We wish to thank Johnson &amp; Johnson, the TB Alliance, Astra Zeneca, Rib-X and Trius Therapeutics for providing bedaquiline, PA-824 (pretomanid), AZD5847, radezolid and tedizolid, respectively. Brendan Prideaux, Matthew Zimmerman, Stephen Juzwin, Emma Rey-Jurado, Nancy Ruel, Leyan Li and Danielle Weiner provided support with MALDI analysis, bioanalytical methods, preparation of the caseum surrogate, chemical synthesis, and isolation of rabbit caseum. This work was conducted with funding from the Bill and Melinda Gates Foundation, award # OPP1044966 and OPP1024050 to V. Dartois, NIH Shared Instrumentation Grant S10OD018072, as well as joint funding from the Bill and Melinda Gates Foundation and Wellcome Trust for A Centre of Excellence for Lead Optimization for Diseases of the Developing World to P. Wyatt.

All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with approval from the Institutional Animal Care and Use Committee of the NIAID (NIH), Bethesda, MD. All studies involving M. tuberculosis were performed in a laboratory with biosafety containment level 3 (BSL-3).
1. Rabbit Infection Model and Caseum Collection
<ol>
	<li>Infect New Zealand white rabbits with M. tuberculosis using a nose-only aerosol exposure system as previously described.12,13 Allow the infection to progress for 12-16 weeks. Sedate the rabbits with 35 mg/kg ketamine and 5 mg/kg xylazine intramuscularly, euthanize the rabbits with 0.22 mL/kg pentobarbital sodium and phenytoin sodium intravenously and proceed with the necropsies.</li>
	<li>Using tweezers and a scalpel, remove lungs from the chest cavity. From each lung lobe, dissect out individual cavities and large necrotic granulomas using a scalpel. Carefully scrape off caseum from the cavity and granuloma walls. Weigh, record and store samples in 2 mL screw-capped tubes at -20 &#176;C until ready for use.</li>
	<li>Gamma-irradiate the infectious caseum samples at 3 MegaRad on dry ice to render them uninfectious and safe for use in a BSL-2 lab.</li>
</ol>
2. In Vitro Generation of Caseum Surrogate from THP-1 Cells
<ol>
	<li>Grow THP-1 monocytes in RPMI 1640 medium (2 mM L-glutamine and 10% fetal bovine serum) in T175 cell culture flasks (80 mL/flask). Incubate the flasks in a 5% CO2 atmosphere at 37 &#176;C for 3-4 days.</li>
	<li>Centrifuge the culture from a T175 flask in two 50 mL conical tubes at 150 x g for 5 min. Discard the supernatant and suspend the pellet in 10 mL of RPMI 1640 media.</li>
	<li>Pipette 5 &#181;L of this culture into a 1.5 mL tube containing 45 &#181;L of trypan blue. Mix thoroughly by pipetting. Transfer 10 &#181;L to a hemocytometer and count the number of viable THP-1 monocytes (unstained) using a light microscope (10X magnification). Calculate the number of viable cells per mL of the culture. Dilute it with RPMI media to the final density of 1.25 x 106 cells/mL.</li>
	<li>Load 40 mL of the culture on a large cell culture plate (50 x 106 cells/plate). Add 40 &#181;L of 100 &#181;M PMA (phorbol 12-myristate13-acetate prepared in ethanol) and allow cells to adhere overnight in the incubator. 
	NOTE: Final concentration of PMA is 100 nM.</li>
	<li>Dilute pure oleic acid (OA) (0.89 g/mL) in ethanol to the concentration of 0.1 M (i.e. 31.7 &#181;L OA in 968.3 &#181;L ethanol). Dilute this solution in fresh pre-warmed RMPI media to a concentration of 10 mM. Dilute this OA suspension to 0.4 mM (final working concentration) in RPMI medium pre-warmed to 37 &#176;C.</li>
	<li>Remove the existing media and non-adhered cells from the cell culture plates and gently add 40 mL of 0.4 mM OA to the THP-1 macrophages (THP-M). Incubate at 37 &#176;C in the incubator overnight.</li>
	<li>Use a light microscope at 40x magnification to visually confirm the presence of numerous lipid body inclusions in each THP-M. Remove all RPMI medium from the cell culture plates and gently wash the adherent cells twice with phosphate buffered saline (PBS) using a 50 mL serological pipette. 
	NOTE: Lipid bodies appear as small, clear, spherical structures in the cytoplasm of the THP-M.</li>
	<li>Add 40 mL of 5 mM ethylenediaminetetraacetic acid (EDTA) in PBS to each plate. Incubate for 15 min at 37 &#176;C.</li>
	<li>Detach the foamy macrophages (FM) by repeatedly pipetting up and down over the surface of the whole plate using a 10-mL serological pipette. Transfer the cell suspension to a 50-mL conical tube and spin down at 150 x g for 5 min.</li>
	<li>Resuspend the cell pellet in 10 mL of PBS (third PBS wash) and transfer to a pre-weighed 15-mL conical tubes. Spin down again at 150 x g for 5 min. Carefully aspirate the supernatant using a serological pipette and discard.</li>
	<li>Subject the FM pellets to 3 freeze-thaw cycles to lyse the cells and incubate them at 75 &#176;C for 20-30 min to denature proteins in the matrix. Store the pellets at -20 &#176;C until ready for use.</li>
</ol>
3. Rapid Equilibration Dialysis (RED) Assay
<ol>
	<li>Prepare 10 mM stock solutions of all test compounds in dimethyl sulfoxide (DMSO). Dilute to 500 &#181;M working solutions in DMSO prior to each assay.</li>
	<li>Weigh the tube containing the caseum surrogate pellet. Subtract the weight of the empty tube to derive the weight of the pellet alone. Add 2-3 metal beads per tube and, using a tissue homogenizer at 1,200 strokes/min for 1 min, disrupt caseum or the surrogate matrix in PBS (1:9 w/v) to achieve 10x diluted suspension of each matrix.</li>
	<li>Spike 6.5 &#181;L of the 500 &#181;M solution of the test compound into 643.5 &#181;L of the homogenate to achieve the final concentration of 5 &#181;M (&#8804;1% DMSO) and vortex.</li>
	<li>Place the RED inserts into the base plate. Add 200 &#181;L of the drug-spiked matrix into the donor chamber (red ring) of each RED insert and 350 &#181;L of PBS into each receiver chamber. Prepare 3 inserts for each test compound (triplicate samples). Seal plate with an adhesive plate seal and incubate at 37 &#176;C on the thermomixer at 200 rpm (1 x g) for 4 h.</li>
	<li>After the incubation, gently mix the contents of the donor and receiver chambers by pipetting up and down 2-3 times. Pipette out 20 &#181;L aliquots of homogenate from the donor chambers and add to 20 &#181;L of clean PBS in a 1.5 mL tube (1:1). Similarly, pipette out 20 &#181;L aliquots of PBS samples from the receiver chambers and add to 20 &#181;L of clean homogenate (matrix matching).8 
	NOTE: Matrix-matching eliminates the need for 2 separate calibration curves (in homogenate and PBS) to be made for quantitative analysis. Contents of the donor chamber may sediment over time. Gently mix the contents by pipetting prior to removing aliquots.</li>
</ol>
4. LC-MS Quantification and Data Analysis
<ol>
	<li>Add 160 &#181;L of 1:1 methanol:acetonitrile containing 500 ng/mL diclofenac or 10 ng/mL verapamil (internal standard) to each tube and vortex to precipitate proteins. Centrifuge at 10,000 x g for 5 min to sediment the precipitate and transfer supernatants into 96-well deep-well plates for liquid chromatography-mass spectrometry (LCMS) analysis.7</li>
	<li>Build calibration curves from 1-1,000 nM for each test compound while keeping the same matrix composition as the samples above. Quantify the concentration of test compound in samples from the donor and receiver chambers using an LC-MS method.</li>
	<li>Calculate the unbound fraction (fu) of the drug in diluted matrix using equation 1. Calculate the fu in undiluted matrix using equation 2 (D = dilution factor of 10).14 
	<img alt="Equation 1" src="/files/ftp_upload/55559/55559eq1.jpg" /> 
	<img alt="Equation 2" src="/files/ftp_upload/55559/55559eq2.jpg" /></li>
	<li>Check the recovery (mass balance) of each compound using equation 3 to identify compounds with stability/metabolism/non-specific binding issues. 
	NOTE: Recovery typically falls between 70% and 130%.15 
	<img alt="Equation 3" src="/files/ftp_upload/55559/55559eq3.jpg" /></li>
</ol>

<ol>
	<li>Sacchettini, J. C., Rubin, E. J., Freundlich, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+versus+bugs:+in+pursuit+of+the+persistent+predator+Mycobacterium+tuberculosis.">Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis.</a> Nat Rev Microbiol. 6 (1), 41-52 (2008).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+and+dormant+tubercle+bacilli+and+latent+tuberculosis.">Persistent and dormant tubercle bacilli and latent tuberculosis.</a> Front Biosci. 1 (9), 1136-1156 (2004).</li><li>Aber, V. R., Nunn, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+term+chemotherapy+of+tuberculosis.+Factors+affecting+relapse+following+short+term+chemotherapy.">Short term chemotherapy of tuberculosis. Factors affecting relapse following short term chemotherapy.</a> Bull Int Union Tuberc. 53 (4), 276-280 (1978).</li><li>Chang, K. C., Leung, C. C., Yew, W. W., Ho, S. C., Tam, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nested+case-control+study+on+treatment-related+risk+factors+for+early+relapse+of+tuberculosis.">A nested case-control study on treatment-related risk factors for early relapse of tuberculosis.</a> Am J Respir Crit Care Med. 170 (10), 1124-1130 (2004).</li><li>Dartois, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+path+of+anti-tuberculosis+drugs:+from+blood+to+lesions+to+mycobacterial+cells.">The path of anti-tuberculosis drugs: from blood to lesions to mycobacterial cells.</a> Nature Rev Microbiol. 12 (3), 159-167 (2014).</li><li>Prideaux, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+sterilizing+activity+and+drug+distribution+into+tuberculosis+lesions.">The association between sterilizing activity and drug distribution into tuberculosis lesions.</a> Nat Med. 21 (10), 1223-1227 (2015).</li><li>Sarathy, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+Drug+Penetration+in+Tuberculosis+Lesions.">Prediction of Drug Penetration in Tuberculosis Lesions.</a> ACS Infect Dis. 2 (8), 552-563 (2016).</li><li>Waters, N. J., Jones, R., Williams, G., Sohal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+rapid+equilibrium+dialysis+approach+for+the+measurement+of+plasma+protein+binding.">Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding.</a> J Pharm Sci. 97 (10), 4586-4595 (2008).</li><li>Singh, J. K., Solanki, A., Maniyar, R. C., Banerjee, D., Shirsath, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Equilibrium+Dialysis+(RED):+an+In-vitro+High-Throughput+Screening+Technique+for+Plasma+Protein+Binding+using+Human+and+Rat+Plasma.">Rapid Equilibrium Dialysis (RED): an In-vitro High-Throughput Screening Technique for Plasma Protein Binding using Human and Rat Plasma.</a> J Bioequiv Availab. 14, 1-4 (2012).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbound+drug+concentration+in+brain+homogenate+and+cerebral+spinal+fluid+at+steady+state+as+a+surrogate+for+unbound+concentration+in+brain+interstitial+fluid.">Unbound drug concentration in brain homogenate and cerebral spinal fluid at steady state as a surrogate for unbound concentration in brain interstitial fluid.</a> Drug Metab Dispos. 37 (4), 787-793 (2009).</li><li>Able, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+localization,+native+tissue+binding+and+ex+vivo+occupancy+for+centrally+penetrant+P2X7+antagonists+in+the+rat.">Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat.</a> Br J Pharmacol. 162 (2), 405-414 (2011).</li><li>Subbian, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+pulmonary+cavitary+tuberculosis+in+rabbits:+a+failed+host+immune+response.">Chronic pulmonary cavitary tuberculosis in rabbits: a failed host immune response.</a> Open Biol. 1 (4), 1-14 (2011).</li><li>Via, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuberculous+Granulomas+are+Hypoxic+in+Guinea+pigs,+Rabbits,+and+Non-Human+Primates.">Tuberculous Granulomas are Hypoxic in Guinea pigs, Rabbits, and Non-Human Primates.</a> Infect Immun. 76 (6), 2333-2340 (2008).</li><li>Kalvass, J. C., Maurer, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+nonspecific+brain+and+plasma+binding+on+CNS+exposure:+implications+for+rational+drug+discovery.">Influence of nonspecific brain and plasma binding on CNS exposure: implications for rational drug discovery.</a> Biopharm Drug Dispos. 23 (8), 327-338 (2002).</li><li>Di, L., Umland, J. P., Trapa, P. E., Maurer, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+recovery+on+fraction+unbound+using+equilibrium+dialysis.">Impact of recovery on fraction unbound using equilibrium dialysis.</a> J Pharm Sci. 101 (3), 1327-1335 (2012).</li><li>Lenaerts, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+persisting+mycobacteria+in+a+Guinea+pig+model+of+tuberculosis+revealed+by+r207910.">Location of persisting mycobacteria in a Guinea pig model of tuberculosis revealed by r207910.</a> Antimicrob Agents Chemother. 51 (9), 3338-3345 (2007).</li><li>Prideaux, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+MALDI-MRM-MS+imaging+of+moxifloxacin+distribution+in+tuberculosis-infected+rabbit+lungs+and+granulomatous+lesions.">High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions.</a> Anal Chem. 83 (6), 2112-2118 (2011).</li></ol>

There are no competing financial interests.

Pulmonary necrotic lesions and cavities in tuberculosis-infected patients contain subpopulations of bacteria that are recalcitrant to drug treatment. The caseous cores of these structures are particularly responsible for harboring these persisters in an extracellular environment.16 Favorable distribution of anti-bacterial agents into these remote locations is believed to be an important determinant of tuberculosis drug efficacy. Prior to the validation of this protocol, there were no available <em...

The treatment of pulmonary tuberculosis disease requires effective distribution of drugs into different types of lesions. Necrotic lesions and cavities contain caseous centers that harbor subpopulations of drug-tolerant or &#39;persistent&#39; bacteria.1,2 The cavitary disease is associated with inferior cure rates and poor prognosis.3,4 Previous studies have shown, using quantitative and imaging techniques, that the ability to penetrate caseum varies significantly from one drug class to another.5,6 These methods, however, require the use of animal infection models that are slow and tedious. An in vitro assay that measures drug binding to ex vivo caseum was designed. This binding was found to inversely correlate with drug penetration in caseous granulomas and, hence, is used as a predictive tool.7
Equilibrium dialysis is regarded as the gold standard approach to plasma protein binding studies. The rapid equilibrium dialysis (RED) device provides a quick, easy-to-use and reliable system for performing such assays.8 The device is made up of two components: single-use, disposable inserts consisting of 2 chambers separated by a vertical cylinder of semi-permeable membrane; and reusable base plates that can hold up to 48 inserts at a time. The dialysis membrane has an 8 kDa molecular weight cut-off (MWCO) that is ideal for drug-macromolecule binding studies. The high surface area-to-volume ratio of the membrane compartment allows rapid dialysis and equilibration. Both the inserts and the base plate have been validated for minimal non-specific binding. The combination of the RED device with bioanalytical techniques provides accurate estimations of the unbound fractions of drugs in plasma.8,9
Although originally designed to measure plasma protein binding, the RED device has been used in several tissue binding studies using homogenates.10,11 In this protocol, we measure drug binding to caseum, the necrotic debris excised from the necrotic lesions and cavities of tuberculosis-infected rabbits. The acellular and non-vascular nature of caseous material makes it easy to homogenize into a homogenous suspension that is compatible with the assay.
Given that caseum is tedious to produce and hard to come by, the protocol has also been validated for use with a surrogate matrix that is prepared from foamy macrophages. THP-1 monocyte-derived macrophages are induced with oleic acid to accumulate multiple lipid bodies that give them their &#39;foamy&#39; appearance. These lipid-loaded cells are harvested and processed to produce a matrix that we use as a surrogate to caseum. This study has shown that drug binding to this surrogate matrix correlates well with binding to caseum, effectively mimicking the in vivo process that hinders drug penetration in the caseous core of granulomas and cavities.

<table border="0" cellpadding="0" cellspacing="0" width="960">
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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

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

Using this protocol, we have tested hundreds of tuberculosis drug development compounds for their predicted efficiency at penetrating caseum. Figure 1 visualizes the basic concepts of the RED assay. The dialysis membrane of the RED inserts allows for unbound small molecules to diffuse from the donor well to the receiver well, finally achieving an equilibrium between both compartments. Small molecules that are bound to macromolecules such as proteins or lipids are trapped ...

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

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

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

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

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11.6K Views.  Rutgers. Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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