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 p...

<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. 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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. 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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...

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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

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Immunology and Infection

11.8K visualizações.  University Medicine Greifswald. O método apresentado oferece um modo para detectar auto-anticorpos funcionais eficazes cardiotrópicos no plasma de pacientes com cardiomiopatia dilatada, independentemente do antigénio específico, por meio da análise do impacto da imunoglobulina paciente isolado no encurtamento celular e os transientes de cálcio intracelular em cardiomiócitos de ratos isolados.