This work is funded by R.J. Reynolds Tobacco Company (RJRT) under a collaborative research agreement with Wake Forest University School of Medicine. G.L. Prasad is a full time employee of RJRT.

NOTE: Written Informed consent to do this study was obtained at a local clinical research unit under IRB approval, per Good Clinical Practices. Processing of blood, isolation of PBMCs and other cell culture experiments are performed under sterile conditions, using microbiologically sterile supplies and reagents.
1. WS-CM Preparation
<ol>
	<li>Generate WS-CM as previously described12.
	<ol>
		<li>Prepare WS-CM by passing smoke from four 3R4F reference cigarettes through Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red using the following smoking regimen: 35-60-2, puff volume in ml, puff interval in sec, and puff duration in sec, respectively. Each preparation generates a 20 ml sample.</li>
	</ol>
	</li>
	<li>Label tube(s) with date, time completed, and cigarette name and number. Store the 500 &#181;l aliquots at -80 &#176;C immediately after smoking is completed.</li>
	<li>Analyze aliquots of frozen WS-CM to determine the levels of nicotine, tobacco specific nitrosamines, and polycyclic aromatic hydrocarbons as previously described12.</li>
</ol>
2. Isolation of PBMCs
<ol>
	<li>Collect fresh blood from healthy donors (who are non-consumers of tobacco products) Isolate PBMCs from fresh blood as described below under a separate approval from Wake Forest Baptist Health IRB15.
	<ol>
		<li>Prior to the arrival of the blood bag, have a 500 ml bottle, scissors, isolation buffer, and Dulbecco&#8217;s phosphate buffered saline (DPBS) (RT) ready in a biosafety level 2 (BSL-2) cell culture hood. Isolation buffer must be protected from light.</li>
		<li>Hold the blood bag upside down and cut the tube just below where it has been clamped leaving at least 3 cm of tubing.</li>
		<li>Remove the cap from the 500 ml bottle and hold the tube above the bottle opening. Pick up the blood bag and invert it to allow the blood to flow freely from the bag into the bottle until the bag is empty. Allow the blood to flow onto the inside wall of the bottle versus straight down as to avoid creating bubbles.</li>
		<li>Pour isolation buffer into the bottle at a 1:5 ratio of isolation buffer to blood. Cap the bottle tightly and gently invert it, end-over-end, 10 times. Leave the bottle in the cell culture hood to incubate with lights off, for 1 hr at RT.</li>
		<li>A light, straw-colored layer will build up above the blood. Remove this layer using a 25 ml serological pipette into 50 ml conical tubes. The collected amount may vary from 50 - 300 ml, depending on the subject who donated blood.</li>
		<li>Centrifuge the tubes at 200 x g for 10 min at RT.</li>
		<li>Aspirate the translucent supernatant, leaving the dark blood-colored pellet. The pellet will be loose but viscous.</li>
		<li>Pipette 3 ml of isolation buffer into 15 ml conical tubes.</li>
		<li>To the resuspend pellet, add 20 ml of DPBS for every 50 ml of straw-colored liquid collected in step 2.1.5. Vortex to mix thoroughly, and consolidate the resuspended liquid from multiple tubes. This liquid contains suspended blood cells.</li>
		<li>With a transfer pipet, transfer 5 ml of the suspended blood cells onto 3 ml of isolation buffer in step 2.1.8. Tilt the 15 ml conical tube that contains the cell suspension slowly and gently to create two separate layers. Centrifuge the tubes at 400 x g for 40 min at RT with minimal acceleration and without brake.</li>
		<li>Use a transfer pipet to remove the resulting cloudy middle layer (buffy coat) containing PBMCs into a 50 ml conical tube. Avoid drawing other clear layers below it. Transfer no more than 25 ml into each 50 ml conical tube.</li>
		<li>Add 25 ml of cold running buffer (or more to fill the entire remaining volume of the 50 ml conical tube) to wash the cells. Centrifuge the cells at 400 x g for 10 min at 4 &#176;C.</li>
		<li>Resuspend the pellet with 10 ml running buffer. This contains PBMCs. Count the cells and use immediately or place on ice for freezing.</li>
	</ol>
	</li>
</ol>
3. Freezing and Thawing the PBMCs
<ol>
	<li>Centrifuge the PBMCs that were collected in step 2.1.13 for 10 min at 400 x g.</li>
	<li>Fill the freezing container with isopropyl alcohol per the manufacturer&#8217;s instructions. CAUTION: Isopropyl alcohol is flammable and acutely toxic.</li>
	<li>Resuspend the pellet with RPMI 1640 medium (4 &#176;C) containing 20% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). This is RPMI 1640 freezing medium. Resuspend the pellet with an amount of freezing medium that will result in a suspension having about 5 x 107 cells/ml. The number of cells available for freezing will vary.</li>
	<li>Dispense 1 ml aliquots of cell suspension into 2 ml cryotubes and place the cryotubes in the freezing container. Place the freezing container with cryotubes in a freezer at -80 &#176;C to store O/N and then remove the cryotubes and transfer to store in a cryogenic freezer between -150 &#176;C to -190 &#176;C.</li>
	<li>Remove the cryotube from cryogenic storage and thaw it rapidly with gentle agitation in a water bath at 37 &#176;C.</li>
	<li>Immediately transfer the thawed PBMCs in the cryotube into a 15 ml conical tube with 10 ml RPMI 1640 complete medium (4 &#176;C) containing 10% FBS, 1% Pen/Strep and 1% L-glutamine. The contents of the thawed cryotube should be transferred as soon as possible to obtain maximal cell viability15.</li>
	<li>Centrifuge the PBMCs at 400 x g for 10 min.</li>
	<li>Resuspend the pellet with 5 ml RPMI 1640 complete medium and count the cells.</li>
	<li>Measure cell viability by established methods such as trypan blue exclusion method. Generally cell viability with this method is about 90 - 95%. The PBMCs are now ready to use in experiments.</li>
</ol>
4. Cell Death Determination

NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be changed accordingly.
<ol>
	<li>Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well, as indicated below.</li>
	<li>Dilute WS-CM to the following concentrations: 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4, and 5 &#181;g/mL of equi-nicotine units (based on the nicotine content in WS-CM)12.</li>
	<li>Dilute nicotine in RPMI medium to the following concentrations: 100, 200, 500, 750, 1000, 2000, and 3000 &#181;g/ml. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs suspended in RPMI complete medium to each well at a concentration of 1 x 106 cells/well. The total volume of cells plus WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>For the purpose of this study, prepare two sets of plates as above. Cover the plates and incubate one plate for 24 hr and one plate for 3 hr at 37 &#176;C and 5% CO2. Adjust time points as necessary.</li>
	<li>Wash the cells at RT by centrifuging at 300 x g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally resuspending cells with 200 &#181;l of ice cold running buffer, and repeat the washing step one more time.</li>
	<li>Add 95 &#181;l of running buffer followed by 5 &#181;l of 7-aminoactinomycin D (7AAD) to each well for a total volume of 100 &#181;l/well. Incubate the plate in the dark at RT for 15 min.</li>
	<li>Add 100 &#181;l of running buffer to cluster tubes. Transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on flow cytometer.</li>
	<li>Determine the percentage of 7AAD-positive cells using flow cytometry analysis software.</li>
</ol>
5. EC50 Determination
<ol>
	<li>The EC50 values of WS-CM and nicotine are determined by 7AAD-positive staining of PBMCs.</li>
	<li>The EC50 value is defined as the concentration at which 50% of the cells were no longer viable in a 24 hr assay, and the values are expressed as &#956;g of equi-nicotine units/ml.</li>
	<li>For the purposes of this study, the EC50 values were determined to be 1.56 &#956;g/ml and 1,650 &#181;g/ml for WS-CM and nicotine, respectively.</li>
</ol>
6. Secreted Cytokines
NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be adjusted accordingly.
<ol>
	<li>Dilute WS-CM in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well at the concentration of 0.3, 1.56, 3, and 5 &#181;g/ml of equi-nicotine units.</li>
	<li>Dilute nicotine to the following concentrations: 100, 200, 500, 750, 1000, 2000, and 3000 &#181;g/mL. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs suspended in RPMI complete medium to each well at a concentration of 1 x 106 cells/well. The total volume of cells plus WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>Cover the plate and incubate for 3 h at 37 &#176;C and 5% CO2.</li>
	<li>Wash the cells at RT by centrifuging at 300 x g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally suspending cells with 200 &#181;l of ice cold running buffer and repeating the washing step one more time.</li>
	<li>Add 200 &#181;l of RPMI complete medium, and repeat the washing step one more time.</li>
	<li>Add 200 &#181;l of 10 &#181;g/ml LPS medium to each well.</li>
	<li>Cover the plate and incubate for 4 hr, 24 hr, 48 hr, or 72 hr at 37 &#176;C and 5% CO2. Adjust incubation times as necessary.</li>
	<li>Centrifuge the plate at 300 &#215; g for 3 min.</li>
	<li>Take 175 &#181;l of supernatant from each well and store in a freezer at -80 &#176;C to perform the assays in steps 7 and 8.</li>
</ol>
7. Cytometric Bead Array Assay
<ol>
	<li>Thaw the cell supernatants prepared from step 6.10 and use in the CBA assay. Perform the cytometric bead array (CBA) assay as per the manufacturer&#8217;s instructions.</li>
</ol>
8. IL-8 ELISA
<ol>
	<li>Thaw the cell supernatants from step 6.10 and use in the IL-8 ELISA. Perform the ELISA assay as per the manufacturer&#8217;s instructions.</li>
</ol>
9. Intracellular Staining and Flow Cytometry
NOTE: Dilutions listed here are for the purpose of this study. The dilutions can be adjusted accordingly.
<ol>
	<li>Dilute WS-CM in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well at the concentration of 0.3, 1.56, 3, and 5 &#181;g/ml of equi-nicotine units.</li>
	<li>In the same plate, dilute nicotine to the following concentrations: 2, 10, 50, 100, 500, 2000, and 4000 &#181;g/ml. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs suspended in RPMI complete medium at a concentration of 1 &#215; 106 cells/well. The total volume of cells plus WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>Cover the plate and incubate for 3 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the cells at RT by centrifuging at 300 &#215; g for 3 min, aspirating the supernatant, vortexing the bottom of the plate with plate covered and finally resuspending cells with 200 &#181;l of ice cold running buffer, and repeat the washing step one more time.</li>
	<li>Add 200 &#181;l of RPMI complete medium to the plate, and repeat the wash step one more time.</li>
	<li>Prepare the working concentrations of 2 &#181;l/ml GolgiPlug and 10 &#181;g/ml LPS using RPMI complete medium and add 200 &#181;l to each well.</li>
	<li>Incubate the plate for 6 hr at 37 &#176;C and 5% CO2.</li>
	<li>At the end of step 9.8, wash the cells with running buffer (4 &#176;C) and spinning at 300 x g for 3 min at 4 &#176;C.</li>
	<li>Add 100 &#181;l of Cytofix to each well and incubate for 20 min at 4 &#176;C.</li>
	<li>Wash the cells 3 times as described in step 9.9 with 1x Permwash (4 &#176;C) at 300 x g for 3 min at 4 &#176;C.</li>
	<li>Add 45 &#181;L of 1x Cytoperm to each well followed by 5 &#181;l of each of one of the following antibodies to each well: TNF-&#945;-Alexa Fluor 488, IFN-&#947; V500, MIP-1&#945; PE. Incubate at 4 &#176;C for 30 min.</li>
	<li>Wash the cells two times as described in step 9.9 with 1x Permwash (4 &#176;C) and one time with running buffer (4 &#176;C) at 300 x g for 3 min at 4 &#176;C.</li>
	<li>Resuspend the cells with 200 &#181;l of 2% paraformaldehyde (4 &#176;C). Transfer the cells into 12 &#215; 75 mm tubes, and analyze the samples on flow cytometer. CAUTION: Paraformaldehyde is corrosive, acutely toxic and a health hazard.</li>
</ol>
10. K562 Killing Assay
NOTE: K562 cells should be grown in culture at 37 &#176;C and 5% CO2 with RPMI complete medium until they reach 80% confluence before the assay.
<ol>
	<li>Prepare a 5 mM carboxyfluorescein succinimidyl ester (CFSE) stock solution by adding 18 &#181;l of DMSO to the vial.</li>
	<li>Dilute WS-CM or nicotine in a 96-well plate using RPMI complete medium to a total volume of 100 &#181;l/well to achieve the desired equi-nicotine units or nicotine concentrations for each well. CAUTION: Nicotine is acutely toxic and environmentally hazardous.</li>
	<li>Add 100 &#181;l of PBMCs into RPMI complete medium at a concentration of 1.5 x 106 cells/well. The total volume of cells with WS-CM or nicotine will be 200 &#181;l/well.</li>
	<li>Cover the plate and incubate for 3 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the K562 cells by adding 10 ml of DPBS and centrifuge at 400 x g for 8 min at RT.</li>
	<li>Resuspend the cells with 10 ml of DPBS and count the K562 cells.</li>
	<li>Prepare CFSE working solution by adding 1 &#181;l of CFSE stock solution to 1 ml DPBS.</li>
	<li>Add 1 ml of the CFSE working solution to 1 ml of the K562 cell suspension containing 1 - 2 x 107 cells. Vortex and incubate precisely for 2 min at RT.</li>
	<li>Immediately add 200 &#956;l of FBS. Vortex and incubate precisely for 2 min at RT.</li>
	<li>Add 10 ml of RPMI complete medium and centrifuge the tube at 400 x g for 8 min at RT.</li>
	<li>Remove the RPMI supernatant and break the pellet and resuspend the cells with 10 ml of RPMI complete medium and count the CFSE-labeled K562 cells.</li>
	<li>Wash the PBMCs by centrifuging the plate at 300 x g for 3 min at RT. Aspirate the supernatant by decanting the liquid. Replace the cover and vortex the bottom of the plate. Add 200 &#181;l of RPMI complete medium and repeat washing step one more time.</li>
	<li>Add CFSE-labeled K562 cells at a ratio of 1:15 (100,000 K562s:1.5 x 106 PBMCs) to each sample well of the PBMC plate and further incubate for 5 hr at 37 &#176;C and 5% CO2.</li>
	<li>Wash the cell mix by centrifuging at 300 x g for 3 min at RT. Aspirate the supernatant by discarding the liquid. Place the cover and vortex the bottom of the plate.</li>
	<li>Add 200 &#181;l of running buffer and repeat the washing step described in step 10.14 one more time.</li>
	<li>Add 95 &#181;l of running buffer followed by 5 &#181;l of 7AAD to each well for a total volume of 100 &#181;l/well. Incubate the plate in the dark at RT for 15 min.</li>
	<li>Add 100 &#181;l of running buffer to each well and transfer the entire volume of cell suspension from each well of the plate to the cluster tubes and acquire the samples on flow cytometer.</li>
	<li>Determine the percentage of 7AAD-positive and CFSE-positive cells using flow cytometry analysis software.</li>
</ol>

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The authors declare that there are no conflicts of interest.

We and others have previously demonstrated that treatment of PBMCs with TPPs suppresses several responses, including expression and secretion of cytokines and functional measures such as target cell killing14. The experimental methods described in the previous work require longer incubation periods and were modest in magnitude14. Given the potential applications of this attractive ex vivo model for basic and applied research, we investigated whether any of the assay parameters in these mult...

A substantial body of knowledge points to the adverse health effects of chronic cigarette smoking, including cardiovascular disease (CVD), chronic obstructive pulmonary disease (COPD) and cancer1,2. Chronic cigarette smoking has been known to cause inflammation and immune suppression, and these alterations are reported to contribute to increased risk of microbial infection and cancer in smokers3. In vitro and ex vivo techniques are useful in elucidating the molecular basis of the pathophysiological effects of cigarette smoke4-9 (Table 1) and are recognized as important tools for guiding the emerging regulation of various tobacco products10,11.
For example, we have demonstrated that combustible tobacco product preparations (TPPs) such as whole smoke-conditioned medium (WS-CM) and total particulate matter (TPM) are far more cytotoxic and damaging to DNA than non-combustible TPPs or nicotine12,13. Consistent with the published work, it was recently reported that combustible TPPs or nicotine12,13. Consistent with the published work, we recently reported that combustible TPPs caused marked immunosuppression. This was evidenced by suppression of Toll- like receptor (TLR)-ligands, stimulated cytokine secretion, and targeted cell (K562) killing by PBMCs in an ex vivo model14. Given the significance of inflammation in cigarette smoke-induced disease processes, further optimization of the assay conditions to evaluate the immune modulatory effects of cigarette smoke is presented in this report.
The ex vivo assays typically measured intracellular and secreted cytokines as well as the cytolytic function of cytotoxic T and NK cells in K562 cell killing assays14. The assays involved pre-incubation with WS-CM and nicotine and subsequent stimulation of PBMCs with TLR agonists over a period of 3 days; the final readouts are performed using enzyme-linked immunosorbent assays (ELISAs) and/or flow cytometry. We utilized bacterial lipopolysaccharide (LPS), which binds to TLR-4 receptors and stimulates PBMCs resulting in the production of intracellular cytokines and secretion of cytokines. In addition to optimization of the various assay steps for evaluating the immunomodulatory effects of TPPs, we also present methods for isolating PBMCs, cell death assays, and IL-8 quantification. These methods may be applied to address other research questions and further refined to evaluate tobacco products in the regulatory context.
Table 1. Published reports of in vitro and ex vivo methods used to study varilus pathophysiological effects of tobacco product preparations. CS, cigarette smoke medium; CSC, cigarette smoke condensate; CSE, cigarette smoke extract; ELISA, enzyme-linked immunosorbent assay; GADPH, glyceraldehyde 3-phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction; RT, real time quantitative polymerase chain reaction; TS, tobacco smoke.
<table>
	<tbody>
		<tr>
			<td>Author (Year of study)</td>
			<td>Laan et al. (2004)</td>
			<td>Moodie et al. (2004)</td>
			<td>Oltmanns et al. (2005)</td>
			<td>Vayssier (1998)</td>
			<td>Witherden et al. (2004)</td>
			<td>Birrell et al. (2008)</td>
		</tr>
		<tr>
			<td>Cells used</td>
			<td>Human bronchial endothelium cells (BEAS-2B), human neutrophils</td>
			<td>Human alveolar epithelial cells (A549)</td>
			<td>Human airway smooth muscle cells (HASMC)</td>
			<td>Human premonocytic U937 cells, human monocytes</td>
			<td>Alveolar type II epithelial cells (ATII)</td>
			<td>Human monocytic cell line (THP-1), human lung macrophages</td>
		</tr>
		<tr>
			<td>TPP used</td>
			<td>CSE</td>
			<td>CSC</td>
			<td>CSE</td>
			<td>TS</td>
			<td>CSE</td>
			<td>CS</td>
		</tr>
		<tr>
			<td>Method used</td>
			<td>ELISA, qPCR, migration, electromobility shift</td>
			<td>Immunohisto-chemistry, electrophoresis, Arrayscan kit, RT-PCR, ELISA</td>
			<td>ELISA, RT-PCR, qPCR, electrophoresis</td>
			<td>Gel-mobility shift</td>
			<td>Light microscopy, electron microscopy, electrophoresis, ELISA</td>
			<td>qPCR, ELISA, E-toxate kit (Sigma), p65 plate assay (TransAM), electrophoresis, various immunoassay kits</td>
		</tr>
		<tr>
			<td>Measure</td>
			<td>IL-8, GM-CF, AP-1, NF-&#954;B, migration</td>
			<td>Histone acetyltransferases, histone deacetylases, NF-&#954;B, IL-8, p-I &#954;B-&#945;, GADPH</td>
			<td>HO-1, GADPH, RANTES, IL-8, eotaxin</td>
			<td>Heat shock/stress proteins (HSP/Hsp70), HF transcription factor, NF-&#954;B, TNF-&#945;</td>
			<td>Surfactant protein (SP-A, SP-C), IL-8, MCP-1, GRO-&#945;, TNF-&#945;, IL-1&#946;, IFN-&#947;</td>
			<td>IL-8, IL-1&#946;, IL-6, TNF-&#945;, MIP1-&#945;, GRO-&#945;, MAPK/JNK/ERK phosphorylation, cJUN:DNA binding, glutathione, p65:DNA binding</td>
		</tr>
		<tr>
			<td>End result</td>
			<td>CSE down-regulates cytokine production via suppression of AP-1 activation.</td>
			<td>H2O2 and CSC enhance acetylation of histone proteins, decrease histone deacetylase activity, differentially regulate proinflammatory cytokine release.</td>
			<td>Cigarette smoke may cause the release of IL-8 from HASMC, enhanced by TNF-&#945;, 20% CSE less IL-8 release, Inhibition of eotaxin and RANTES by cigarette smoke.</td>
			<td>TS activated HF transcription factor, which was associated with Hsp70 overexpression and inhibition of NFkB binding activity and TNF-&#945; release.</td>
			<td>Reduced ATII cell-derived chemokine levels compromise alveolar repair, contributing to cigarette smoke-induced alveolar damage and emphysema.</td>
			<td>Data provide mechanistic explanation for why smokers have increased respiratory infections. Suppression of the innate response is accompanied by an increase in IL-8.</td>
		</tr>
	</tbody>
</table>

<table border="0" cellpadding="0" cellspacing="0" width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:223px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">12 X 75 tubes</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">2 mL Microtubes</td>
			<td style="width:148px;">Axygen</td>
			<td style="width:119px;">MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">3R4F reference cigarettes&#160;&#160;</td>
			<td style="width:148px;">Univ. of Kentucky, College of Agriculture</td>
			<td style="width:119px;">3R4F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">50 ml conical tubes</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430828</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">500 ml bottle</td>
			<td style="width:148px;">Corning</td>
			<td style="width:119px;">430282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">7AAD</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">559925</td>
			<td style="width:223px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well flat bottom plate</td>
			<td>Termo Nunc</td>
			<td>439454</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">96 well round bottom plates</td>
			<td style="width:148px;">BD Falcon</td>
			<td style="width:119px;">353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell culture hood</td>
			<td style="width:148px;">Thermo Scientific</td>
			<td style="width:119px;">1300 Series A2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Centrifuge</td>
			<td style="width:148px;">Eppendorf</td>
			<td style="width:119px;">58110R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">CFSE</td>
			<td style="width:148px;">Molecular Probes Life Technologies</td>
			<td style="width:119px;">C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cluster tubes</td>
			<td style="width:148px;">Corning</td>
			<td>4401</td>
			<td>Harmful if swallowed, carcinogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cytofix/Cytoperm (Permwash)</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555028</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DMSO (Dimethyl sulfoxide )</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">DPBS</td>
			<td style="width:148px;">Lonza</td>
			<td style="width:119px;">17-512F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FBS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">F2442</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">FCAP Array</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">652099</td>
			<td>Software analyzes CBA data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Filter unit</td>
			<td style="width:148px;">Nalgene</td>
			<td style="width:119px;">156-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Canto II</td>
			<td>&#160;8 colors, at Ex 405 and Em785.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Flow Cytometer</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">FACS Calibur</td>
			<td>&#160;4 colors at Ex 495 and Em 785.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Flow cytometry analysis software</td>
			<td style="width:148px;">Tree Star</td>
			<td style="width:119px;">FlowJo</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Freezing Container</td>
			<td style="width:148px;">Nalgene</td>
			<td>5100-0001</td>
			<td>Contains DMSO,&#160; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">GogliPlug</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">555029</td>
			<td>Carcinogen, Irritant, Corrosive&#160;</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:223px;">H2SO4</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">339741</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Human Inflammatory Cytokine Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IFN-&#947; V-500 Antibody</td>
			<td style="width:148px;">BD Horizon</td>
			<td style="width:119px;">561980</td>
			<td>&#160;skin sensitizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">IL-8 ELISA Kit</td>
			<td style="width:148px;">R and D Systems</td>
			<td style="width:119px;">DY208</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Isolation Buffer</td>
			<td style="width:148px;">Isolymph, CTL Scientific Corp.</td>
			<td style="width:119px;">1114868</td>
			<td>Flammable liquid, Irritant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Isopropyl alcohol</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">L-Glutamine</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">LPS</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td>L2630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">MIP1-&#945; PE Antibody</td>
			<td style="width:148px;">BD Pharmingen</td>
			<td style="width:119px;">554730</td>
			<td>Acute toxicity, Oral</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Monensin</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">M5273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">NaCl</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotine</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">N3876</td>
			<td>Acute toxicity, Environmental hazard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Parafilm</td>
			<td style="width:148px;">Bemis</td>
			<td style="width:119px;">&#8220;M&#8221;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Paraformaldehyde</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>Flammable, Skin irritation&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">Pen/strep</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">RPMI 1640</td>
			<td style="width:148px;">Gibco Life Technologies</td>
			<td style="width:119px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:223px;">Running buffer</td>
			<td style="width:148px;">MACS Running Buffer, Miltenyi Biotech</td>
			<td style="width:119px;">130-091-221</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Th1/Th2 CBA Kit</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:119px;">551809</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:223px;">TNF-&#945; Alexa Fluor 488 Antibody</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:119px;">502915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Transfer pipette</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Tris Base</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to evaluate cytotoxicity and immunosuppression of combustible tobacco product preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

The results were presented as mean &#177; standard error of the mean (four donor samples). The student&#8217;s t-test between treated and untreated control samples was performed using Excel software as well as t-test comparisons for all treatments with their corresponding controls. The statistical significance was indicated by: *, P &#60; 0.05; **, P &#60; 0.005; ***, P &#60; 0.0005.
To measure the effect of exposure to WS-CM and nicotine, PBMCs were treated with different concentrations of WS...

Watch this Scientific Journal Video about Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations at JoVE.com

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

methods-to-evaluate-cytotoxicity-immunosuppression-combustible

Research

JoVE Journal

Immunology and Infection

11.3K Views.  Wake Forest University Health Sciences. Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure. 

Video: Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...