Ce travail a été financé en partie par une subvention d&#39;examen du mérite du ministère des Anciens Combattants, par des subventions du NIH AI045540, AI067874, AI076233-01 et AI080801 (MEW), et par AI29646 (SMB). Le travail a été effectué en partie au cours de financement du CT et JG par le NIH T32 AI07511.

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	<li>Capul, A. A., Barron, T., Dobson, D. E., Turco, S. J., Beverley, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+functionally+divergent+UDP-Gal+nucleotide+sugar+transporters+participate+in+phosphoglycan+synthesis+in+Leishmania+major.">Two functionally divergent UDP-Gal nucleotide sugar transporters participate in phosphoglycan synthesis in Leishmania major.</a> J Biol Chem. 282, 14006-14017 (2007).</li><li>LeBowitz, J. H., Coburn, C. M., McMahon-Pratt, D., Beverley, S. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priming+of+a+beta-galactosidase+(beta-GAL)-specific+type+1+response+in+BALB/c+mice+infected+with+beta-GAL-transfected+Leishmania+major.">Priming of a beta-galactosidase (beta-GAL)-specific type 1 response in BALB/c mice infected with beta-GAL-transfected Leishmania major.</a> Infect Immun. 68, 809-814 (2000).</li><li>Sacks, D. L., Perkins, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+infective+stage+of+Leishmania+promastigotes.">Identification of an infective stage of Leishmania promastigotes.</a> Science. 223, 1417-1419 (1984).</li><li>da Silva, R., Sacks, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metacyclogenesis+is+a+major+determinant+of+Leishmania+promastigote+virulence+and+attenuation.">Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.</a> Infect Immun. 55, 2802-2806 (1987).</li><li>Spath, G. F., Beverley, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lipophosphoglycan-independent+method+for+isolation+of+infective+Leishmania+metacyclic+promastigotes+by+density+gradient+centrifugation.">A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation.</a> Exp Parasitol. 99, 97-103 (2001).</li><li>Scott, P., Caspar, P., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+Leishmania+major+in+BALB/c+mice+by+adoptive+transfer+of+a+T+cell+clone+recognizing+a+low+molecular+weight+antigen+released+by+promastigotes.">Protection against Leishmania major in BALB/c mice by adoptive transfer of a T cell clone recognizing a low molecular weight antigen released by promastigotes.</a> J Immunol. 144, 1075-1079 (1990).</li><li>Belkaid, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+interleukin+(IL)-10+in+the+persistence+of+Leishmania+major+in+the+skin+after+healing+and+the+therapeutic+potential+of+anti-IL-10+receptor+antibody+for+sterile+cure.">The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antibody for sterile cure.</a> J Exp Med. 194, 1497-1506 (2001).</li><li>Wilson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+suppression+of+IFN-gamma+in+hepatic+granulomas+correlates+with+tissue-specific+replication+of+Leishmania+chagasi.">Local suppression of IFN-gamma in hepatic granulomas correlates with tissue-specific replication of Leishmania chagasi.</a> J Immunol. 156, 2231-2239 (1996).</li><li>McElrath, M. J., Murray, H. W., Cohn, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+granuloma+formation+in+experimental+visceral+leishmaniasis.">The dynamics of granuloma formation in experimental visceral leishmaniasis.</a> J Exp Med. 167, 1927-1937 (1988).</li><li>Ato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dendritic+cell+migration+and+impaired+resistance+to+Leishmania+donovani+infection+in+mice+deficient+in+CCL19+and+CCL21.">Loss of dendritic cell migration and impaired resistance to Leishmania donovani infection in mice deficient in CCL19 and CCL21.</a> J Immunol. 176, 5486-5493 (2006).</li><li>Ahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intradermal+infection+model+for+pathogenesis+and+vaccine+studies+of+murine+visceral+leishmaniasis.">Intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis.</a> Infect Immun. 71, 401-410 (2003).</li><li>Kamala, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hock+immunization:+a+humane+alternative+to+mouse+footpad+injections.">Hock immunization: a humane alternative to mouse footpad injections.</a> J Immunol Methods. 328, 204-214 (2007).</li><li>Lang, T., Goyard, S., Lebastard, M., Milon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+Leishmania+expressing+luciferase+for+rapid+and+high+throughput+screening+of+drugs+acting+on+amastigote-harbouring+macrophages+and+for+quantitative+real-time+monitoring+of+parasitism+features+in+living+mice.">Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote-harbouring macrophages and for quantitative real-time monitoring of parasitism features in living mice.</a> Cell Microbiol. 7, 383-392 (2005).</li><li>Brittingham, A., Miller, M. A., Donelson, J. E., Wilson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+GP63+mRNA+stability+in+promastigotes+of+virulent+and+attenuated+Leishmania+chagasi.">Regulation of GP63 mRNA stability in promastigotes of virulent and attenuated Leishmania chagasi.</a> Mol Biochem Parasitol. 112, 51-59 (2001).</li><li>Roy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Episomal+and+stable+expression+of+the+luciferase+reporter+gene+for+quantifying+Leishmania+spp.+infections+in+macrophages+and+in+animal+models.">Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.</a> Mol Biochem Parasitol. 110, 195-206 (2000).</li><li>Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18, 593-603 (1995).</li><li>Hardy, J., Margolis, J. J., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+Biliary+Excretion+of+Listeria+monocytogenes.">Induced Biliary Excretion of Listeria monocytogenes.</a> Infect. Immun. 74, 1819-1827 (2006).</li><li>Lecoeur, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Topical+Therapy+for+Leishmania+major+Localized+Cutaneous+Leishmaniasis+Using+a+Reliable+C57BL/6+Model.">Optimization of Topical Therapy for Leishmania major Localized Cutaneous Leishmaniasis Using a Reliable C57BL/6 Model.</a> PLoS Negl Trop Dis. 1, e34-e34 (2007).</li><li>Lang, T., Lecoeur, H., Prina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Leishmania+development+in+their+host+cells.">Imaging Leishmania development in their host cells.</a> Trends Parasitol. 25, 464-473 (2009).</li></ol>

Le système d&#39;imagerie in vivo (IVIS) fournit une méthode pour l&#39;imagerie des animaux entiers ou dans des modèles d&#39;imagerie in vivo l&#39;infection expérimentale de différentes formes de leishmaniose. 18,16 Le spp Leishmania. parasites peuvent être modifiées pour exprimer la luciférase de luciole à un niveau qui est détectée in vivo avec la technologie d&#39;imagerie IVIS. Un des principaux avantages de cette méthode est qu&#39;elle...

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		<th>Material Name</th>
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<td>D-Luciferin Potassium Salt </td>
<td>Reagent</td>
<td>Caliper LifeSciences (Formerly Xenogen)</td>
<td>122796</td>
<td>&nbsp;</td>
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<td>IVIS Imaging System 200 Series</td>
<td>Equipment</td>
<td>Caliper LifeSciences</td>
<td>&nbsp;</td>
<td>Other IVIS models that can be used include: Lumina II, Lumina XR, Kinetic, and Spectrum. </td>
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in vivo imaging of transgenic leishmania parasites in a live host

Espèces distinctes de<em&gt; Leishmania,</em&gt; Un parasite protozoaire de la famille<em&gt; Trypanosomatidae</em&gt;, Causent généralement différentes manifestations de la maladie humaine. Les formes les plus courantes de maladie sont la leishmaniose viscérale (LV) et la leishmaniose cutanée (LC). Des modèles murins de la leishmaniose sont largement utilisés, mais la quantification des charges parasites lors de la maladie nécessite des souris murin d&#39;être euthanasiés à différents moments après l&#39;infection. Charges parasitaires sont ensuite mesurées soit par microscopie, en limitant la dilution d&#39;essai, ou d&#39;amplification PCR quantitative de l&#39;ADN parasitaire. L&#39;<em&gt; In vivo</em&gt; Système d&#39;imagerie (IVIS) a un logiciel intégré qui permet la détection d&#39;un signal bioluminescent associés à des cellules dans les organismes vivants. Tant pour minimiser l&#39;utilisation d&#39;animaux et de suivre l&#39;infection longitudinalement dans les individus, dans des modèles in vivo pour l&#39;imagerie<em&gt; Leishmania</em&gt; Spp. provoquant VL ou CL ont été établis. Les parasites ont été conçues pour exprimer la luciférase, et ces souris ont été introduites dans deux intradermique ou intraveineuse. Les mesures quantitatives de la bioluminescence de la luciférase de la conduite du transgénique<em&gt; Leishmania</em&gt; Parasites dans la souris ont été faites en utilisant IVIS. Souris individuelles peuvent être imagées à plusieurs reprises au cours des études longitudinales, nous permettant d&#39;évaluer la variation inter-animal dans l&#39;inoculum parasitaire expérimentale initiale, et d&#39;évaluer la multiplication des parasites dans les tissus de la souris. Les parasites sont détectés avec une grande sensibilité dans les endroits cutanés. Bien qu&#39;il soit très probable que le signal (photons / seconde / parasite) est plus faible dans les organes viscéraux profond que la peau, mais les comparaisons quantitatives des signaux dans superficielle par rapport sites profonds n&#39;ont pas été faites. Il est possible que le nombre de parasites entre les sites de l&#39;organisme ne peut pas être directement comparés, bien que la charge parasitaire dans les mêmes tissus peuvent être comparés entre les souris. Exemples d&#39;une espèce visceralizing (<em&gt; L. infantum chagasi</em&gt;) Et une espèce causant la leishmaniose cutanée (<em&gt; L. mexicana</em&gt;) Sont affichés. La procédure IVIS peut être utilisé pour surveiller et analyser les modèles de petits animaux d&#39;une grande variété de<em&gt; Leishmania</em&gt; Espèces causant des différentes formes de leishmaniose humaine.

Watch this Scientific Journal Video about In vivo Imaging of Transgenic Leishmania Parasites in a Live Host at JoVE.com

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Une<em&gt; In vivo</em&gt; Système d&#39;imagerie est utilisée pour générer des mesures quantitatives de l&#39;infection murin avec le protozoaire trypanosomatides<em&gt; Leishmania</em&gt;. C&#39;est une méthode non invasive et non létales pour la détection des parasites exprimant la luciférase dans de nombreux tissus tout au long de chroniques<em&gt; Leishmania</em&gt; Spp. l&#39;infection.

L&#39;imagerie in vivo des parasites Leishmania transgéniques dans un hôte en direct

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

in-vivo-imaging-of-transgenic-leishmania-parasites-in-a-live-host

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JoVE Journal

Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

15.8K vues.  University of Iowa, and the VA Medical Center. Une In vivo Système d'imagerie est utilisée pour générer des mesures quantitatives de l'infection murin avec le protozoaire trypanosomatides Leishmania. C'est une méthode non invasive et non létales pour la détection des parasites exprimant la luciférase dans de nombreux tissus tout au long de chroniques Leishmania Spp. l'infection.

Vidéo: In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

L&#39;imagerie non invasive des Homing leucocytes et des migrations In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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Immunologie et infection

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

L&#39;utilisation d&#39;un piège optique pour l&#39;étude des interactions hôtes pathogènes pour l&#39;imagerie cellulaire dynamique en direct

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

L&#39;induction de la réaction du greffon contre l&#39;hôte et des maladies<em&gt; In Vivo</em&gt; Suivi des lymphocytes T utilisant un modèle murin MHC-apparié

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Cultures placentaire et déciduale organe humain pour l&#39;étude des infections à l&#39;interface materno-fœtale

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

Quantification de la croissance à l’intérieur de Macrophages intracellulaire est un rapide et une méthode fiable pour évaluer la Virulence de Leishmania

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

In Vivo Imagerie des espèces réactives de l’oxygène dans un murin plaie modèle

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Monocouches de Colonoid humain pour étudier les Interactions entre les pathogènes, germes commensaux et hôte épithélium Intestinal

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to visceral lesions. Currently, 12 million people are estimated to be infected with Leishmania worldwide and over 1 billion people live at the risk of infection. Leishmania amazonensis is endemic in Central and South America and usually leads to the cutaneous form of the disease, which can be directly visualized in an animal model. Therefore, L. amazonensis strains are good models for cutaneous leishmaniasis studies because they are also easily cultivated in vitro. C57BL/6 mice mimic the L. amazonensis-driven disease progression observed in humans and are considered one of the best mice strains model for cutaneous leishmaniasis. In the vertebrate host, these parasites inhabit macrophages despite the defense mechanisms of these cells. Several studies use in vitro macrophage infection assays to evaluate the parasite infectivity under different conditions. However, the in vitro approach is limited to an isolated cell system that disregards the organism&#39;s response. Here, we compile an in vivo murine infection method that provides a systemic physiological overview of the host-parasite interaction. The detailed protocol for the in vivo infection of C57BL/6 mice with L. amazonensis comprises parasite differentiation into infective amastigotes, mice footpad cutaneous inoculation, lesion development, and parasite load determination. We propose this well-established method as the most adequate method for physiological studies of the host immune and metabolic responses to cutaneous leishmaniasis.

In Vivo Infection with Leishmania amazonensis to Evaluate Parasite Virulence in Mice

Here, we present a compiled protocol to evaluate the cutaneous infection of mice with Leishmania amazonensis. This is a reliable method for studying parasite virulence, allowing a systemic view of the vertebrate host response to the infection.

Infection in vivo avec l’amazonense de Leishmania pour évaluer la virulence parasite chez les souris

Leishmania spp. are protozoan parasites that cause leishmaniases, diseases that present a wide spectrum of clinical manifestations from cutaneous to ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Imagerie en direct des récepteurs de chimiokine dans les neutrophiles du poisson-zèbre pendant les réponses des plaies

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infection récurrente des voies urinaires à Escherichia coli déclenchée par Gardnerella vaginalis Exposition à la vessie chez la souris

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...