This work was supported by the Sonderforschungsbereich Transregio 19 (SFB/TR19, C2) of the Deutsche Forschungsgemeinschaft (DFG), the Federal Ministry of Economics and Technology project ZIM-KF 2727801MD0 and the Centre for Innovation Competence - Humoral Immune Responses in Cardiovascular Diseases (ZIK-HIKE, BMBF FKZ 03Z2CN12) of the Federal Ministry of Education and Research, Germany. Housing and experiments with animals were performed in accordance with the recommendations of the Society for Laboratory Animal Science (Gesellschaft f&uuml;r Versuchstierkunde, GV-SOLAS) and the Federation of European Laboratory Animal Science Association (FELASA).

1. IgG Isolation from Patient Samples
<ol>
	<li>Prepare mini-immunoadsorption columns by filling 3 ml anti-IgG sepharose per 2 ml patient EDTA plasma into an empty Econo Pac column. IgG isolation requires at least 2 ml of patient plasma.</li>
	<li>Place a filter on top of the anti-IgG sepharose, cut open the lower tip of the column and press the filter to reach a flow rate of approximately 1 drop/sec. Let the preservation solution run out of the column.</li>
	<li>Wash the column with 3 volumes 0.9% NaCl per volume of plasma.</li>
	<li>Pipette the plasma on the column. When the plasma has completely immersed in the anti-IgG sepharose, wash with the same volume 0.9% NaCl.</li>
	<li>Pipette one plasma volume of 0.2 M glycine HCl, pH 2.8 on the column and let immerse into the sepharose. Add another volume of glycine HCl onto the column. Collect the flow through in a 15 ml tube and adjust the pH to 7.3 with 0.5 M Tris-HCl, pH 8.0.</li>
	<li>To regenerate the column, wash with 3 plasma volumes glycine HCl. After a final wash with 2 volumes 0.9% NaCl, the column may be used for the next plasma sample. Alternatively, the column can be filled with preservation solution and stored at 4 - 8 &deg;C for up to 4 weeks.</li>
	<li>For use on cardiomyocytes, the collected IgG fraction has to be dialyzed against experimental buffer (EB). For preparation of EB, add 117 mM NaCl, 2.8 mM KCl, 0.6 mM MgCl2, 1.2 mM KH2PO4, 1.2 mM CaCl2,10 mM HEPES and 20 mM glucose to 1 L distilled water on a magnetic stirrer and adjust pH to 7.3.</li>
	<li>Fill the IgG fraction to a cellulose ester dialysis membrane tube with a molecular weight cut off of 100 kDa. Put the tube in 1 L EB and stir slowly with a magnetic stirrer at 4 &deg;C. After 8 hr, transfer the dialysis tube into 3 L of fresh EB and dialyze for another 20 hr at 4 &deg;C.</li>
	<li>Following dialysis, heat the sample for 30 min at 57 &deg;C in a water bath to inactivate complement factors. Determine total IgG concentration and prepare aliquots containing 330 &mu;g IgG each. When adding the sample to cardiomyocytes for measurement (step 4.3), fill up the aliquots to 1 ml with EB. Undiluted aliquots can be stored at -20 &deg;C until further use.</li>
</ol>
2. Isolation of Rat Cardiomyocytes
<ol>
	<li>For preparation of Ca2+-free isolation buffer (IB), add 110 mM NaCl, 2.6 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM HEPES and 11 mM glucose to 1 L distilled water using a magnetic stirrer. Adjust pH to 7.4 at room temperature and filter through a 0.2 &mu;m bottle top filter for sterilization.</li>
	<li>Fill 80 ml of IB in the upper reservoir of a thermostated Langendorff perfusion system. Adjust thermostat settings to maintain a buffer temperature of 37 &deg;C and aerate buffer with 95% O2/5% CO2 for 30 min.</li>
	<li>Weigh approximately 10,000 - 12,000 U of collagenase type II, 175 mg fatty acid free BSA and dissolve in 20 ml IB containing 22.5 &mu;l of a 100 mM CaCl2 stock solution. Control the pH of the isolation buffer in the upper reservoir and adjust if required.</li>
	<li>Anaesthetize a Wistar rat of 175 - 200 g with 375 &mu;g/kg body weight thiopental. Add 2500 IU Heparin to thiopental solution. After thoracotomy, excise the heart carefully with an intact aortic arch and transfer it into ice-cold 0.9% NaCl. Clean the heart from blood and surrounding tissue and transfer to fresh 0.9% NaCl. Expose the aorta, open the flow reducer of the Langendorff system to obtain a dropping speed of 2-3/sec and attach the heart to the cannula.</li>
	<li>Perfuse the heart for up to 3 min with a flow rate of 1 drop/sec to wash out remaining blood. Start to circulate the IB in the Langendorff system. Remove 20 ml buffer from the upper reservoir, fill in the collagenase solution and refill as much buffer as required to reach 80 ml. Circulate the collagenase solution for another 27 min. Flow rate should be controlled periodically and maintained at 1 drop/sec using the flow reducer.</li>
	<li>Detach the heart from the cannula, clean from atria and aorta and chop into small pieces. Allow the collagenase solution to run into the lower reservoir, reduce the volume to a maximum of 40 ml and further digest the chopped ventricles for 10 - 15 min under continuous aeration with 95% O2/5% CO2. Afterwards, filter the cell suspension through a 200 &mu;m mesh size gauze into a 50 ml tube.</li>
	<li>Restore the Ca2+ content of the cells in 3 steps: centrifuge the cell suspension at 43 x g for 2 min at room temperature and resuspend cells in 10 ml IB containing 200 &mu;M CaCl2. Centrifuge again and resuspend cells in 10 ml IB with 500 &mu;M CaCl2. After a final centrifugation resuspend cardiomyocytes in sterile filtered EB (see 1.8) to a density of 50,000 cells/ml.</li>
</ol>
3. Fura-2 Staining of Cardiomyocytes
<ol>
	<li>Coat a 4 well chambered coverglass with 10 &mu;g laminin per well and wait until it is fully dried.</li>
	<li>Fill 1 ml of the cardiomyocyte suspension into each well using a micropipette with a cut tip. Allow the cells to adhere for 1 hr at room temperature.</li>
	<li>Dilute 10 &mu;l of the 1 mg/ml Fura-2 AM stock solution in 5 ml EB. Keep the staining solution in the dark.</li>
	<li>Take off buffer and unattached cells from the coverglass and pipette 0.5 ml of the staining solution into each well. Incubate the cells for 10 min under moderate shaking. Subsequently, take off the staining solution, wash cells once with 1 ml EB and finally fill 0.5 ml EB into each well. Avoid direct light during the staining process and always keep the stained cells in the dark.</li>
</ol>
4. Recording of Cell Shortening and Ca2+ Transients
<ol>
	<li>Place the chambered coverglass on an inverse fluorescence microscope connected to a myocyte calcium and contractility recording system. Position a custom made electrode with an influx and efflux tube in the first well. Superfuse cardiomyocytes with 1 ml/min EB and start the electric stimulation (20 V, 1 Hz, 5 msec duration). Allow the cells to adapt to the stimulation for about 2 min.</li>
	<li>Choose an intact rod shaped cardiomyocyte with clear striation and constant contraction. Position the cell in the center of the visual field and open the camera channel. Orientate the cell horizontally within the video area by rotating the cell framing adapter and moving the microscope stage.</li>
	<li>Adjust the edge detecting control elements of the video area (Figure 2), open the fluorescent channel and record 10 - 15 contractions to obtain the initial cell shortening and Ca2+ transient.</li>
	<li>Close the microscope light channel to avoid bleaching of the fluorescence stain. Switch the flow through from EB to the sample bypass with a 3-way valve and superfuse cardiomyocytes with 1 ml of patient IgG. Stop the pump just before air reaches the well.</li>
	<li>Open the light channel and record another 10 - 15 contractions to obtain the acute cell response. Pause the recording and close the light channel again. Repeat recording after 2 and 5 min.</li>
	<li>Take out the electrode of the well. Clean the tubes thoroughly with EB and continue with the next well of the chambered coverglass.</li>
	<li>Analyze the cell shortening and Ca2+ transient with the IonWizard software using the "Edge-Length/Length" and the "fura 2-Numeric subtracted/Ratio" window, respectively. Calculate the percent change from the initial cell shortening and Ca2+ transient of the peak height referred to the baseline (bl%peak h) for the acute, the 2 min and the 5 min response.</li>
</ol>

<ol>
	<li>Roger, V. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+disease+and+stroke+statistics--2011+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2011 update: a report from the American Heart Association.</a> Circulation. 123 (4), e18 (2011).</li><li>Caforio, A. L., Vinci, A., Iliceto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-heart+autoantibodies+in+familial+dilated+cardiomyopathy.">Anti-heart autoantibodies in familial dilated cardiomyopathy.</a> Autoimmunity. 41 (6), 462 (2008).</li><li>Yoshikawa, T., Baba, A., Nagatomo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+mechanisms+underlying+dilated+cardiomyopathy.">Autoimmune mechanisms underlying dilated cardiomyopathy.</a> Circ. J. 73 (4), 602 (2009).</li><li>Felix, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+effects+of+immunoadsorption+and+subsequent+immunoglobulin+substitution+in+dilated+cardiomyopathy:+Three-month+results+from+a+randomized+study.">Hemodynamic effects of immunoadsorption and subsequent immunoglobulin substitution in dilated cardiomyopathy: Three-month results from a randomized study.</a> Journal of the American College of Cardiology. 35 (6), 1590 (2000).</li><li>Staudt, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+immunoglobulin+G3+subclass+in+dilated+cardiomyopathy:+results+from+protein+A+immunoadsorption.">Role of immunoglobulin G3 subclass in dilated cardiomyopathy: results from protein A immunoadsorption.</a> Am. Heart J. 150 (4), 729 (2005).</li><li>Nikolaev, V. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fluorescence+method+for+the+rapid+detection+of+functional+beta1-adrenergic+receptor+autoantibodies+in+heart+failure.">A novel fluorescence method for the rapid detection of functional beta1-adrenergic receptor autoantibodies in heart failure.</a> J. Am. Coll. Cardiol. 50 (5), 423 (2007).</li><li>Jahns, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+activating+human+beta1-adrenergic+receptors+are+associated+with+reduced+cardiac+function+in+chronic+heart+failure.">Autoantibodies activating human beta1-adrenergic receptors are associated with reduced cardiac function in chronic heart failure.</a> Circulation. 99 (5), 649 (1999).</li><li>Gocayne, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+structure+of+rat+cardiac+beta-adrenergic+and+muscarinic+cholinergic+receptors+obtained+by+automated+DNA+sequence+analysis:+further+evidence+for+a+multigene+family.">Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.</a> Proceedings of the National Academy of Sciences. 84 (23), 8296 (1987).</li><li>Jaenicke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+sequence+of+the+human+beta-myosin+heavy+chain+gene+and+a+comparative+analysis+of+its+product.">The complete sequence of the human beta-myosin heavy chain gene and a comparative analysis of its product.</a> Genomics. 8 (2), 194 (1990).</li><li>Piper, H. M., Volz, A., Schwartz, P., Piper, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Culture Techniques in Heart and Vessel Research. , (1990).</li><li>Kubin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+endothelial+cells+remodel+cultured+adult+cardiomyocytes+and+increase+their+survival.">Microvascular endothelial cells remodel cultured adult cardiomyocytes and increase their survival.</a> Am. J. Physiol. 276 (6 Pt. 2), H2179 (1999).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J. Biol. Chem. 260 (6), 3440 (1985).</li><li>Magnusson, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+idiopathic+dilated+cardiomyopathy.+Characterization+of+antibodies+against+the+beta+1-adrenoceptor+with+positive+chronotropic+effect.">Autoimmunity in idiopathic dilated cardiomyopathy. Characterization of antibodies against the beta 1-adrenoceptor with positive chronotropic effect.</a> Circulation. 89 (6), 2760 (1994).</li><li>Wakefield, I. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+application+of+in+vitro+methods+to+safety+pharmacology.">The application of in vitro methods to safety pharmacology.</a> Fundam. Clin. Pharmacol. 16 (3), 209 (2002).</li><li>Kajzar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+physiological+conditions+for+cell+analyses:+forces+of+heart+muscle+cells+suspended+between+elastic+micropillars.">Toward physiological conditions for cell analyses: forces of heart muscle cells suspended between elastic micropillars.</a> Biophys. J. 94 (5), 1854 (2008).</li></ol>

No conflicts of interest declared.

The presented method offers a suitable way to detect functionally effective cardiac autoantibodies in patients with DCM of unclear origin. In comparison to other methods, e.g. the detection of functional active antibodies against the &beta;1-adrenoceptor by their impact on cAMP levels6, our method is independent of a specific epitope. Of course there are other epitope independent assays described in the literature, i.e. counting the beating rate of neonatal cardiomyocytes13, ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Immunosorba preservation solution</td>
 <td>Fresenius Medical Care</td>
 <td>903609211</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase type 2</td>
 <td>Cell System</td>
 <td>LS004176</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA, fatty acid free</td>
 <td>Sigma-Aldrich</td>
 <td>A6003</td>
 <td></td>
 </tr>
 <tr>
 <td>Laminin</td>
 <td>Sigma-Aldrich</td>
 <td>L2020</td>
 <td></td>
 </tr>
 <tr>
 <td>Fura-2 AM</td>
 <td>Sigma-Aldrich</td>
 <td>F0888</td>
 <td>1 mg/ml in DMSO</td>
 </tr>
 <tr>
 <td>Econo Pac Chromatography Columns</td>
 <td>BioRad</td>
 <td>732-1010</td>
 <td></td>
 </tr>
 <tr>
 <td>Spectra/Por Biotech Cellulose Ester Dialysis Membrane</td>
 <td>Spectrumlabs</td>
 <td>131417</td>
 <td></td>
 </tr>
 <tr>
 <td>4 well Chambered Coverglass</td>
 <td>Nunc</td>
 <td>155383</td>
 <td></td>
 </tr>
 <tr>
 <td>Myocyte Calcium and Contractility Recording System</td>
 <td>IonOptix</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>IonWizard 6.0 analysis software</td>
 <td>IonOptix</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of antibody effects on cellular function of isolated cardiomyocytes

Dilated cardiomyopathy (DCM) is one of the main causes for heart failure in younger adults1. Although genetic disposition and exposition to toxic substances are known causes for this disease in about one third of the patients, the origin of DCM remains largely unclear. In a substantial number of these patients, autoantibodies against cardiac epitopes have been detected and are suspected to play a pivotal role in the onset and progression of the disease2,3. The importance of cardiac autoantibodies is underlined by a hemodynamic improvement observed in DCM patients after elimination of autoantibodies by immunoadsorption3-5. A variety of specific antigens have already been identified2,3 and antibodies against these targets may be detected by immunoassays. However, these assays cannot discriminate between stimulating (and therefore functionally effective) and blocking autoantibodies. There is increasing evidence that this distinction is crucial6,7. It can also be assumed that the targets for a number of cardiotropic antibodies are still unidentified and therefore cannot be detected by immunoassays. Therefore, we established a method for the detection of functionally active cardiotropic antibodies, independent of their respective antigen. The background for the method is the high homology usually observed for functional regions of cardiac proteins in between mammals8,9. This suggests that cardiac antibodies directed against human antigens will cross-react with non-human target cells, which allows testing of IgG from DCM patients on adult rat cardiomyocytes. Our method consists of 3 steps: first, IgG is isolated from patient plasma using sepharose coupled anti-IgG antibodies obtained from immunoadsorption columns (PlasmaSelect, Teterow, Germany). Second, adult cardiomyocytes are isolated by collagenase perfusion in a Langendorff perfusion apparatus using a protocol modified from previous works10,11. The obtained cardiomyocytes are attached to laminin-coated chambered coverglasses and stained with Fura-2, a calcium-selective fluorescent dye which can be easily brought into the cell to observe intracellular calcium (Ca2+) contents12. In the last step, the effect of patient IgG on the cell shortening and Ca2+ transients of field stimulated cardiomyocytes is monitored online using a commercial myocyte calcium and contractility monitoring system (IonOptix, Milton, MA, USA) connected to a standard inverse fluorescent microscope.

Two examples for the measurement of cellular inotropy in field stimulated adult cardiomyocytes (Figure 2) using a myocyte Ca2+ and contractility recording system are given below. Figure 3 gives an impression of a control measurement, while in Figure 4 the effect of cardiodepressive antibodies of a patient with DCM is shown.
In both examples, initial cell shortening and Ca2+ transient during superfusion with EB exhibi...

Watch this Scientific Journal Video about Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes at JoVE.com

Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Dilated cardiomyopathy  (DCM) is one of the main causes for heart failure in younger adults1.  Although genetic disposition and exposition to toxic ...

measurement-antibody-effects-on-cellular-function-isolated

Research

JoVE Journal

Immunology and Infection

11.8K Views.  University Medicine Greifswald. The presented method offers a way to detect functional effective cardiotropic autoantibodies in the plasma of patients with dilated cardiomyopathy, irrespective of the specific antigen, by analysing the impact of isolated patient immunoglobulin on cellular shortening and intracellular calcium transients in isolated rat cardiomyocytes.

Video: Measurement of Antibody Effects on Cellular Function of Isolated Cardiomyocytes

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that may facilitate the development of vaccines and peptide drugs. Peptide scanning is a simple and efficient method that straightforwardly maps the linear epitope recognized by a monoclonal antibody (mAb). Here, the authors present an epitope determination methodology involving serially truncated recombinant proteins, synthetic peptide design, and dot-blot hybridization for the antigenic recognition of nervous necrosis virus coat protein using a neutralizing mAb. This technique relies on the dot-blot hybridization of synthetic peptides and mAbs on a polyvinylidene fluoride (PVDF) membrane. The minimum antigenic region of a viral coat protein recognized by the RG-M56 mAb can be narrowed down by step-by-step trimmed peptide mapping onto a 6-mer peptide epitope. In addition, alanine scanning mutagenesis and residue substitution can be performed to characterize the binding significance of each amino acid residue making up the epitope. The residues flanking the epitope site were found to play critical roles in peptide conformation regulation. The identified epitope peptide may be used to form crystals of epitope peptide-antibody complexes for an x-ray diffraction study and functional competition, or for therapeutics.

Here, the authors present a simple and efficient protocol to define a linear antigenic epitope using a purified monoclonal antibody and peptide scanning through dot-blot hybridization. The identified epitope can then be used in therapeutic and diagnostic applications.

The identification of an antigenic epitope by the immune system allows for the understanding of the protective mechanism of neutralizing antibodies that ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb development since its first report in 1975. As an alternative technology, phage display methods for mAb development are increasingly attractive since Humira, the first phage-derived antibody and one of the best-selling mAbs, was approved for clinical treatment of rheumatoid arthritis in 2002. As a non-animal based mAb development technology, phage display bypasses antigen immunogenicity, humanization, and animal maintenance that are required from traditional hybridoma technology based antibody development. In this protocol, we describe a method for construction of synthetic phage-displayed Fab libraries with diversities of 109-1010 obtainable with a single electroporation. This protocol consists of: 1) high-efficiency electro-competent cell preparation; 2) extraction of uracil-containing single-stranded DNA (dU-ssDNA); 3) Kunkel&#39;s method based oligonucleotide-directed mutagenesis; 4) electroporation and calculation of library size; 5) protein A/L-based enzyme-linked immunosorbent assay (ELISA) for folding and functional diversity evaluation; and 6) DNA sequence analysis of diversity.

This protocol describes a detailed procedure for the construction of a phage-displayed synthetic antibody library with tailored diversity. Synthetic antibodies have broad applications from basic research to disease diagnostics and therapeutics.

Demand for monoclonal antibodies (mAbs) in basic research and medicine is increasing yearly. Hybridoma technology has been the dominant method for mAb ...