Name: sexual transmission of american trypanosomes from males and females to naive mates
Uploaded: 2019-01-27T13:00:00
Description: Watch this Scientific Journal Video about Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates at JoVE.com

We acknowledge the laboratory facilities and the critical comments of Izabela Dourado, Carla Araujo, and Clever Gomes and the technical assistance of Bruno Dalago and Rafael Andrade. We are indebted to the Foundation for the Advancement of Science (FAPDF), The National Research Council, Ministry of Science and Technology (CNPq/MCT), and The Agency for Training Human Resources, Ministry of Education (CAPES/ME), Brazil, for supporting these investigations.

The Human and the Animal Research Committees of the Faculty of Medicine of the University of Brasilia approved all the procedures with human subjects and laboratory animals, respectively, in research protocols 2500.167567 and 10411/2011. The Ethics Committee of the Public Foundation Hospital Gaspar Vianna (protocol n&#186; 054/2009 and CONEP 11163/2009) approved the free consent forms for the field study, with extension to the Ministry of Health National Commission on Human Research (CONEP 2585/04). The protocol was adju...

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Herein, we discuss a family-based research protocol that answered the question of whether human Chagas disease stems from sexually transmitted intraspecies T. cruzi infections. Early studies could not provide evidence of the sexual transmission of T. cruzi infections, probably because the available data and information on Chagas disease were obtained separately from the individual3,4,5,<sup class="xr...

The protozoan parasite Trypanosoma cruzi belonging to the family Trypanosomatidae undergoes trypomastigote and amastigote life cycle stages in mammalian hosts and exists as epimastigotes in the insect-vector&#39;s (Reduviid: Triatominae) gut and in axenic culture. In recent decades, several studies have shown the presence of Chagas disease in countries on four continents considered triatomine bug free1,2,3,4,5,6,7,8,9,10,11,12,13; the dispersion of American trypanosomes was initially attributed to Latin American immigrants to the Northern Hemisphere, but the possibility that some are autochthonous cases of Chagas disease can no longer be denied3,4,5,6,7,8,9,10,11,12,13,14. The only recognizable endogenous source of T. cruzi transmission has been ascribed to the chagasic mother&#39;s transfer of the parasite to the offspring in approximately 10% of pregnancies15; the male partner&#39;s contribution to in utero&#160;infections through semen ejaculates has remained unrecognized.
Over one century ago, investigators16,17 observed intracellular T. cruzi amastigotes in the theca cells of the ovary and in the germ line cells of the testicles of acute cases of Chagas disease. The nests and clumps of T. cruzi trypomastigotes and amastigotes in theca cells of the ovary, in goniablasts and in the lumen of seminiferous tubules (Figure 1) of fatal acute Chagas disease cases develop immune privilege in the organs of reproduction in the absence of inflammatory infiltrates18,19. In recent decades, a few experimental studies have shown nests of the round amastigote forms of T. cruzi in the seminiferous tubule, epididymis, and vas deferens as well as in the uterus, tubes and ovary theca cells of acutely infected mice1,20,21,22. Furthermore, in the course of family studies to document the transfer of protozoan&#160;mitochondrial DNA from parental Chagas patients to their descendants, T. cruzi nuclear DNA (nDNA) was verified in human haploid germ line cells23, and parasite life cycle stages were observed in the ejaculates of chagasic mice24. These findings are in agreement with reports on the immune tolerance attained by the progeny of T. cruzi-infected hosts in the absence of the specific antibody1,25,26. Additionally, epidemiological reports that suggested the spread of endemic Chagas disease to the other continents3,4,5,6,7,8,9,10,11,12,13 are now supported by experimental studies showing that Chagas disease can be transmitted sexually1. The present investigation presents an epidemiologic family study protocol and shows that T. cruzi infection propagates by sexual intercourse.

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		<tr height="20">
			<td height="20" style="height:20px;width:336px;">BCIP and NBT redox system</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">681 451 001</td>
			<td style="width:65px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Blood DNA Purification columns</td>
			<td style="width:183px;">Amersham Biosciences</td>
			<td style="width:102px;">27-9603-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">d-ATP, [&#945;-32P], 250 &#181;Ci.</td>
			<td style="width:183px;">&#160; Perkin Elmer&#160;&#160;</td>
			<td style="width:102px;">BLU012H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">DNA, Solution Salt Fish Sperm</td>
			<td style="width:183px;">AMRESCO</td>
			<td style="width:102px;">064-10G</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:336px;">dNTP Set, 100 mM Solutions</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">28-4065-51</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Eco&#160;RI</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">15202-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Goat anti-human IgG- alkaline phosphatase conjugated</td>
			<td style="width:183px;">Southern Biotech</td>
			<td style="width:102px;">&#160;&#160;&#160;&#160;&#160;&#160; 2040-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Goat anti-human IgG- FITC conjugated</td>
			<td style="width:183px;">Biocompare</td>
			<td style="width:102px;">MB5198020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Hybond &#8211; N+ nylon membrane</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">RPN303B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Hybridization oven</td>
			<td style="width:183px;">Thomas Scientific</td>
			<td style="width:102px;">95-0031-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Micro imaging software cell Sens software</td>
			<td style="width:183px;">Olympus, Japan</td>
			<td style="width:102px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Molecular probes labeling System</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">700-0030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Nsi I</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">R5584 1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Plasmid Prep Mini Spin Kit</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">28-9042-70</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Plate reader&#160;</td>
			<td style="width:183px;">Bio-Tek GmBH</td>
			<td style="width:102px;">2015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-chicken IgG-alkaline phosphatase conjugated</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">A9171</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-chicken IgG-FITC conjugated</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">F8888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-mouse IgG- alkaline phosphatase conjugated</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:102px;">A2418&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-mouse IgG-FITC conjugated</td>
			<td style="width:183px;">Biorad</td>
			<td style="width:102px;">MCA5787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Spin Columns for radio labeled DNA purification, Sephadex G-25, fine</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">G25DNA-RO&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Taq DNA Polymerase Recombinant</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">11615-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Thermal cycler system</td>
			<td style="width:183px;">Biorad, USA</td>
			<td style="width:102px;">1709703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Vector Systems</td>
			<td style="width:183px;">Promega</td>
			<td style="width:102px;">A1380</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This research, conducted according to the protocol, aimed to detect acute cases of Chagas disease by clinical and parasitological exams. Venous blood samples were subjected to direct microscopic examination and in vitro culture for parasite growth. Twenty-one acute cases of Chagas disease showed T. cruzi in blood. The research protocol secured the isolation of T. cruzi ECI1-to ECI21 from acute Chagas disease, and the DNA samples exhibited positive DNA footprints in the r...

Watch this Scientific Journal Video about Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates at JoVE.com

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

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 ...

The trypanosoma cruzi agent of Chagas disease may produce long-lasting symptomatic infections that may break open into clinical recognized pathology. The finding of the Trypanosoma cruzi nests in the seminiferous tube of a boy that died of Chagas disease suggests the research protocol to unravel the possible sexual transmission of the Chagas parasites. Deliver health care for the period of five years and recruit the families, showing participants with clinical symptoms of the acute Chagas disease, ACG.Obtained 15 ml of venous blood of family participants on three occasions one year apart, divided in three, five mil aliquots and store at four Centigrades. Collected two ml of the serum ejaculate from adult volunteers show the T.cruzi in the blood of the ACG cases to validate the molecular assays. Isolate the T.cruzi from the ACG blood aliquot is stored in sterile 10 ml tube with anticoagulant.Inoculate five ml of the blood into 50 ml tube slants plus five ml of liver infusion tryptose medium and incubate in a shaker at 27 Centigrades for three months. Search microscopy for the T.cruzi in 100 microliter of the supernatant on the glass slide. Harvest the flagellates from the medium centrifuged at 1, 000 x G for 10 min and washed in PBS pH 7.4.Inoculate 75 ml L6 muscle cell culture flask each with 10 to the sixth T.cruzi isolates. Feed the culture cells with 15 ml of Dulbecco Minimal Essential Medium, DMEM at 37 Centigrades. Grow the parasite isolates and the positive control Berenice T.cruzi.Grow the T.cruzi relative Leishmania braziliensis in DMEM as a negative control. Use 10 x 10 to the sixth tissue culture derived T.cruzi trypomastigotes to infect mice. Search for the nests of the T.cruzi in the sections of the muscles and organs of reproduction after one month.Use the blood aliquots in the EDTA Corning sterile 10 ml tube and collect the white cells by density gradient centrifugation at 3, 000 x G for 45 min. Wash the cells twice with PBS and centrifuge at 1, 500 x G for 10 min at a different tube. Extract the DNA from the isolate sent from the Berenice T.cruzi from L.braziliensis from 109 family subjects and from the spermatozoa from 21 participants as in the step 3.3 of the protocol.Measure the DNA concentrations on 0.8%agarose gel and absorbed at 206 nanometers. Use the T.cruzi TCZ one dash two primers anneal to the 188 nucleotide probe in the PCR analysis. Make the PCR mix with 10 nanogram template DNA, 0.4 micromolar of each pair of primers, two units stock DNA polymerase, 0.2 micromolar DNA-TPs, and 5 micromolar magnesium chloride in a 25 microliter final volume.Conduct the DNA amplification as in step 3.5.2 and show the DNA bands in the gel. Use the T.cruzi 188 nucleotide probe anneals to date primers label with alpha 32P dATP and separate the PCR product on a 1.3%agarose gel and transfer to a positively charged nylon membrane. Wash the membrane and now expose to X-rays field for variable periods.Grow the clones selected from the X-rays field and sequence commercially with the TCZ one dash two primers. Conduct a three independent immunofluorescence assays and triplicate one to 100 serum dilution in PBS from 109 study participants. Centrifuge the five ml aliquot of the enclosed blood at 1, 000 x G for 30 min and store the serum at minus 20 Centigrades.Run independent immunofluorescence serum dilutions from the study samples obtained on three occasions one year apart. Use 5 microliter of the formally incubated T.cruzi amastigotes or L.braziliensis promastigotes, 10 parasites per microliter in PBS suspensions onto glass slide and let it dry overnight, as in step 5.3.3. Incubate the parasite on the glass with 100 microliters of the one to 100 serum dilutions and cover with slip in a moist chamber for one hour at 37 Centigrades, then wash twice with PBS.Incubate the air-dried glass slides with one to 1, 000 dilution of a fluorescein labeled rabbit antibody to human IgG, wash and dry as well. Mount the glass slide with a cover slip and examine it under the UV light microscope. Detect the T.cruzi and the L.braziliensis soluble antigen one microgram in 100 microliter of carbonate buffer pH 9.6.Incubate with 100 microliter of the one to 100 serum dilution in triplicate quartered wells. Wash the plates twice with PBS-Tween solutions and wait to dry. Incubate with 100 microliter of the one to 1, 000 dilutions of alkaline phosphatase conjugate, goat anti-rabbit IgG.Wash the plates twice with PBS-Tween, add the substrate para-nitrophenylphospate and wait for the color development. Read the optical densities at 630 nanometers in a multi-mode plate reader. Transilluminate fertile chicken eggs and make a hole in the shell at their bubble.Inoculate 100 trypomastigotes on each shell culture medium in the mock control eggs. Seal the hole with adhesive tape. Incubate the eggs at 37 Centigrades, 65%humidity for 21 days.Grow the chicks hatched from live control eggs group A from the mock controls, group B and from the T.cruzi inoculated eggs, group C until their adult life. Challenge the chickens of group B and C twice with 10 x 10 to the seventh formally incubated trypomastigote weekly. Obtain two ml of the venous blood from a wing vein of the A, B, and C chickens five weeks after challenge.Separate the serum and run immunofluorescence analyzer to detect the T.cruzi antibody. Secure animal welfare with excellent housing, food, and water supply. Use the semen ejaculate from an adult, and a APCR positive Chagas disease case, and from a control subject as in step 1.5 of the protocol.Instill 100 microliter aliquots of semen into the peritoneal and into the vagina of mice. The sexual transmission of the T.cruzi infection in parental and progeny mice. The footprint of the Trypanosoma cruzi from the acute Chagas disease showed by the nDNA-PCR products that form in advance with the radio labeled probe.The phenotype of the T.cruzi revealed with the immunofluorescence assay. The anti-T. cruzi antibody does not recognize L.braziliensis.Graphic representation of the ELISA and of the nDNA-PCR assays. Groups one and two respectively, negative and positive control. Group three, positive nDNA-PCR and the specific antibody assays.Group four with the T.cruzi infections detected by the nDNA-PCR but in the absence of the specific antibody. Group five, infection free participants. The heredograms and mapping of the family population showed discrepancies among the ratios of the anti-T.cruzi antibody and those of the nDNA-PCR assays. Open is square and circle, negative male and female. Red with anti-T.cruzi antibody and nDNA-PCR positive. Black, positive nDNA-PCR alone. The immune tolerance in chickens hatched from T.cruzi inoculated eggs.A, mock control, B, live controlled chickens challenged with the T.cruzi antigen. C, chickens hatched from the T.cruzi inoculated eggs after challenge with the specific antigen. The infectivity of the T.cruzi showed upon semen instills into the peritoneal cavity and into the vagina.The mice sacrificed three weeks of these stills showed the amastigote nest in the heart, in the skeletal muscle, and in the vas deferens and in the uterine tube. The sexual transmission of the T.cruzi infections in the mouse model system. The T.cruzi infected males and females, transmitted the infection to naive mates.The T.cruzi infection vertically transferred from the parental to the progeny mice. The Southern blotting revealed the parasite anginate band in a majority of the F1 litters. The immunoperoxidase stain T.cruzi amastigotes showed engoniablast and in the interstitial of the epididymitis and into the lumen of seminiferous tubes of the progeny mice.The parasitological demonstration of the protozoan in 21 acute cases of Chagas disease was crucial to validate the T.cruzi footprint in all the infected subjects. The discrepancies among the ratios of the parasite antibody assays and those of the nucleic acids tests revealed the majority of the footprints in the antibody-free patients. The broad difference well explains in the chicken model reflected to the T.cruzi after the first week of the embryo development.The immune tolerance adult chickens showed the parasite footprints but with the absence of the specific antibody. The vital anti-cruzi in Chagas patient ejaculates initiated widespread infections upon instills into the mouse vagina and as well into the peritoneal cavity. The pathology showed the T.cruzi amastigote nests in the heart and skeletal muscles and in the vas deferens and the uterine tube.The breeding of Chagas mice with naive mates revealed the infected females and males transmitted the T.cruzi to the uninfected naive mates and the majority of their litters acquired the infection vertically transferred to the progeny. The family-based protocol addressed the nucleic acids assay in order to perform reliable blood transfusions and epidemiology inquiries and to elucidate the potential ongoing sexually transmitted Chagas disease.

sexual-transmission-american-trypanosomes-from-males-females-to-naive

Research

JoVE Journal

Immunology and Infection

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...