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: 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

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...

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

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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7.4K 조회수.  University of Erlangen-Nuremberg. 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.

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

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.

세균성 접착 연구에 전단 응력을 소개합니다

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

연구

면역학 및 감염병학

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.

골수 이식으로 마우스 대장염에서 면역 및 비 면역 세포에서 유전자 발현의 기능 역할을 차별화

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).

Genetic Manipulation in &lt;em&gt;&amp;Delta;ku80&lt;/em&gt; Strains for Functional Genomic Analysis of &lt;em&gt;Toxoplasma gondii&lt;/em&gt;

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.

유전 조작<em&gt; Δku80</em의 기능 게놈 분석을위한&gt; 균주<em&gt; 톡소 포자충</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

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.

정량<em&gt; 체외</em정적 조건에서 활성화 된 기본 인간의 미세 혈관 내피 세포에 호중구 부착을 측정하는&gt; 분석

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

생물 발광 박테리아를 사용하여 실시간으로 급성 폐렴 마우스 모델에서 폐렴 구균 독성 요인의 영향에 따라

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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.

인간 호중구 유량 용기 접착 분석

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 림프구에있는 인테그린 활성화의 연구에 대한 정적 접착 분석

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

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 interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

구조화 조명 현미경 데이터의 시공간적 이미지 상관 분광학에 의해 정량화 T 세포에서 대뇌 피질 굴지 흐름

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

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.

T 림프구 - 접착 분자 상호 작용의 연구에 대한 접착에서 전단 응력의 분석

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.