Name: parasite induced genetically driven autoimmune chagas heart disease in the chicken model
Uploaded: 2012-07-29T14:00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model at JoVE.com

We are indebted to Nancy R. Sturm, Department of Immunology, Microbiology and Molecular Biology, David Geffen School of Medicine, University of California at Los Angeles, for critical reading the manuscript. The National Council of Research-CNPq, and the Foundation for Research Development-FAPDF, Brazil, supported the study. We thank the technical aid of Alessandro O. Souza, Maria C. Guimaro, Ciro Cordeiro, Ana de Cassia Rosa, Roseneide Alves, and Rafael Andrade, from the University of Brasilia, Brazil.

<ol>
	<li>Teixeira, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+chagas'+disease:+parasite+persistence+and+autoimmunity.">Pathogenesis of chagas' disease: parasite persistence and autoimmunity.</a> CMR. 24, 592-630 (2011).</li><li>Teixeira, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chagas+disease.">Chagas disease.</a> Postg. Med. J. 82, 788-798 (2006).</li><li>Teixeira, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+cruzi+in+the+chicken+model:+Chagas-like+heart+disease+in+the+absence+of+parasitism.">Trypanosoma cruzi in the chicken model: Chagas-like heart disease in the absence of parasitism.</a> PLoS Negl. Trop. Dis. 5, e1000 (2011).</li><li>Hecht, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+DNA+transferred+from+American+trypanosomes+to+human+hosts.">Inheritance of DNA transferred from American trypanosomes to human hosts.</a> PLoS One. 5, e9181 (2010).</li><li>Xing, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+ERK+MAPK+in+the+regulation+of+proinflammatory+and+apoptotic+responses+in+chicken+macrophages+infected+with+H9N2+avian+influenza+virus.">Roles of the ERK MAPK in the regulation of proinflammatory and apoptotic responses in chicken macrophages infected with H9N2 avian influenza virus.</a> J. Gen. Virol. 91, 343-351 (2010).</li><li>Kim, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIK+and+IKKbeta+interdependence+in+NF-kappaB+signalling--flux+analysis+of+regulation+through+metabolites.">NIK and IKKbeta interdependence in NF-kappaB signalling--flux analysis of regulation through metabolites.</a> Biosystems. 99, 140-149 (2010).</li><li>Karakhanova, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK/p38+MAP-kinases+and+PI3K+are+involved+in+the+differential+regulation+of+B7-H1+expression+in+DC+subsets.">ERK/p38 MAP-kinases and PI3K are involved in the differential regulation of B7-H1 expression in DC subsets.</a> Eur. J. Immunol. 40, 254-266 (2010).</li><li>Finsterer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heart+in+human+dystrophinopathies.">The heart in human dystrophinopathies.</a> Cardiology. 99, 1-19 (2003).</li><li>Nitz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+integration+of+kDNA+minicircle+sequences+from+Trypanosoma+cruzi+into+the+avian+genome:+insights+into+human+Chagas+disease.">Heritable integration of kDNA minicircle sequences from Trypanosoma cruzi into the avian genome: insights into human Chagas disease.</a> Cell. 118, 175-186 (2004).</li><li>Simpson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetoplast+DNA+in+trypanosomid+flagellates.">Kinetoplast DNA in trypanosomid flagellates.</a> Int. Rev. Cytol. 99, 1-19 (1986).</li><li>Bonney, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-killed+Trypanosoma+cruzi+induces+acute+cardiac+damage+and+polyantigenic+autoimmunity.">Heat-killed Trypanosoma cruzi induces acute cardiac damage and polyantigenic autoimmunity.</a> PLoS One. 6, e14571 (2011).</li><li>Moser, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Trypanosoma+cruzi+by+DNA+amplification+using+the+polymerase+chain+reaction.">Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction.</a> J. Clin. Microbiol. 27, 1477-1482 (1989).</li><li>Sturm, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+and+schizodeme+classification+of+Trypanosoma+cruzi+cells+by+amplification+of+kinetoplast+minicircle+DNA+sequences:+use+in+diagnosis+of+Chagas'+disease.">Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease.</a> Mol. Biochem. Parasitol. 33, 205-214 (1989).</li><li>Teixeira, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+pathology+in+chagas+disease--a+review.">Evolution and pathology in chagas disease--a review.</a> Mem. Inst. Oswaldo Cruz. 101, 463-491 (2006).</li><li>Yurchenko, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Leishmania+minicircle+kinetoplast+DNA+classes.">Structure of Leishmania minicircle kinetoplast DNA classes.</a> J. Clin. Microbiol. 37, 1656-1657 (1999).</li><li>Simpson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+organization+of+guide+RNA+genes+in+kinetoplastid+protozoa:+several+conundrums+and+their+solutions.">The genomic organization of guide RNA genes in kinetoplastid protozoa: several conundrums and their solutions.</a> Mol. Biochem. Parasitol. 86, 133-141 (1997).</li><li>Thomas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-universal+transcription+factor?+The+Leishmania+tarentolae+TATA+box-binding+protein+LtTBP+associates+with+a+subset+of+promoters.">A non-universal transcription factor? The Leishmania tarentolae TATA box-binding protein LtTBP associates with a subset of promoters.</a> Int J. Parasitol. 36, 1217-1226 (2006).</li></ol>

No conflicts of interest declared.

In contrast to mammals susceptible to life-long T. cruzi infections, chickens are refractory to T. cruzi infection. The major advantage of the chicken model system is the elimination of the infection early in the development of the embryonic immune system. Thus, the only parasite DNA remaining within the chicken body is integrated at several loci.
Use of the optimal quantity of virulent T. cruzi trypomastigotes to inoculate fertile egg is the critical step towards obtaining integration of the kDNA minicircles into the chicken embryo genome. The rate of live chicks hatching from eggs inoculated with 100 trypomastigotes is four-fold higher than that obtained with 500 parasites. Care should be taken to inoculate the parasites suspension in 10 &mu;L of culture medium into the egg air chamber. There should be no leakage of egg white. Under optimal conditions, the intracellular parasitic infection takes place within a few hours after incubation and parasite multiplication inside the host cells proceeds for one week; thereafter the infection is eliminated by the innate immunity. The kDNA integration requires a living infection, and the inoculation of naked minicircles into early embryo chicken eggs does not result in integration. The kDNA-positive embryos and controls should be housed under controlled conditions at 37.5 &deg;C and 65% humidity. The chicks are kept in cages for two weeks at 33 &deg;C room temperature. Thereafter the chickens are kept in cages on suspended racks separated by 1.5 meter width aisles in a room at 22 &deg;C with filtered air and positive pressure under constant exhaustion to secure animal welfare conditions. The adults are fed chicken-chow and drink potable running water to achieve full growth and maturity, laying eggs at five months of age. Maintenance of hygienic procedures are essential for reproducibility of results when working with T. cruzi inoculated into fertile chicken eggs.
In the chicken model system the T. cruzi infections are eradicated after development of the immune system in the early period of embryo growth. Additionally to being infection-free, the chicks that hatch from T. cruzi inoculated eggs, in lack of specific antibodies, are tolerant to the parasite antigens. The rejection of the target heart cells by cytotoxic lymphocytes (minimal rejection unit, Figure 3) is seen in the kDNA-mutated chickens showing genotype modifications and breakdown of immunological surveillance 3. The genotypically modified T-cells present accelerated rejection of self tissues in the body. The main lesion site is the heart, which is a hallmark of Chagas disease. The passage from a physiologic (surveillance) to a physiopathologic state is seen in the kDNA-mutated chicken showing clonally proliferation of cytotoxic lymphocytes 3.
This transkingdom model system shows a parasite-induced, genetically-driven autoimmune disease (Figure 6), stemming from genome modifications by the T. cruzi kDNA minicircle integrations. These modifications are not seen in chicks hatched from Lb-inoculated eggs. 
This phenomenon suggests that experimental treatment of the inflammatory autoimmune cardiomyopathy in kDNA-mutated chickens may require drug suppression of bone marrow progenitor of specific T-cell phenotype infiltrating the myocardium, and transplantation of histocompatible healthy bone marrow to prevent the rejection of self-tissue.

<table border="1">
 <tbody>
 <tr>
 <td>Name 				of the reagent</td>
 <td>Company</td>
 <td>Catalogue 				number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Taq DNA Polymerase 				Recombinant</td>
 <td>Invitrogen</td>
 <td>11615-010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Platinum Taq DNA 				Polymerase</td>
 <td>Invitrogen</td>
 <td>10966-030</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Random Primers DNA 				Labeling System </td>
 <td>Invitrogen</td>
 <td>18187-013</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>EcoRI </td>
 <td>Invitrogen</td>
 <td>15202-021</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td> MboI </td>
 <td>Invitrogen</td>
 <td>15248-016</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>dNTP Set, 100mM 				Solutions </td>
 <td>GE 				Healthcare</td>
 <td>28-4065-51</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Amersham 				Hybond &#x2013; N+ -&#x2013; Cat n.</td>
 <td>GE 				Healthcare</td>
 <td>RPN303B</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>PlasmidPrep Mini Spin 				kit </td>
 <td>GE 				Healthcare</td>
 <td>28-9042-70</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>NsiI </td>
 <td>SIGMA 				-ALDRICH</td>
 <td>R5884 1KU</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>DNA, Sodium Salt Fish 				Sperm</td>
 <td>AMRESCO</td>
 <td>0644-10G</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Mouse 				anti-chicken Bu-1b</td>
 <td>SouthernBiotech</td>
 <td>8370-02</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Mouse anti-chicken CD45</td>
 <td>SouthernBiotech</td>
 <td>8270-08</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Mouse anti-chicken TCR&gamma;&delta;</td>
 <td>SouthernBiotech</td>
 <td>8230-08</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Mouse anti-chicken CD8&alpha;</td>
 <td>SouthernBiotech</td>
 <td>9220-02</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Mouse anti-chicken 				monocyte/macrophage</td>
 <td>SouthernBiotech</td>
 <td>8420-02</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>MyCycle Termocycler </td>
 <td>Bio-Rad 				Laboratories</td>
 <td>580BR 5501</td>
 <td>&nbsp; </td>
 </tr>
 </tbody>
</table>

parasite induced genetically driven autoimmune chagas heart disease in the chicken model

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases show Chagas disease up to three decades or later. Autoimmunity and parasite persistence are competing theories to explain the pathogenesis of Chagas disease 1, 2. To separate roles played by parasite persistence and autoimmunity in Chagas disease we inoculate the T. cruzi in the air chamber of fertilized eggs. The mature chicken immune system is a tight biological barrier against T. cruzi and the infection is eradicated upon development of its immune system by the end of the first week of growth 3. The chicks are parasite-free at hatching, but they retain integrated parasite mitochondrial kinetoplast DNA (kDNA) minicircle within their genome that are transferred to their progeny. Documentation of the kDNA minicircle integration in the chicken genome was obtained by a targeted prime TAIL-PCR, Southern hybridizations, cloning, and sequencing 3, 4. The kDNA minicircle integrations rupture open reading frames for transcription and immune system factors, phosphatase (GTPase), adenylate cyclase and phosphorylases (PKC, NF-Kappa B activator, PI-3K) associated with cell physiology, growth, and differentiation 3, 5-7, and other gene functions. Severe myocarditis due to rejection of target heart fibers by effectors cytotoxic lymphocytes is seen in the kDNA mutated chickens, showing an inflammatory cardiomyopathy similar to that seen in human Chagas disease. Notably, heart failure and skeletal muscle weakness are present in adult chickens with kDNA rupture of the dystrophin gene in chromosome 1 8. Similar genotipic alterations are associated with tissue destruction carried out by effectors CD45+, CD8&gamma;&delta;+, CD8&alpha; lymphocytes. Thus this protozoan infection can induce genetically driven autoimmune disease.

Watch this Scientific Journal Video about Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model at JoVE.com

Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model

The inoculation of Trypanosoma cruzi in fertile eggs prior to incubation renders the parasite kDNA minicircle integration in embryo cells genome. Crossbreeding reveals the vertical transfer of the mutations to progeny. The kDNA integrates into coding regions at several chromosomes and the chickens die with an inflammatory autoimmune heart disease.

The Trypanosoma cruzi acute infections acquired in infancy and childhood seem asymptomatic, but approximately one third of the chronically infected cases ...

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