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

CN、IA、最惠国和深圳的研究是由临床研究交叉学科中心 (IZKF) 和大学埃尔兰根-纽伦堡, 其他 Kröner 费森尤斯基金会, 弗里茨弯机基金会, 德国克罗恩和结肠炎基金会 (DCCV), 德国研究理事会的临床研究小组 CEDER (DFG), DFG 专题计划在微生物组, 新兴的领域主动性和 DFG 合作研究中心 643, 796 和1181。

以下各节所述的研究是根据埃尔兰根-亚历山大大学的道德委员会的批准进行的。
1. 毛细血管的制备
<ol><li>将矩形毛细血管连接到橡胶管材上, 用小剪刀将油管一侧拉伸, 并小心地将毛细管 (大约0.5 厘米) 插入管内。用塑料石蜡膜密封毛细管与管的连接。</li>
	<li>毛细管与20µL 的 addressin (重组 Fc 嵌合体; 5 µg/毫升 addressin,如MAdCAM-1, 在涂层缓冲器 (150 毫米氯化钠 + 1 毫米 HEP...

<ol>
	<li>von Andrian, U. H., Mackay, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Cell+Function+and+Migration+-+Two+Sides+of+the+Same+Coin.">T-Cell Function and Migration - Two Sides of the Same Coin.</a> New England Journal of Medicine. 343 (14), 1020-1034 (2000).</li><li>Zundler, 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=The+&#945;4&#946;1+Homing+Pathway+Is+Essential+for+Ileal+Homing+of+Crohn's+Disease+Effector+T+Cells+In+Vivo.">The &#945;4&#946;1 Homing Pathway Is Essential for Ileal Homing of Crohn's Disease Effector T Cells In Vivo.</a> Inflammatory Bowel Diseases. 23 (3), 379-391 (2017).</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. , (2018).</li><li>Feagan, B. G., 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+as+induction+and+maintenance+therapy+for+ulcerative+colitis.">Vedolizumab as induction and maintenance therapy for ulcerative colitis.</a> The New England Journal of Medicine. 369 (8), 699-710 (2013).</li><li>Sandborn, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vedolizumab+as+induction+and+maintenance+therapy+for+Crohn's+disease.">Vedolizumab as induction and maintenance therapy for Crohn's disease.</a> The New England Journal of Medicine. 369 (8), 711-721 (2013).</li><li>Baumgart, D. 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. J., Shattil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-based+therapeutics:+biological+basis,+clinical+use+and+new+drugs.">Integrin-based therapeutics: biological basis, clinical use and new drugs.</a> Nature Reviews. 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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenic+T+cell+subsets+in+allergic+and+chronic+inflammatory+bowel+disorders.">Pathogenic T cell subsets in allergic and chronic inflammatory bowel disorders.</a> Immunological Reviews. 278 (1), 263-276 (2017).</li><li>Zundler, S., Becker, E., Weidinger, C., Siegmund, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-Adhesion+Therapies+in+Inflammatory+Bowel+Disease-Molecular+and+Clinical+Aspects.">Anti-Adhesion Therapies in Inflammatory Bowel Disease-Molecular and Clinical Aspects.</a> Frontiers in Immunology. 8, (2017).</li><li>Zhou, Y., Kucik, D. F., Szalai, A. J., Edberg, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Neutrophil+Flow+Chamber+Adhesion+Assay.">Human Neutrophil Flow Chamber Adhesion Assay.</a> Journal of Visualized Experiments: JoVE. (89), (2014).</li><li>Zundler, 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=Blockade+of+&#945;E&#946;7+integrin+suppresses+accumulation+of+CD8(+)+and+Th9+lymphocytes+from+patients+with+IBD+in+the+inflamed+gut+in+vivo.">Blockade of &#945;E&#946;7 integrin suppresses accumulation of CD8(+) and Th9 lymphocytes from patients with IBD in the inflamed gut in vivo.</a> Gut. 66 (11), 1936-1948 (2017).</li></ol>

上述协议描述了一种有效的方法来研究人体免疫细胞对内皮配体的动态黏附力。通过涂层配体的变化, 灌注细胞类型或亚群, 孵化与附加刺激或不同中和抗体, 它有几乎无限的潜在应用。因此, 这种动态黏附力的检测可能有助于回答基本研究的基本问题, 以及翻译查询, 可以帮助开发和优化临床治疗与药物干扰粘连的过程。
这项试验的效用, 以调查临床抗粘连治疗的效果最近已经...

细胞运动是对多细胞生物体的发展和功能必不可少的严格调控的过程, 但也牵涉到多种疾病的发病机制1。近年来, 从血流到外周组织的免疫细胞的归巢过程越来越受到重视, 因为它有助于免疫介导疾病炎症组织中致病细胞的补充和扩张2 ,3。特别是, 在炎症性肠病 (IBD) 中, 归巢已被证明具有平移相关性。治疗 anti-α4β7整合素抗体 vedolizumab 干预肠道归巢已显示疗效的大型临床试验4,5 , 已成功地用于实际临床实践6,7,8. 进一步的化合物可能会跟随9,10。同样, 治疗 anti-α4整合素抗体, natalizumab, 用于治疗多发性硬化症 (MS)11。
然而, 我们对整个归巢过程的功能理解, 特别是这种治疗抗体的作用机制仍然是有限的。这是很好的确定, 归巢包括几个步骤, 包括细胞的结合和滚动的后续细胞黏附导致坚定的逮捕后, 反式内皮移植12,13。上述抗体中和整合在细胞表面防止与 addressins 的相互作用的血管壁内皮。这被认为阻碍牢固的细胞黏附力14,15。然而, 我们只是开始了解特定整合对细胞归一化的差异相关性。此外, 抗整合素抗体对不同细胞亚群和剂量反应关联的影响在很大程度上是未知的, 导致肠内归巢和抗粘连治疗在 IBD 领域的许多开放性问题。
因此, 迫切需要解决这些问题的方便工具。抗整合素抗体对整合蛋白-addressin 相互作用的影响迄今主要通过评估结合效能/束缚抑制与流式细胞术或通过静态黏附力测定16,17 ,18,19,20, 因而以明显的简单化和偏离从生理情况。我们最近建立了动态粘附试验研究整合素依赖性的人体细胞对 addressins 和抗整合酶抗体在剪切应力下的作用2。该技术的原理已被证明与小鼠细胞21,22。在这里, 它的适应和发展, 以解决上述的翻译问题, 开辟了新的途径, 以更好地了解治疗机制的抗整合素抗体在体内。

<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>
</table>

dynamic adhesion assay for the functional analysis of anti-adhesion therapies in inflammatory bowel disease

免疫细胞的肠道归巢对炎症性肠病 (IBD) 发病有重要意义。整合素依赖细胞黏附 addressins 是这一过程中的关键步骤, 并成功地建立了干扰粘连的治疗策略。anti-α4β7整合素抗体, vedolizumab, 用于临床治疗克罗恩病 (CD) 和溃疡性结肠炎 (UC) 和进一步的化合物可能会跟随。
由于该领域功能研究的有限可用技术, 抗整合素抗体的黏附程序和作用机制的细节在许多方面仍然不清楚。
在此, 我们提出了动态粘附试验的功能分析的人细胞黏附在流动条件下的影响, 抗整合素疗法的背景下, IBD。它的基础上, 通过 addressin 涂层超薄玻璃毛细血管灌注的原发性人类细胞和实时显微分析。该检测提供了各种改进和修改的机会, 并持有机械发现和转化应用的潜力。

本论文提出的方法旨在模拟人体细胞粘附到内皮壁的体内过程, 尽可能地对细胞黏附力和干扰抗体的作用进行功能性评估。因此, 超薄毛细血管涂上 addressins, 用灌注泵将荧光标记的人类细胞灌注。使用活细胞成像可以实时观察人体细胞与 addressins 的黏附力 (图 1)。
该方法特别适用于炎症性肠病的抗?...

Watch this Scientific Journal Video about Dynamic Adhesion Assay for the Functional Analysis of Anti-adhesion Therapies in Inflammatory Bowel Disease at JoVE.com

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

动态粘附法在炎症性肠病抗粘连治疗功能分析中的作用

免疫细胞对血管壁的动态黏附是肠道归巢的前提。在这里, 我们提出了一个功能性的体外检测的协议, 以影响分析抗整合素抗体, 趋化因子或其他因素的细胞动态细胞黏附的人体细胞使用 addressin 涂层毛细血管。

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

这种方法可以帮助回答基本免疫生物学和炎症性肠病研究有关免疫细胞的粘附级联，需要到周围器官的关键问题。该技术的主要优点是，它是在流条件下细胞粘附的上下文中回答功能问题的一种方便而直接的方法。虽然该方法是为探索炎症性肠病中细胞细胞性抗蛋白抗体的机理而开发的，但可以对其进行修改，以分析其他炎症性疾病环境中的类似问题。首先用小剪刀将一块橡胶管一侧拉伸，以便将长方形毛细管插入约 0.5 厘米的管子中。然后用石蜡膜密封连接。用20微升的息片覆盖毛细管，用塑料石蜡薄膜密封未连接到管子的一侧，在37摄氏度下至少孵育一小时。在孵化结束时，取出塑料石蜡膜，让液体从毛细管中浸泡到纸巾上，以清空管子。然后在37摄氏度下用20微升的阻滞溶液阻断毛细管至少一小时。去除涂层后，直接对毛细管施加阻尼非常重要，以避免毛细管干燥，因为这可能损害编码蛋白的功能。当毛细管被阻塞时，从50毫升管中加入18毫升的抗凝全身血液，用PBS稀释血液到总体积为35毫升。在血液溶液下10毫升密度梯度介质，通过密度梯度离心分离外周血单核血细胞。在分离结束时，将外周血单核细胞从密度梯度介质的界面转移到新的50毫升管。使用新鲜的 PBS 使管子中的总体积达到 50 毫升。离心后，将颗粒重新在 10 毫升新鲜 PBS 中重新供应，根据制造商的说明，用合适的细胞跟踪染料对细胞进行计数和染色。在染色孵育结束时，通过离心收集细胞，并在每毫升完全RPMI中等浓度的1.5倍至6个细胞中重新下粒。然后将一毫升的细胞播种到48孔板的井中，因为有条件。并在37摄氏度下将适当体积的抗内黄蛋白抗体加入到适当的井中，进行一小时的孵育。接下来使用P1000移液器多次重新给细胞，然后再将细胞转移到单独的两毫升管中。用一毫升 PBS 冲洗每口井，将洗涤液池在适当的两毫升管中。计数后，通过离心对细胞进行颗粒，并在适当的粘附缓冲量中重新悬浮细胞，获得每毫升悬浮液的10至第6个细胞的1.5倍。然后将适当的氯化锰体积添加到每个管中，达到一毫摩尔的最终浓度。当细胞准备就绪时，打开显微镜并选择适当的过滤器和激光器、目标和采集模式。从毛细管尖端取下塑料石蜡膜，用剪刀拉伸一块五厘米的橡胶管，使毛细管连接到较小的管子上。用塑料石蜡膜密封毛细管和管之间的连接。将毛细管安装在固定托盘上。将托盘放在显微镜的托盘支架上，使毛细管对焦。并将橡胶管插入近位泵的相应槽中。将管的未连接端放入包含细胞悬浮液的第一个样品管中。将毛细管另一侧的短管插入15毫升管中，收集流量。让管子以每分钟 500 微升的流量填充，直到细胞悬浮液几乎到达毛细管。然后让毛细管以每分钟 100 微升的流速填充。当毛细管充满细胞时，将流速调整为每分钟10微升，并每两秒钟沿地址涂层墙内缓慢移动的细胞的时差图像，共三分钟。一旦细胞悬浮液到达毛细管内部，就避免流量中断至关重要，因为这将导致静态整流地址相互作用，从而可能使测量偏置。在丢弃之前，通过显微镜的目镜流过整个毛细管，以确保屏幕上观察到的部分具有代表性。将油管从泵中释放，让剩余的液体回两毫升管中。然后断开毛细管和管子与固定托盘的连接，并像刚才演示的一样对下一个毛细管进行成像。要评估时间间隔记录，请打开相应分析软件中的第一个序列，然后将第一个和最后三个图像导出为 TIFF 文件。打开 ImageJ 中的三个开始图像，然后选择图像、类型和 32 位以将图像转换为 32 位。然后选择图像、颜色和合并通道将图像合并到一个图像中，并将复合图像转换为 RGB 以保存为 TIFF 文件。以相同方式转换最后三个图像后，计算开头和结尾复合图像中可见的白单元格。并从结束图像中的白细胞数中减去开始图像中的白细胞数。涂有 ICAM-1、VCAM-1、MAdCAM-1 的毛细管灌注，但与人类 PBMC 的 Isotype Fc 嵌合不上，当存在三个地址之一时，会导致明显的粘附。相比之下，使用中和抗体抑制蛋白对丁相互作用通常会导致动态粘附的显著减少，而当添加等型控制抗体时，这种粘附力是不存在的。此外，与未经治疗的人类细胞相比，使用特异性化疗治疗CD4人类T细胞可增加粘附性。在尝试此程序时，重要的是要记住，各种因素有助于体内的动态细胞粘附，而这种分析不能完全概括。但是，可以调整检测方法，以解决非常复杂的问题。为了解决测试细胞类型的差分粘附行为的其他问题，还可以对标有不同细胞跟踪染料的不同细胞类型进行竞争性粘附测定。在开发后，该技术使我们能够探索用于治疗炎症性肠病的不同抗内黄蛋白抗体的作用。

Research

JoVE Journal

Immunology and Infection

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

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

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

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

科研

免疫与感染

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

到哺乳动物的上皮细胞在体外细菌粘附的含量

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

在细菌粘附的研究剪应力

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

区分功能基因表达的作用，通过骨髓移植免疫和非免疫细胞在小鼠结肠炎

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

Genetic Manipulation in ÃƒÅ½Ã¢â‚¬Âku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

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

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

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

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

人中性粒细胞流动室粘附实验

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

静电粘附实验对整合素激活的T细胞研究

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

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

皮层肌动蛋白流通过结构照明显微镜数据的时空图像相关光谱量化性T细胞

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

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

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

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