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 ...

parasite-induced-genetically-driven-autoimmune-chagas-heart-disease

Research

JoVE Journal

Immunology and Infection

13.7K vistas.  University of Brasilia. La inoculación de Trypanosoma cruzi En los huevos fértiles antes de la incubación hace que el parásito de KDNA minicircle la integración en el genoma de las células del embrión. Cruzamiento revela la transferencia vertical de las mutaciones a la descendencia. El KDNA se integra en las regiones de codificación en varios cromosomas, y los pollos mueren de una enfermedad inflamatoria autoinmune del corazón.

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

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a&#160;chagasic mother to her offspring, but the male partner&#39;s contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody.&#160;Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...