Name: passive administration of monoclonal antibodies against h. capsulatum and others fungal pathogens
Uploaded: 2011-02-14T17:00:00
Description: Watch this Scientific Journal Video about Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens at JoVE.com

1. Growth of H. Capsulatum
<ol>
	<li>Prepare a biosafety cabinet for work with the fungus by cleaning the cabinet with 10% bleach followed by 20 min UV irradiation. H. capsulatum in the yeast phase requires Biosafety level (BSL) II practices, while the mould form requires BSLIII. Use yeast form in the following steps.</li>
	<li>Next prepare a 15 ml conical tube with 5 ml of PBS. Harvest a colony of H. capsulatum (strain Hc ATCC G217B) from a BHI blood agar plate [g...

<ol>
	<li>Casadevall, A., Mukherjee, J., Scharff, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+based+ELISAs+for+cryptococcal+polysaccharide.">Monoclonal antibody based ELISAs for cryptococcal polysaccharide.</a> J. Immunol. Methods. 154, 27-35 (1992).</li><li>Guimaraes, A. J., Frases, S., Gomez, F. J., Zancope-Oliveira, R. M., Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+to+heat+shock+protein+60+alter+the+pathogenesis+of+Histoplasma+capsulatum.+Infect+Immun+77.">Monoclonal antibodies to heat shock protein 60 alter the pathogenesis of Histoplasma capsulatum. Infect Immun 77.</a> , 1357-1367 (2009).</li><li>Guimaraes, A. J., Hamilton, A. J., de M Guedes, H. L., Nosanchuk, J. D., Zancope-Oliveira, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+function+and+molecular+mapping+of+M+antigen+in+yeast+phase+of+Histoplasma+capsulatum.">Biological function and molecular mapping of M antigen in yeast phase of Histoplasma capsulatum.</a> PLoS One. 3, e3449-e3449 (2008).</li><li>Nosanchuk, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+antibodies+and+endemic+dimorphic+fungi.">Protective antibodies and endemic dimorphic fungi.</a> Curr Mol Med. 5, 435-442 (2005).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Jr, D. e. e. p. e., &amp;amp, G. S., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J Clin Invest. 112, 1164-1175 (2003).</li><li>Nosanchuk, J. D., Steenbergen, J. N., Shi, L., Deepe, G. S., , ., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+a+cell+surface+histone-like+protein+protect+against+Histoplasma+capsulatum.">Antibodies to a cell surface histone-like protein protect against Histoplasma capsulatum.</a> J. Clin. Invest. 112, 1164-1175 (2003).</li><li>Smith, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+laboratory+animals+to+some+injectable+anaesthetics.">Responses of laboratory animals to some injectable anaesthetics.</a> Lab Anim. 27, 30-39 (1993).</li></ol>

The protocol presented here demonstrates that mAb's to H. capsulatum can modify the course of experimental murine histoplasmosis. MAbs to cell surface antigens of pathogens can modify the complex dynamics that occur during the interplay between a host and a pathogen. This study establishes that mAbs mediate protection in a murine model of lethal histoplasmosis when injected intraperitoneally, and it suggests candidate proteins for vaccine development, such as H2B, M antigen (surface catalase)(3) and Hsp60. In vi...

<table border="1">
<tbody>
 <tr>
 <th>Name of Material</th>
 <th>Company</th>
 </tr>
 <tr>
 <td>Biological safety cabinet (BSL2)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 mL conical tubes </td>
 <td>Falcon, BD</td>
 </tr>
 <tr>
 <td>HAM F-12 medium</td>
 <td>Gibco</td>
 </tr>
 <tr>
 <td>37&deg;C shaker</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL conical tubes</td>
 <td>Falcon, BD</td>
 </tr>
 <tr>
 <td>26G1/2 needle </td>
 <td>BD</td>
 </tr>
 <tr>
 <td>10 mL syringe</td>
 <td>BD</td>
 </tr>
 <tr>
 <td>250 mL flasks</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FPLC (Fast protein liquid chromatography) system</td>
 <td>GE Helthcare</td>
 </tr>
 <tr>
 <td>ELISA reader </td>
 <td>BioTek</td>
 </tr>
 <tr>
 <td>Anesthesia (ketamine and Xylazine)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Column stands</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nylon string</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heat lamp</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>70&mu;m cell strainers</td>
 <td>Falcon, BD</td>
 </tr>
 <tr>
 <td>BHI agar plates </td>
 <td>Gibco</td>
 </tr>
 </tbody>
</table>

passive administration of monoclonal antibodies against h. capsulatum and others fungal pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Watch this Scientific Journal Video about Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens at JoVE.com

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

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