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 capillary with 20 &#181;L of addressin (recombinant Fc chimera; 5 &#181;g/mL addressin, e.g. MAdCAM-1, in coating buffer (150 mM NaCl + 1 mM HEPES)) or the appropriate isotype control solution using a p20 pipette (fluid will soak itself into the capillary). Then, seal the side not connected to the tubing with the plastic paraffin film and incubate for at least 1 h at 37 &#176;C. Use a capillary filled only with the coating buffer or with the appropriate isotype control as negative control.</li>
	<li>Remove the plastic paraffin film from the tip of the capillary, empty the coating by letting the liquid soak out of the capillary on a paper towel and block capillaries for at least 1 h with 20 &#181;L of blocking solution (1x PBS with 5% BSA) at 37 &#176;C. Seal the open side of the capillary with plastic paraffin film. Do not remove the blocking solution before proceeding to microscopy.</li>
</ol>
2. Cell Isolation, Treatment and Preparation for Microscopy
NOTE: These steps can be performed during the incubation periods necessary for the capillary preparation.
<ol>
	<li>Collect 18 mL of anticoagulated full human blood with an aseptic blood collection set from the cubital vein of volunteering persons (e.g., IBD patients and/or healthy controls) after informed written consent according to the regulations of the local ethics committee. 
	NOTE: This step should only be performed by trained and experienced medical staff according to national and/or local regulations.</li>
	<li>Fill the blood into a 50 mL tube and dilute to a volume of 35 mL with 1x PBS. Then underlay the blood-PBS mixture with 10 mL of density gradient medium.</li>
	<li>Perform density gradient centrifugation for 15 min at 800 x g and 24 &#176;C without brake to separate cells.</li>
	<li>Collect the peripheral blood mononuclear cells (PBMCs) by aspirating the white cell layer right on top of the density gradient medium layer (clear middle layer). Transfer PBMCs into a fresh 50 mL tube, fill up to 50 mL with 1x PBS and centrifuge for 10 min at 300 x g and 10 &#176;C.</li>
	<li>Discard the supernatant, resuspend the cell pellet in 10 mL of 1x PBS and subsequently count cell numbers, e.g., by using a Neubauer cell counting chamber.</li>
	<li>Optional: To study the adhesion of granulocytes perform the following procedure. Otherwise, proceed with step 2.7 or 2.8.
	<ol>
		<li>Collect the granulocyte layer (bottommost layer after density gradient centrifugation containing also the erythrocytes). Resuspend cells in 40 mL of 1x PBS. Then add 8 mL of 6% Dextran in 1x PBS, mix gently and let cells sediment for 30 min at room temperature. 
		NOTE: The erythrocytes will sink to the bottom of the tube, while the granulocytes will stay in suspension.</li>
		<li>After 30 min, collect the supernatant, transfer into a fresh 50 mL tube and centrifuge for 6 min at 300 x g and 4 &#176;C. Discard the supernatant and lyse the remaining erythrocytes by adding 5 mL of 0.2% NaCl in ddH2O. Incubate for 1 min.</li>
		<li>Add 5 mL of 1.4% NaCl in ddH2O and incubate for another minute. Stop the hypotonic lysis by adding 20 mL of 1x PBS and centrifuge for 6 min at 300 x g and 4 &#176;C.</li>
		<li>Resuspend the cell pellet in 10 mL of 1x PBS. Then, count cell numbers by using a Neubauer cell counting chamber.</li>
	</ol>
	</li>
	<li>Optional: To study the adhesion of PBMC subsets, purify different cell types or subsets (e.g., CD4+ and CD8+ T cells or CD19+ B cells) using microbeads according to the manufacturer&#8217;s protocol or by fluorescence-activated cell sorting (FACS). Otherwise, proceed with step 2.8.</li>
	<li>Centrifuge the cell suspension for 10 min at 300 x g and 10 &#176;C. Discard the supernatant and stain cells with cell tracking dye (e.g., CFSE) according to the manufacturer&#8217;s instructions.</li>
	<li>Subsequently, centrifuge the cells for 10 min at 300 x g, and discard supernatant. Resuspend in 1x PBS, determine the cell number and centrifuge again for 10 min at 300 x g.</li>
	<li>Discard the supernatant and resuspend the stained cell pellet in the appropriate amount of adhesion buffer (150 mM NaCl + 1 mM HEPES + 1 mM MgCl2 + 1 mM CaCl2) to obtain a suspension of 1.5 x 106 cells/mL. Then, add the appropriate amount of 100 mM MnCl2 to obtain a final concentration of 1 mM. 
	NOTE: These cells are now ready for microscopy.</li>
	<li>Optional: Treat cells with cytokines, neutralizing antibodies or other compounds. Otherwise, proceed with step 3.
	<ol>
		<li>Omit step 2.10 and resuspend the stained cell pellet from step 2.9 in RPMI medium (with 10% FBS and 1% Penicillin/Streptomycin) to obtain a concentration of 1.5 x 106 cells/mL.</li>
		<li>Pipette 1 mL of this cell suspension into as many wells of a 48-well plate as there are conditions to be analyzed. Always prepare one well with untreated cells to be perfused through an uncoated capillary as negative control and one well with untreated cells to be perfused through an addressin-coated capillary as positive control.</li>
		<li>For the treatment with anti-integrin antibodies, add the appropriate amount of antibody (e.g., 10 &#181;g/mL vedolizumab) to the cell suspension. Incubate for 1 h at 37 &#176;C. For treatment with cytokines, add the appropriate amount of recombinant cytokine (e.g., 10 ng/mL CXCL10) to the cell suspension. Incubate for 5 min at 37 &#176;C.</li>
		<li>After incubation, harvest cells by resuspending cells several times using a p1000 pipette. Transfer cells into a 2 mL tube, rinse well again with 1 mL of 1x PBS and add to the 2 mL tube. Determine the cell number. 
		NOTE: Incubation of adherent cell populations (e.g., monocytes) might require additional measures (e.g., treatment with trypsin) to detach cells from the plate.</li>
	</ol>
	</li>
	<li>Centrifuge cells for 10 min at 300 x g at 10 &#176;C. Discard the supernatant and resuspend the cell pellet in the appropriate amount of adhesion buffer to obtain a suspension of 1.5 x 106 cells/mL. Then, add the appropriate amount of 100 mM MnCl2 to obtain a final concentration of 1 mM. When pretreating with chemokines do not add MnCl2 to the cell suspension. These cells are now ready for microscopy. 
	NOTE: The minimum volume used for the assay is dependent on the pump and tubing used. In this study, using the peristaltic pump and the tubing specified in the Table of Materials, a minimum volume of 400 &#181;L was required. If desired and applicable (depending on cell type and yield), the cell concentration in the suspension can be increased to measure higher adhesion in a shorter period of time.</li>
	<li>Potential modification: Competitive dynamic adhesion assay
	<ol>
		<li>Perform the above-mentioned steps with different cell populations to be compared to each other (e.g., regulatory and effector T cells isolated through magnetic bead isolation following manufacturer&#8217;s instructions as per step 2.7).</li>
		<li>Stain each population with a different dye (e.g., CFSE and FarRed) as described in steps 2.8&#8211;2.9 to be able to differentiate cell populations during microscopy.</li>
		<li>Discard the supernatant and resuspend the stained cell pellets in the appropriate amount of adhesion buffer (150 mM NaCl + 1 mM HEPES + 1 mM MgCl2 + 1 mM CaCl2) to obtain a suspension of 1.5 x 106 cells/mL.</li>
		<li>Mix equal volumes of the cell suspensions (e.g., 500 &#181;L of regulatory T cells and 500 &#181;L of effector T cells) to obtain a final mixed cell concentration of 1.5 x 106 cells/mL. Then, add the appropriate amount of 100 mM MnCl2 to obtain a final concentration of 1 mM. Such suspensions can subsequently be used for competitive analysis of the adhesion process by perfusing different cell populations through the same capillary.</li>
	</ol>
	</li>
</ol>
3. Live Cell Imaging of Dynamic Adhesion
<ol>
	<li>Turn on the microscope and select the appropriate filter or laser to excite the cell tracking dye(s) (e.g., 488 nm laser for CFSE), an appropriate objective (e.g., 40X) and an acquisition mode enabling time lapse imaging.</li>
	<li>Remove the plastic paraffin film from the tip of the capillary and connect the capillary with a short piece of tubing (approximately 5 cm of length) 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 the capillary and tube with the plastic paraffin film.</li>
	<li>Mount the capillary on a fixation tray (e.g., lid from a 6-well plate with a rectangular cut-out slightly shorter than the length of the capillary) and place the fixed capillary on the tray holder of the microscope, so that the capillary is in focus and ready for microscopy.</li>
	<li>Insert the rubber tubing into the corresponding notch of the peristaltic pump and place the end not attached to the capillary into the according sample tube containing the cell suspension. Insert the short tube on the other side of the capillary into a 15 mL tube to collect flow-through.</li>
	<li>Let the tubing fill with a flow rate of 500 &#181;L per minute until the cell suspension almost reaches the capillary. Then let the capillary fill with a flow rate of 100 &#181;L/min. 
	NOTE: A quick filling up of the capillary ensures a regular distribution of the cell suspension inside the capillary.</li>
	<li>When the capillary is filled with the cell suspension, adjust flow rate to 10 &#181;L/min. 
	NOTE: These flow rates may vary, when capillaries of a different size or different peristaltic pumps are used. In this study, the flow rate of 10 &#181;L/min was optimized for the pump and capillaries to mimic in vivo blood flow.</li>
	<li>Capture time-lapse images of the cells moving slowly through the capillary along the addressin-coated inside wall every 2 s for a total of 3 min. 
	NOTE: Intervals and total time may vary depending on cell type and question to be addressed.</li>
	<li>Before discarding the capillary, screen the whole capillary through the eye piece of the microscope to make sure that the section shown on the video is representative. If necessary, make a second recording.</li>
	<li>Free the tubing from the pump and let the remaining fluid run back into the 2 mL tube, then disconnect the capillary and tubing from the fixation tray and continue with the next capillary.</li>
</ol>
4. Evaluation and Cell Counting
<ol>
	<li>Open the time-lapse sequence with appropriate software and export the first three images (&#8220;Beginning&#8221;) and the last three images (&#8220;End&#8221;) as Tiff files.</li>
	<li>Open the three &#8220;Beginning&#8221; images in ImageJ, convert to 32 bit (&#39;Image | Type | 32 Bit&#39;) and merge pictures (&#39;Image | Color | Merge channels&#39;; assign the colors red, green and blue to the different pictures). Convert composite image into RGB (&#39;Image | Type-RGB&#39;) and save as Tiff file. Repeat with the &#8220;End&#8221; images. 
	Note: In competitive dynamic adhesion assays with cells stained with different colors, first split the channels of the images to analyze adhesion of the different cell populations separately.</li>
	<li>Count the white cells visible in the &#8220;Beginning&#8221; and &#8220;End&#8221; composite and subtract the number of white cells in the &#8220;Beginning&#8221; from the white cells in the &#8220;End&#8221;. 
	Note: The difference represents the number of newly adhering cells in the 3 min time interval. In the composite image, cells that moved are visible in the specific color that was assigned to the respective frame since they localize in another spot in the previous and following frame. For cells that are adherent, the red, green and blue signals in the same spot overlap to white.</li>
	<li>To better visualize the results, compile a video from the time-lapse sequences, e.g., using ImageJ.</li>
</ol>

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M.F.N. has served as an advisor for Pentax, Giuliani, MSD, Abbvie, Janssen, Takeda and Boehringer. M.F.N. and S.Z. received research support from Takeda and Roche.

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: 150 mM NaCl + 1 mM HEPES + 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt; + 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Blocking solution: 1x PBS in ddH&lt;sub&gt;2&lt;/sub&gt;O + 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>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>2382</td><td></td></tr><tr><td>Capillaries: Rectangle Boro Tubing 0.20 mm x 2.00 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&amp;trade; 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&amp;amp;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&amp;amp;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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Roth</td><td></td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;amp;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)&amp;nbsp;</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, 1.02 mm 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

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 ...

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7.5K Views.  University of Erlangen-Nuremberg. Deze methode kan helpen bij het beantwoorden van belangrijke vragen van fundamentele immunobiologie en inflammatoire darmziekte onderzoek met betrekking tot de adhesie cascade van immuuncellen die nodig zijn voor homing aan perifere organen. Het belangrijkste voordeel van deze techniek is dat het een handige en eenvoudige aanpak is voor het beantwoorden van functionele vragen in de context van celhechting onder stroomomstandigheden. Hoewel deze methode is ontwikkeld voor het verkennen van het...

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

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioengineering

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Medicine

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 ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Biology

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of plasma inflammatory markers and bacterial translocation to the spleen and lymph nodes, the gold standard directly quantifies the ability of selected molecules to traverse the gut mucosal layer toward systemic circulation. This article uses a non-invasive, cost-effective, and low-burden technique to quantify and follow in real time the intestinal permeability in mice using fluorescein-isothiocyanate-labeled dextran (FITC-dextran). Prior to oral supplementation with FITC-dextran, the mice are fasted. They are then gavaged with FITC-dextran diluted in phosphate-buffered saline (PBS). One hour after the gavage, the mice are subjected to general anesthesia using isoflurane, and the in vivo fluorescence is visualized in an imaging chamber. This technique aims to assess residual fluorescence in the abdominal cavity and the hepatic uptake, which is suggestive of portal migration of the fluorescent probe. Blood and stool samples are collected 4 h after oral gavage, and the mice are sacrificed. Plasma and fecal samples diluted in PBS are then plated, and the fluorescence is recorded. The concentration of FITC-dextran is then calculated using a standard curve. In previous research, in vivo imaging has shown that fluorescence rapidly spreads to the liver in mice with a weaker gut barrier induced by a low-fiber diet, while in mice supplemented with fiber to strengthen the gut barrier, the fluorescent signal is retained mostly in the gastrointestinal tract. In addition, in this study, control mice had elevated plasma fluorescence and reduced fluorescence in the stool, while inversely, inulin-supplemented mice had higher levels of fluorescence signals in the gut and low levels in the plasma. In summary, this protocol provides qualitative and quantitative measurements of intestinal permeability as a marker for gut health.

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

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

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