Name: dynamic adhesion assay for the functional analysis of anti-adhesion therapies in inflammatory bowel disease
Uploaded: 2018-09-20T13:00:00
Description: Watch this Scientific Journal Video about Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease at JoVE.com

The research of CN, IA, MFN and SZ was supported by the Interdisciplinary Center for Clinical Research (IZKF) and the ELAN program of the University Erlangen-Nuremberg, the Else Kr&#246;ner-Fresenius-Stiftung, the Fritz-Bender-Stiftung, the German Crohn&#39;s and Colitis Foundation (DCCV), the Clinical Research Group CEDER of the German Research Council (DFG), the DFG topic program on Microbiota, the Emerging Field Initiative and the DFG Collaborative Research Centers 643, 796 and 1181.

The studies described in the following sections have been performed according to approval of the Ethics committee of the Friedrich-Alexander University Erlangen-Nuremberg.
1. Preparation of Capillaries
<ol>
	<li>Connect rectangular capillaries to the rubber tubing by stretching the tubing on one side with small scissors and carefully inserting the capillary (approximately 0.5 cm) into the tube. Seal the connection between capillary and tube with plastic paraffin film.</li>
	<li>Coat the capi...

<ol>
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C., Bokemeyer, B., Drabik, A., Stallmach, A., Schreiber, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vedolizumab+Germany+Consortium+Vedolizumab+induction+therapy+for+inflammatory+bowel+disease+in+clinical+practice--a+nationwide+consecutive+German+cohort+study.">Vedolizumab Germany Consortium Vedolizumab induction therapy for inflammatory bowel disease in clinical practice--a nationwide consecutive German cohort study.</a> Alimentary Pharmacology &amp; Therapeutics. 43 (10), 1090-1102 (2016).</li><li>Kopylov, U., 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=Efficacy+and+Safety+of+Vedolizumab+for+Induction+of+Remission+in+Inflammatory+Bowel+Disease-the+Israeli+Real-World+Experience.">Efficacy and Safety of Vedolizumab for Induction of Remission in Inflammatory Bowel Disease-the Israeli Real-World Experience.</a> Inflammatory Bowel Diseases. 23 (3), 404-408 (2017).</li><li>Amiot, A., 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=Effectiveness+and+Safety+of+Vedolizumab+Induction+Therapy+for+Patients+With+Inflammatory+Bowel+Disease.">Effectiveness and Safety of Vedolizumab Induction Therapy for Patients With Inflammatory Bowel Disease.</a> Clinical Gastroenterology and Hepatology: The Official Clinical Practice Journal of the American Gastroenterological Association. 14 (11), 1593-1601 (2016).</li><li>Vermeire, 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=Etrolizumab+as+induction+therapy+for+ulcerative+colitis:+a+randomised,+controlled,+phase+2+trial.">Etrolizumab as induction therapy for ulcerative colitis: a randomised, controlled, phase 2 trial.</a> Lancet. 384 (9940), 309-318 (2014).</li><li>Vermeire, 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=Anti-MAdCAM+antibody+(PF-00547659)+for+ulcerative+colitis+(TURANDOT):+a+phase+2+randomised,+double-blind,+placebo-controlled+trial.">Anti-MAdCAM antibody (PF-00547659) for ulcerative colitis (TURANDOT): a phase 2 randomised, double-blind, placebo-controlled trial.</a> Lancet. 390 (10090), 135-144 (2017).</li><li>Miller, D. H., 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=A+Controlled+Trial+of+Natalizumab+for+Relapsing+Multiple+Sclerosis.">A Controlled Trial of Natalizumab for Relapsing Multiple Sclerosis.</a> New England Journal of Medicine. 348 (1), 15-23 (2003).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nature Reviews. Immunology. 7 (9), 678-689 (2007).</li><li>Ley, K., Rivera-Nieves, J., Sandborn, W. 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Drug Discovery. 15 (3), 173-183 (2016).</li><li>Wyant, T., Yang, L., Fedyk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+assessment+of+the+effects+of+vedolizumab+binding+on+peripheral+blood+lymphocytes.">In vitro assessment of the effects of vedolizumab binding on peripheral blood lymphocytes.</a> mAbs. 5 (6), 842-850 (2013).</li><li>Fischer, A., 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=Differential+effects+of+&#945;4&#946;7+and+GPR15+on+homing+of+effector+and+regulatory+T+cells+from+patients+with+UC+to+the+inflamed+gut+in+vivo.">Differential effects of &#945;4&#946;7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo.</a> Gut. 65 (10), 1642-1664 (2016).</li><li>Wyant, T., Estevam, J., Yang, L., Rosario, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+receptor+occupancy+pharmacodynamic+assays+used+in+the+clinical+development+of+the+monoclonal+antibody+vedolizumab.">Development and validation of receptor occupancy pharmacodynamic assays used in the clinical development of the monoclonal antibody vedolizumab.</a> Cytometry. Part B, Clinical Cytometry. 90 (2), 168-176 (2016).</li><li>Tidswell, M., 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=Structure-function+analysis+of+the+integrin+beta+7+subunit:+identification+of+domains+involved+in+adhesion+to+MAdCAM-1.">Structure-function analysis of the integrin beta 7 subunit: identification of domains involved in adhesion to MAdCAM-1.</a> Journal of Immunology. 159 (3), 1497-1505 (1997).</li><li>Soler, D., Chapman, T., Yang, L. -. L., Wyant, T., Egan, R., Fedyk, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Binding+Specificity+and+Selective+Antagonism+of+Vedolizumab,+an+Anti-&#945;4&#946;7+Integrin+Therapeutic+Antibody+in+Development+for+Inflammatory+Bowel+Diseases.">The Binding Specificity and Selective Antagonism of Vedolizumab, an Anti-&#945;4&#946;7 Integrin Therapeutic Antibody in Development for Inflammatory Bowel Diseases.</a> Journal of Pharmacology and Experimental Therapeutics. 330 (3), 864-875 (2009).</li><li>Parikh, A., 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=Vedolizumab+for+the+treatment+of+active+ulcerative+colitis:+a+randomized+controlled+phase+2+dose-ranging+study.">Vedolizumab for the treatment of active ulcerative colitis: a randomized controlled phase 2 dose-ranging study.</a> Inflammatory Bowel Diseases. 18 (8), 1470-1479 (2012).</li><li>Wendt, E., White, G. E., Ferry, H., Huhn, M., Greaves, D. R., Keshav, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocorticoids+Suppress+CCR9-Mediated+Chemotaxis,+Calcium+Flux,+and+Adhesion+to+MAdCAM-1+in+Human+T+Cells.">Glucocorticoids Suppress CCR9-Mediated Chemotaxis, Calcium Flux, and Adhesion to MAdCAM-1 in Human T Cells.</a> The Journal of Immunology. 196 (9), 3910-3919 (2016).</li><li>Nussbaum, C., 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=Sphingosine-1-phosphate+receptor+3+promotes+leukocyte+rolling+by+mobilizing+endothelial+P-selectin.">Sphingosine-1-phosphate receptor 3 promotes leukocyte rolling by mobilizing endothelial P-selectin.</a> Nature Communications. 6, (2015).</li><li>Pruenster, M., 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=Extracellular+MRP8/14+is+a+regulator+of+&#946;2+integrin-dependent+neutrophil+slow+rolling+and+adhesion.">Extracellular MRP8/14 is a regulator of &#946;2 integrin-dependent neutrophil slow rolling and adhesion.</a> Nature Communications. 6, (2015).</li><li>Binder, M. -. T., 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=Similar+Inhibition+of+Dynamic+Adhesion+of+Lymphocytes+From+IBD+Patients+to+MAdCAM-1+by+Vedolizumab+and+Etrolizumab-s.">Similar Inhibition of Dynamic Adhesion of Lymphocytes From IBD Patients to MAdCAM-1 by Vedolizumab and Etrolizumab-s.</a> Inflammatory Bowel Diseases. 24 (6), 1237-1250 (2018).</li><li>Wang, 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=Vessel+Sampling+and+Blood+Flow+Velocity+Distribution+With+Vessel+Diameter+for+Characterizing+the+Human+Bulbar+Conjunctival+Microvasculature.">Vessel Sampling and Blood Flow Velocity Distribution With Vessel Diameter for Characterizing the Human Bulbar Conjunctival Microvasculature.</a> Eye &amp; Contact Lens. 42 (2), 135-140 (2016).</li><li>House, S. D., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diameter+and+blood+flow+of+skeletal+muscle+venules+during+local+flow+regulation.">Diameter and blood flow of skeletal muscle venules during local flow regulation.</a> The American Journal of Physiology. 250 (5 Pt 2), H828-H837 (1986).</li><li>Zundler, S., Neurath, M. 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The above protocol describes a useful technique to study dynamic adhesion of human immune cells to endothelial ligands. Through variation of the coated ligands, the perfused cell types or subsets, incubation with additional stimuli or different neutralizing antibodies, it has almost unlimited potential applications. Therefore, such dynamic adhesion assays may be useful to answer both fundamental questions of basic research as well as translational queries that might help to develop and optimize clinical therapy with drug...

Cell motion is a tightly regulated process indispensable for the development and function of multi-cellular organisms, but is also implicated in the pathogenesis of a multitude of diseases1. Recently, the homing process of immune cells from the blood stream to the peripheral tissues has gained increasing attention, since it contributes to replenishment and expansion of pathogenic cells in inflamed tissues in immunologically mediated diseases2,3. In particular, homing has been shown to have translational relevance in inflammatory bowel diseases (IBD). The therapeutic anti-&#945;4&#946;7 integrin antibody vedolizumab interfering with gut homing has shown efficacy in large clinical trials4,5 and has been successfully used in real-world clinical practice6,7,8. Further compounds are likely to follow9,10. Similarly, the therapeutic anti-&#945;4 integrin antibody, natalizumab, is used for the treatment of multiple sclerosis (MS)11.
However, our functional understanding of the homing process in general and the mechanism of action of such therapeutic antibodies in particular is still limited. It is well established that homing consists of several steps including cell tethering and rolling with subsequent cell adhesion leading to firm arrest followed by trans endothelial migration12,13. The above-mentioned antibodies neutralize integrins on the cell surface preventing interaction with addressins on the endothelium of the vessel wall. This is thought to impede firm cell adhesion14,15. Yet, we are only beginning to understand the differential relevance of specific integrins for cell homing of distinct cell subsets. Moreover, the effects of anti-integrin antibodies on different cell subsets and dose-response associations are largely unknown leading to lots of open questions in the field of gut homing and anti-adhesion therapies in IBD.
Therefore, convenient tools to address such questions are desperately needed. The effect of anti-integrin antibodies on integrin-addressin interaction has so far predominantly been evaluated by assessing binding efficacy/binding inhibition with flow cytometry or through static adhesion assays16,17,18,19,20, thus with apparent simplification and deviation from the physiological situation. We recently established a dynamic adhesion assay to study integrin-dependent adhesion of human cells to addressins and the effects of anti-integrin antibodies under shear stress2. The principle of the technique has earlier been demonstrated with mouse cells21,22. Here, it was adapted and developed to address the above mentioned translational questions, opening novel avenues to better understand the mechanisms of therapy with anti-integrin antibodies in vivo.

<table>
	<tbody>
		<tr>
			<td>48-Well plate</td>
			<td>Sarstedt</td>
			<td>833,923</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesion buffer: 150mM NaCl + 1mM HEPES + 1mM MgCl2 + 1mM CaCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution: 1x PBS in ddH2O + 5 % BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum albumin (BSA)</td>
			<td>Applichem</td>
			<td>A1391,0100</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck</td>
			<td>2382</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillaries: Rectangle Boro Tubing 0,20x2.00 mm ID, 50 mm length</td>
			<td>CM Scientific</td>
			<td>3520-050</td>
			<td></td>
		</tr>
		<tr>
			<td>CCL-2, human</td>
			<td>Immunotools</td>
			<td>11343384</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-Microbeads, human</td>
			<td>Miltenyi Biotec</td>
			<td>130-045-101</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrace&#8482; CFSE Cell Proliferation Kit</td>
			<td>ThermoFischer Scientific</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (Rotixa 50 RS)</td>
			<td>Hettrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Coating buffer: 150 mM NaCl + 1 mM HEPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope (TCS SP8)</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CXCL-10, human</td>
			<td>Immunotools</td>
			<td>11343884</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran 500</td>
			<td>Roth</td>
			<td>9219.3</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA KE/9 ml Monovette</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcons (50 mL)</td>
			<td>Sarstedt</td>
			<td>62,547,004</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc chimera isotype control</td>
			<td>R&#38;D Systems</td>
			<td>110-HG</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Rates Peristaltic Pump (LabV1)</td>
			<td>Baoding Shenchen Precision Pump Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>J848-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IgG Isotype Control</td>
			<td>ThermoFischer Scientific</td>
			<td>31154</td>
			<td></td>
		</tr>
		<tr>
			<td>Intercellular Adhesion Molecule 1 (ICAM-1) Fc chimera</td>
			<td>R&#38;D Systems</td>
			<td>720-IC-050</td>
			<td></td>
		</tr>
		<tr>
			<td>LS-Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Roth</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mouse IgG isotype control</td>
			<td>Miltenyi Biotec</td>
			<td>130-106-545</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucosal Vascular Addressin Cell Adhesion Molecule 1 (MAdCAM-1) Fc chimera</td>
			<td>R&#38;D Systems</td>
			<td>6056-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Roth</td>
			<td>3957.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Natalizumab</td>
			<td>Biogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer Counting chamber</td>
			<td>Roth</td>
			<td>T729.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pancoll, human</td>
			<td>PAN Biotech</td>
			<td>P04-601000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)&#160;</td>
			<td>Biochrom</td>
			<td>L 182-10</td>
			<td>w/o Mg and Ca</td>
		</tr>
		<tr>
			<td>Plastic paraffin film: Parafilm (PM-996)</td>
			<td>VWR</td>
			<td>52858-000</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-human CD18</td>
			<td>Biolegend</td>
			<td>302102</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Gibco Life Technologies</td>
			<td>61870-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber tubing: SC0059T 3-Stop LMT-55 Tubing, 1.02mm ID, 406.4 mm length</td>
			<td>Ismatec</td>
			<td>SC0059</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipetts</td>
			<td>Sarstedt</td>
			<td>861,254,025</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Roth</td>
			<td>CN76.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular Cell Adhesion Molecule 1 (VCAM-1) Fc chimera</td>
			<td>Biolegend</td>
			<td>553706</td>
			<td></td>
		</tr>
		<tr>
			<td>Vedolizumab (Entyvio)</td>
			<td>Takeda</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
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dynamic adhesion assay for the functional analysis of anti-adhesion therapies in inflammatory bowel disease

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a crucial step in this process and therapeutic strategies interfering with adhesion have been successfully established. The anti-&#945;4&#946;7 integrin antibody, vedolizumab, is used for the clinical treatment of Crohn&#39;s disease (CD) and ulcerative colitis (UC) and further compounds are likely to follow.
The details of the adhesion procedure and the action mechanisms of anti-integrin antibodies are still unclear in many regards due to the limited available techniques for the functional research in this field.
Here, we present a dynamic adhesion assay for the functional analysis of human cell adhesion under flow conditions and the impact of anti-integrin therapies in the context of IBD. It is based on the perfusion of primary human cells through addressin-coated ultrathin glass capillaries with real-time microscopic analysis. The assay offers a variety of opportunities for refinements and modifications and holds potentials for mechanistic discoveries and translational applications.

The method presented in this manuscript aims to simulate the in vivo process of human cell adhesion to the endothelial wall as closely as possible to functionally assess cell adhesion and the role of interfering antibodies. Therefore, ultrathin capillaries are coated with addressins and perfused with fluorescently labeled human cells of interest using a perfusion pump. Using live cell imaging the adhesion of human cells to the addressins can be observed in real time (<strong clas...

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Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease

Dynamic adhesion of immune cells to the vessel wall is a prerequisite for gut homing. Here, we present a protocol for a functional in vitro assay for the impact analysis of anti-integrin antibodies, chemokines or other factors on the dynamic cell adhesion of human cells using addressin-coated capillaries.

Gut homing of immune cells is important for the pathogenesis of inflammatory bowel diseases (IBD). Integrin-dependent cell adhesion to addressins is a ...

This method can help answering key questions of basic immunobiology and inflammatory bowel disease research regarding the adhesion cascade of immune cells required for homing to peripheral organs. The main advantage of this technique is that it is a convenient and straightforward approach for answering functional questions in the context of cell adhesion under flow conditions. Although this method was developed for exploring the mechanism of cell poietic anti-integrin antibodies in inflammatory bowel diseases, it can be modified to analyze similar issues in other inflammatory disease settings.Begin by stretching a piece of rubber tubing on one side with small scissors to allow the insertion of a rectangular capillary approximately 0.5 centimeters into the tube. Then seal the connection with paraffin film. Coat the capillary with 20 microliters of the addressin of interest and seal the side not connected to the tubing with plastic paraffin film for at least one hour incubation at 37 degrees Celsius.At the end of the incubation, remove the plastic paraffin film and let the liquid soak out of the capillary onto a paper towel to empty the tubing. Then block capillary with 20 microliters of blocking solution for at least one hour at 37 degrees Celsius. It is very important to apply the blocking to the capillary directly after removing the coating to avoid drying up of the capillary as this may impair the functionality of the coded proteins.While the capillary is being blocked, add 18 milliliters of anticoagulated whole human blood from a 50 milliliter tube and dilute the blood to a total volume of 35 milliliters with PBS. Layer 10 milliliters of density gradient medium under the blood solution and isolate the peripheral blood mononuclear blood cells by density gradient centrifugation. At the end of the separation, transfer the peripheral blood mononuclear cells from the interface of the density gradient medium to a new 50 milliliter tube.And bring the total volume in the tube up to 50 milliliters with fresh PBS. After centrifugation, resuspend the pellet in 10 milliliters of fresh PBS for counting and stain the cells with a suitable cell tracking dye according to the manufacturer's instructions. At the end of the staining incubation, collect the cells by centrifugation and resuspend the pellet at a 1.5 times 10 to the six cells per milliliter of complete RPMI medium concentration.Then seed one milliliter of cells into as many wells of a 48 well plate as there are conditions. And add the appropriate volume of anti-integrin antibody to the appropriate wells for a one hour incubation at 37 degrees Celsius. Next use a P1000 pipette to resuspend the cells several times before transferring the cells into individual two milliliter tubes.Rinse each well with one milliliter of PBS and pool the washes in the appropriate two milliliter tubes. After counting, pellet the cells by centrifugation and resuspened the cells in the appropriate volume of adhesion buffer to obtain 1.5 times ten to the sixth cells per milliliter suspensions. Then add the appropriate volume of manganese chloride to each tube to a one millimolar final concentration.When the cells are ready, turn on the microscope and select the appropriate filters and lasers, objective and acquisition mode. Remove the plastic paraffin film from the tip of the capillary and use scissors to stretch a five centimeter piece of rubber tubing to allow the capillary to be connected to the smaller tube. Seal the connection between the capillary and the tube with plastic paraffin film.And mount the capillary on a fixation tray. Place the tray on the tray holder of the microscope so the capillary is in focus. And insert the rubber tubing into the corresponding notch of the peristaltic pump.Place the unattached end of the tubing into the first sample tube containing the cell suspension. And insert the short tube on the other side of the capillary into a 15 milliliter tube to collect flow through. Let the tubing fill at a 500 microliters per minute flow rate until the cell suspension almost reaches the capillary.Then let the capillary fill at a flow rate of 100 microliters per minute. When the capillary is filled with cells, adjust flow rate to 10 microliters per minute and capture time lapse images of the cells moving slowly through the capillary along the inside of the addressin coated wall every two seconds for a total of three minutes. Once the cell suspension reaches the inside of the capillary, it is critical to avoid any interruption of the flow as this will lead to static integrin addressin interaction potentially biasing the measurement.Before discarding stream the whole capillary through the eyepiece of the microscope to make sure that the section observed on the screen is representative. Free the tubing from the pump and let the remaining fluid run back into the two milliliter tube. Then disconnect the capillary and the tubing from the fixation tray and image the next capillary as just demonstrated.To evaluate the time lapse recordings, open the first sequence in the appropriate analysis software and export the first and last three images as TIFF files. Open the three beginning images in ImageJ and select image, type, and 32-bit to convert the images to 32-bit. Then select image, color and merge channels to merge the images into one image and convert the composite image to RGB for saving as a TIFF file.After converting the last three images in the same way, count the white cells visible in the beginning and ending composite images. And subtract the number of white cells in the beginning image from the number of white cells in the ending image. The perfusion of capillaries coated with ICAM-1, VCAM-1, MAdCAM-1, but not Isotype Fc chimera with human PBMCs leads to marked adhesion when one of the three addressins is present.In contrast, the inhibition of integrin addressin interactions using neutralizing antibodies typically leads to a marked decrease of dynamic adhesion that is absent when isotype control antibodies are added. Further, treatment of CD4 human T cells with specific chemokines leads to an increased adhesion to addressins compared to untreated human cells. While attempting this procedure, it is very important to remember that various factors contribute to dynamic cell adhesion in vivo that cannot completely be recapitulated in this assay.However, it is possible to adjust the assay to address very complex questions. In order to address additional questions on the differential adhesion behavior of testing cell types, it is also possible to perform competitive adhesion assays with different cell types labeled with different cell tracking dyes. After its development, this technique enabled us to explore the effects of the different anti-integrin antibodies used or currently in development for the treatment of inflammatory bowel diseases.

dynamic-adhesion-assay-for-functional-analysis-anti-adhesion

Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

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 ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.