılmış RMPI ortamında 10 mM&#39;lik bir konsantrasyona kadar seyreltin. Bu OA süspansiyonunu önceden 37 ° C&#39;ye ısıtılmış RPMI ortamında 0,4 mM&#39;ye (son çalışma konsantrasyonu) seyreltin. </li><li> Mevcut medyayı çıkarın veYapışmış hücreleri hücre kültür plakalarından hafifçe ilave edin ve THP-1 makrofajlarına (THP-M) 40 mL 0.4 mM OA ekleyin. Gece inkübatörde 37 ° C&#39;de inkübe edin. </li><li> Her bir THP-M&#39;de sayısız lipid cesedi kapanımının varlığını teyit etmek için 40x büyütmede hafif bir mikroskop kullanın. Hücre kültürü plakalardan tüm RPMI orta çıkarın ve 50 ml serolojik pipet kullanarak fosfat tamponlu salin (PBS) ile yapışkan hücreleri hafifçe yıkayın. NOT: THP-M&#39;nin sitoplazmasındaki lipit cisimler küçük, net, küresel yapılar olarak görünür. </li><li> PBS içinde 40 mL 5 mM etilendiamintetraasetik asit (EDTA) her bir tabağa ilave edilir. 37 ° C&#39;de 15 dakika inkübe edin. </li><li> Köpüklü makrofajları (FM), 10 mL&#39;lik bir serolojik pipet kullanarak tüm plakanın yüzeyinde tekrar tekrar pipetleme ile ayırın. Hücre süspansiyonunu 50 mL&#39;lik bir konik tüpe aktarın ve 5 dakika boyunca 150 xg&#39;de aşağı doğru döndürün. </li><li> 10 mL PBS (üçüncü PBS yıkama) içinde hücre topağını tekrar süspansiyon haline getirin.nd önceden tartılmış 15 mL&#39;lik konik tüplere aktarın. 5 dakika boyunca 150 x g hızında tekrar Spin. Dikkatle bir serolojik pipet ve imha edilmesini kullanarak süpernatant aspire. </li><li> hücreler lize edildi ve matris proteinleri denatüre etmek için 20-30 dakika süreyle 75 ° C&#39;de inkübe etmek 3 dondurma-çözme döngüsüne FM pelet Konu. Kullanım için hazır olana kadar -20 ° C &#39;de pelet saklayın. </li></ol> 3. hızlı bir dengeleme Diyaliz (kırmızı) Deneyi <ol><li> dimetil sülfoksit tüm test bileşikleri (DMSO) içinde 10 mM stok çözelti hazırlayın. Her bir deney öncesinde DMSO içinde çözüm çalışan 500 uM&#39;ye seyreltilir. </li><li> caseum vekil pelet içeren tüp tartılır. tek başına pelet ağırlığı elde etmek için boş bir borunun ağırlığı çıkarılır. (: Ağırlık / hacim 1: 9), her matrisin 10x seyreltilmiş süspansiyonu elde etmek için caseum veya PBS ile taşıyıcı matris bozabilir 1 dakika / dakika 1200 vuruş bir doku homojenleştirici kullanılarak, tüp başına 2-3 metal boncuk ekleyin ve. </li><li> 500 6.5 uL başakΜM solüsyonu, 5 uM (≤% 1 DMSO) nihai konsantrasyonunu elde etmek için 643.5 uL homojenata ve vorteks içine yerleştirin. </li><li> KIRMIZI insertleri taban plakasına yerleştirin. Her alıcı bölmesine her RED girişi ve 350 μL PBS verici bölmesine (kırmızı halka) ilaç-spiked matris 200 mcL ekleyin. Her bir test bileşiği için üç eklenti hazırlayın (üçlü örnekler). Plakayı yapışkan plakalı bir conta ile yapıştırın ve 4 saat boyunca 200 rpm&#39;de (1 xg) termomiksör üzerinde 37 ° C&#39;de inkübe edin. </li><li> Kuluçka işleminden sonra verici ve alıcı odalarının içindekileri hafifçe 2-3 defa yukarı ve aşağı pipetleyerek karıştırın. Donör odalardan 20 μL&#39;lik homojenat kısımları pipetleyin ve 1.5 mL&#39;lik bir tüpe (1: 1) 20 μL temiz PBS ekleyin. Benzer şekilde, alıcı odalardan PBS örnekleri 20 mcL alikotları pipetle ve temiz homojenat (matris eşleme) 20 mcL ekleyin. 8 NOT: Matris eşleştirme işlemi,Kantitatif analiz için yapılacak 2 ayrı kalibrasyon eğrisi (homojenat ve PBS&#39;de) için. Donör odasının içeriği zamanla sedimantasyon yapabilir. Alikotları çıkarmadan önce içeriğini pipetle hafifçe karıştırın. </li></ol> 4. LC-MS Niceleme ve Veri Analizi <ol><li> Her bir tüpe 500 ng / mL diklofenak veya 10 ng / mL verapamil (iç standart) içeren 160 uL 1: 1 metanol: asetonitril ekleyin ve proteinleri çökeltmek için girdap ekleyin. Çökeltiyi tortu haline getirmek ve süpernatantları sıvı kromatografi-kütle spektrometresi (LCMS) analizi için 96 oyuklu derin plakalara aktarmak için 5 dakika boyunca 10,000 xg&#39;de santrifüjleyin. 7 </li><li> Yukarıdaki numunelerle aynı matris bileşimini koruyarak, her test bileşiği için 1-1000 nM&#39;den kalibrasyon eğrileri oluşturun. Bir LC-MS yöntemi kullanarak verici ve alıcı odalarından alınan numunelerdeki test bileşiğinin konsantrasyonunu hesaplayın. </li><li> Bağsız fraksiyonu hesapla ( f u ) of denklem 1 kullanılarak seyreltilmiş matris içinde ilaç denklem 2 (D = 10 seyreltme faktörü) kullanılarak seyreltilmemiş matris içinde f u hesaplayın. 14 <img alt="Denklem 1" src="/files/ftp_upload/55559/55559eq1.jpg" /> <img alt="Denklem 2" src="/files/ftp_upload/55559/55559eq2.jpg" /></li><li> stabilite / metabolizma / spesifik olmayan bağlanma konularla bileşimleri belirlemek için Denklem 3 kullanılarak her bileşiğin kurtarma (kütle dengesi) kontrol edin. NOT: Kurtarma tipik% 70 ve% 130 arasında düşer. 15 <img alt="Denklem 3" src="/files/ftp_upload/55559/55559eq3.jpg" /></li></ol>

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Rakip finansal çıkarlar yoktur.

Tüberkülozla enfekte hastalarda pulmoner nekrotik lezyonlar ve boşluklar, ilaç tedavisine dirençli bakteri alt popülasyonlarını içerir. Bu yapıların kazeöz çekirdeği, özellikle, bu persisörleri ekstraselüler bir ortamda barındırmaktan sorumludur. Anti-bakteriyel maddelerin bu uzak yerlere olumlu dağılımının tüberküloz ilaç etkinliğinin önemli bir belirleyicisi olduğuna inanılmaktadır. Bu protokolün onaylanmasından önce, konstitüsyonel lezyonlarda ilaç keşfi bileşiklerinin dağılı...

akciğer tüberkülozu hastalığının tedavisi lezyonlar farklı içine ilaç etkin dağıtım gerektirir. Nekrotik lezyonlar ve boşluklar ilaç toleranslı veya &#39;kalıcı&#39; bakteri alt-popülasyonunu liman peynir merkez içerir. 1, 2 kaviter hastalık alt kür oranları ve kötü prognoz ile ilişkilidir. 3, 4 önceki çalışmalar caseum nüfuz kabiliyeti önemli ölçüde bir ilaç sınıfı diğerine değişir, nicel ve görüntüleme teknikleri kullanılarak, göstermiştir. 5, 6 Ancak bu yöntemler, yavaş ve zahmetlidir hayvan enfeskiyon modellerinden kullanılmasını gerektirir. Ex vivo olarak caseum bağlanma ilaç ölçen bir in vitro deney tasarlanmıştır. Bu ters nedenle, peynir granülomada ilaç nüfuzunun ile ilişkili ve bulunmuştur bağlanma, birTahmin edici bir araç olarak kullanılmıştır. 7 Dengeleme diyalizi, plazma protein bağlama çalışmalarına altın standart yaklaşım olarak görülür. Hızlı denge diyalizi (RED) cihazı, bu tür testleri gerçekleştirmek için hızlı, kullanımı kolay ve güvenilir bir sistem sağlar. 8 Cihaz iki bileşenden oluşur: yarı geçirgen zarın dikey bir silindiri ile ayrılmış 2 hazneden oluşan tek kullanımlık, tek kullanımlık uçlar; Ve aynı anda 48 ek parça tutabilen tekrar kullanılabilir taban plakaları. Diyaliz membranı, ilaç-makromolekül bağlama çalışmaları için ideal olan 8 kDa moleküler ağırlıklı bir kesime (MWCO) sahiptir. Membran kompartmanının yüksek yüzey alanı / hacim oranı, hızlı diyaliz ve dengeleme sağlar. Hem ekler hem de taban plakası, asgari spesifik olmayan bağlanma için onaylanmıştır. RED cihazının biyoanalitik tekniklerle kombinasyonu, ilaçların bağlanmamış fraksiyonlarının kesin tahminlerini plasma. 8 , 9 Başlangıçta plazma protein bağlanmasını ölçmek için tasarlanmış olmasına rağmen, RED cihazı homojenatları kullanarak çeşitli doku bağlama çalışmalarında kullanılmıştır. 10 , 11 Bu protokolde, tüberkülozla enfekte tavşanların nekrotik lezyonlarından ve boşluklarından eksize edilen nekrotik çöpler, kaseum ile ilacın bağlanmasını ölçüyoruz. Caseus materyalinin asellüler ve non-vascular özelliği, tahlil ile uyumlu homojen bir süspansiyona homojenleştirilmesini kolaylaştırır. Kaseum üretmek için sıkıcı ve zor gelmesi nedeniyle, protokol de köpüklü makrofajlardan hazırlanan bir vekil matris ile kullanım için onaylanmıştır. THP-1 monosit türevi makrofajlar, onlara &#39;köpüklü&#39; görünüm veren çoklu lipid cisimcikleri biriktirmek için oleik asit ile indüklenir. Bu lipit yüklü hücreler hasat edilir veBiz caseum için bir ikame olarak kullanmak, bir matris üretmek üzere işlenir. Bu çalışma, bu taşıyıcı matris etkin bir granülomlar ve boşlukların peynir çekirdek ilacın nüfuz etmesine engel in vivo işlemi taklit caseum bağlanma ile iyi bir korelasyon ile bağlanan ilaç göstermiştir.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">New Zealand White Rabbits</td>
			<td style="width:203px;">Covance</td>
			<td style="width:147px;">-</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">HN878 Mycobacterium tuberculosis</td>
			<td style="width:203px;">BEI Resources</td>
			<td style="width:147px;">NR-13647&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ketathesia (Ketamine) 100mg/mL C3N</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">56344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Anased (Xylazine) 100mg/mL</td>
			<td style="width:203px;">Henry Schein Animal Health</td>
			<td style="width:147px;">33198</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Euthasol (pentobarbital sodium and phenytoin sodium) Solution&#160;</td>
			<td style="width:203px;">Virbac</td>
			<td style="width:147px;">710101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">THP-1 monocytic cell line</td>
			<td style="width:203px;">ATCC</td>
			<td style="width:147px;">ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">175cm&#178; TC-Treated Flask (T175)</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-3400-175</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 media w/o glutamine</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>MT-15-040-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone Fetal Bovine Serum, Gamma irradiated&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3091003IR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hyclone L-glutamine, 200mM</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>SH3003401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cellstar TC dish, 145x20mm, vented</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>T-2881-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phorbol 12-myristate13-acetate (PMA)&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>BP685-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:203px;">Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oleic acid&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>ICN15178101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Reusable Base Plate</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED Device Inserts, 50/box&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>PI-89809</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pierce RED insert removal tool&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>89812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adhesive plate seal</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>08-408-240</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS, pH7.4, 10 X 500 mL (Gibco)&#160;</td>
			<td style="width:203px;">Life Technologies&#160;</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">DMSO</td>
			<td style="width:203px;">Sigma</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Acetonitrile</td>
			<td style="width:203px;">Sigma</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Methanol</td>
			<td style="width:203px;">Sigma</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Verapamil hydrochloride</td>
			<td style="width:203px;">Sigma</td>
			<td>V4629</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Diclofenac sodium salt</td>
			<td style="width:203px;">Sigma</td>
			<td>93484</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-250-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Ethanol, 200 proof</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>04-355-451</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">2010 Geno/Grinder</td>
			<td style="width:203px;">SPEX SamplePrep</td>
			<td style="width:147px;">2010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Bead Mill Homogenizer Accessory, Metal Bulk Beads</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td>15-340-158</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">484R Cobalt 60 Irradiator</td>
			<td style="width:203px;">JL Shepard</td>
			<td style="width:147px;">7810-484-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">INCYTO&#160;C-Chip Disposable Hemacytometers</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:147px;">22-600-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Upright Light Microscope</td>
			<td style="width:203px;">Leica</td>
			<td style="width:147px;">DM1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Binary Liquid Chromatography system</td>
			<td style="width:203px;">Agilent</td>
			<td style="width:147px;">1260</td>
			<td>Multi-compenent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">Mass spectrometer</td>
			<td style="width:203px;">AB Sciex</td>
			<td>4000</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro caseum binding assay that predicts drug penetration in tuberculosis lesions

Tüberküloz hastalığının ortadan kaldırılması, kompleks pulmoner lezyonların çok katmanına nüfuz edebilen ilaç rejimleri gerektirir. Çukurların ve lezyonların kazeöz damarlarındaki ilaç dağılımı, özellikle persistan olarak da adlandırılan uyuşturucuya dirençli bakterilerin alt popülasyonlarını barındırdığı için çok önemlidir. Tüberküloz lezyonlarında ilaç penetrasyonunun ölçülmesine ilişkin mevcut metotlar, biyoanalitik veya görüntüleme teknikleriyle birleştirilen yüksek maliyetli ve zaman alan in vivo farmakokinetik çalışmaları içerir. Kozmetik makromoleküllere bağlanan ilacın in vitro ölçümü, bu tür teknikler için bir alternatif olarak önerildi çünkü bu bağlanma, ilaç moleküllerinin kaseum yoluyla pasif difüzyonunu engelledi. Hızlı denge diyalizi plazma proteini ve doku bağlama çalışmalarında hızlı ve güvenilir bir sistemdir. Bu protokolde, kazanın homojenatlarına ilaç bağlanmasını ölçmek için hızlı denge diyaliz (RED) cihazı kullandık.Tüberküloz ile enfekte tavşanların lezyonlarından ve boşluklarından türetilmiştir. Protokol ayrıca, kaseum yerine kullanılacak lipid yüklü THP-1 makrofajlarından bir vekil matrisi nasıl oluşturulacağını açıklar. Bu kaseum / vekil bağlanma testi, tüberküloz ilaç keşfi için önemli bir araçtır ve lezyonlarda veya apselerde diğer hastalıkların yol açtığı ilaç dağılımının araştırılmasına yardımcı olmak için uyarlanabilir.

Bu protokolü kullanarak, yüzlerce tüberküloz ilaç geliştirme bileşiğini penetran kaseumda tahmin edilen etkinlikleri için test ettik. Şekil 1 , RED testinin temel kavramlarını görüntüler. RED eklentilerinin diyaliz zarları bağlanmamış kük moleküllerin donör kuyudan alıcı kuyusuna dağılmasına ve son olarak da her iki bölme arasında denge elde edilmesine olanak tanır. Proteinler veya lipidler gibi makromoleküllere bağlı olan küçük molek?...

Watch this Scientific Journal Video about An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions at JoVE.com

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Burada pulmoner tüberküloz lezyonlarından ve boşluklardan alınan kaseum ile ilacın bağlanmasını ölçmek için hızlı bir denge diyaliz (RED) yöntemi anlatılmaktadır. Protokol, aynı zamanda, kaseum için etkili bir vekil olan köpüklü makrofaj kaynaklı bir matriste de kullanılır.

bir<em&gt; In vitro</emTüberküloz Lezyonlarında İlaç Penetrasyonunu Öngören Caseum Bağlanma Testi

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

an-vitro-caseum-binding-assay-that-predicts-drug-penetration

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Pharmacokinetics and Pharmacodynamics

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Unit 1

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Nonlinear Pharmacokinetics

Introduction to Respiratory System Drugs

SCIENTISTS IN ACTION

11.6K Görüntüleme.  Rutgers.  Burada pulmoner tüberküloz lezyonlarından ve boşluklardan alınan kaseum ile ilacın bağlanmasını ölçmek için hızlı bir denge diyaliz (RED) yöntemi anlatılmaktadır. Protokol, aynı zamanda, kaseum için etkili bir vekil olan köpüklü makrofaj kaynaklı bir matriste de kullanılır. 

Video: An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8-iyi Odası Slayt in vitro Biyofilm Oluşumu

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

İmmünoloji ve Enfeksiyon

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Kolaylaştırılması İlaç Keşfi: Zebra balığı An Otomatik Yüksek içeriği Enflamasyon Assay

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Nicel<em&gt; In vitro</emStatik Koşullarında Account İlköğretim İnsan mikrovasküler endotel hücreleri için Nötrofil yapışma Tedbir&gt; Testi

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Doğal İletim üzerine Tüberküloz-Sıtma Koenfeksiyonu Çalışması için Deneysel Bir Model Mycobacterium tuberculosis Ve Plasmodium berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Bir kullanma Pnömokok Kolonizasyon üzerinde Probiyotiklerin Etkileri incelenmesi<em&gt; In Vitro</em&gt; Yapışma Deneyi

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Çok ilaca dirençli Demonstrating<em&gt; Mycobacterium tuberculosis</em&gt; Amplifikasyon Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Dalbavansin için Başvuru sıvı mikrodilüsyon yöntemi<em&gt; İn Vitro</emAerobik Olarak Büyüyen Bakterilerin&gt; Duyarlılık Testi

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Modüle c-di-GMP sinyal İşte Küçük Moleküller Tarama için Yüksek verim Kur kurulması<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Bir Yüksek verim Uyumlu Deneyi Makrofaj, pasajlamaya karşı ilaç etkinliğini değerlendirmektir<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Terapötik Antikorlar Arasındaki Hedef Bağlanma ve CDC İndüksiyonunu Karşılaştırmak İçin İn Vitro Yöntemler: Biyoeşdeğerlik Analizindeki Uygulamalar

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...