עבודה זו מומנה בחלקה על ידי מענק ההצטיינות סקירה מהמחלקה לענייני יוצאי צבא, על ידי מענקים NIH AI045540, AI067874, AI076233-01 ו AI080801 (מיו), ועל ידי AI29646 (SMB). העבודה בוצעה בחלקה במהלך המימון של CT ו JG ידי NIH T32 AI07511.

<ol>
	<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+stable+Leishmania+expression+vector+and+application+to+the+study+of+parasite+surface+antigen+genes.">Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes.</a> Proc Natl Acad Sci U S A. 87, 9736-9740 (1990).</li><li>Mureev, S., Kushnir, S., Kolesnikov, A. A., Breitling, R., Alexandrov, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+analysis+of+Leishmania+tarentolae+transgenic+strains+free+of+selection+markers.">Construction and analysis of Leishmania tarentolae transgenic strains free of selection markers.</a> Mol Biochem Parasitol. 155, 71-83 (2007).</li><li>Chakkalath, H. 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>

אין ניגודי אינטרסים הכריז.

In vivo מערכת הדמיה (IVIS) מספק שיטה הדמיה חיה כולו או מודלים vivo הדמיה זיהום ניסיוני של צורות שונות של leishmaniasis. 18,16 spp שושנת יריחו. הטפילים יכולים להיות מהונדסים להביע בלוציפראז גחלילית ברמה כי הוא זוהה in vivo עם הטכנולוגיה IVIS הדמיה. אחד היתרונות ?...

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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

מינים שונים של<em&gt; שושנת יריחו,</em&gt; טפיל protozoan המשפחה<em&gt; Trypanosomatidae</em&gt;, בדרך כלל על ביטויים שונים לגרום מחלות אנושיות. הצורות הנפוצות ביותר של המחלה הם leishmaniasis הקרביים (VL) ואת leishmaniasis עורית (CL). מודלים של עכבר leishmaniasis נמצאים בשימוש נרחב, אבל הכימות של הנטל טפיל במהלך המחלה בעכברים Murine דורש להיות מורדמים בזמנים שונים לאחר ההדבקה. המון טפיל נמדדים מכן על ידי מיקרוסקופ, הגבלת assay דילול, או qPCR הגברה של DNA הטפיל.<em&gt; In vivo</em&gt; מערכת הדמיה (IVIS) יש חבילת תוכנה משולבת המאפשרת זיהוי של אות bioluminescent הקשורים תאים אורגניזמים חיים. הן כדי למזער את השימוש בבעלי חיים כדי לעקוב זיהום longitudinally אצל אנשים, במודלים vivo עבור דימות<em&gt; שושנת יריחו</em&gt; Spp. VL גרימת או CL הוקמו. טפילים היו שהונדסו בלוציפראז, ואלה הוכנסו עכברים או intradermally או לווריד. מדידות כמותיות של פליטת אור הנהיגה בלוציפראז של מהונדס<em&gt; שושנת יריחו</em&gt; טפילים בתוך העכבר נעשו באמצעות IVIS. עכברים בודדים ניתן הדמיה מספר פעמים במהלך מחקרים ארוכי טווח, מה שמאפשר לנו להעריך את השונות בין בעלי חיים inocula טפיל הראשונית הניסוי, וכדי להעריך את כפל של טפילים ברקמות העכבר. טפילים מזוהים עם רגישות גבוהה במקומות עורית. למרות זאת הוא מאוד סביר להניח כי את האות (פוטונים / שנייה / טפיל) נמוך באיברים הקרביים עמוק יותר בעור, אך השוואות כמותיות של אותות שטחית לעומת אתרי עמוק לא נעשו. יתכן כי המספרים טפיל בין אתרים הגוף לא ניתן להשוות באופן ישיר, אם כי נטען טפיל ברקמות אותו ניתן להשוות בין העכברים. דוגמאות של מין אחד visceralizing (<em&gt; ל infantum chagasi</em&gt;) אחד המינים גורמת leishmaniasis עורית (<em&gt; ל מקסיקנה</em&gt;) מוצגים. ההליך IVIS יכול לשמש לניטור וניתוח של מודלים של בעלי חיים קטנים של מגוון רחב של<em&gt; שושנת יריחו</em&gt; מינים גורם צורות שונות של leishmaniasis האדם.

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

<em&gt; In vivo</em&gt; מערכת הדמיה משמש להפקת מדידות כמותיות של זיהום Murine עם protozoan Trypanosomatid<em&gt; שושנת יריחו</em&gt;. זו שיטה לא פולשנית ולא קטלני לאיתור טפילים להביע בלוציפראז בתוך רקמות רבות ברחבי במהלך כרוני<em&gt; שושנת יריחו</em&gt; Spp. זיהום.

בתחום ההדמיה vivo של טפילים טראנסגנטי שושנת יריחו שורה חיים

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

Research

JoVE Journal

Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

15.8K Views.  University of Iowa, and the VA Medical Center.  In vivo מערכת הדמיה משמש להפקת מדידות כמותיות של זיהום Murine עם protozoan Trypanosomatid שושנת יריחו. זו שיטה לא פולשנית ולא קטלני לאיתור טפילים להביע בלוציפראז בתוך רקמות רבות ברחבי במהלך כרוני שושנת יריחו Spp. זיהום.

Video: 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.

לא פולשני הדמיה של ביות ליקוציט והגירה 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 ...

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.

השימוש מלכודת אופטית לחקר מארח הפתוגן אינטראקציות עבור הדמיה דינמי תא חי

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.

אינדוקציה של מחלת שתל נגד מארח ו<em&gt; In Vivo</em&gt; ניטור תא T תוך שימוש במודל Murine MHC התאמה

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 ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

לומד קהילות חיידקים<em&gt; In vivo</em&gt;: דגם של אינטראקציה בתיווך בין מארח<em&gt; קנדידה אלביקנס</em&gt; ו<em&gt; Pseudomonas aeruginosa</em&gt; באיירווייס

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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.

שלית Decidual אדם תרבויות איברים ללמוד זיהומים בבית הממשק האם והעובר

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.

כימות של צמיחה תאיים בתוך המקרופאגים הוא מהיר, שיטה אמינה להערכת של התקפה אלימה של טפילים לישמניה

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.

אין ויוו הדמיה של מינים חמצן תגובתי ב מאתר פצע מודל

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

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.

ב Vivo זיהום עם לישמניה מלכותית כדי להעריך טפיל התקפה אלימה בעכברים

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

הדמיה חיה של קולטני כימוקין בנויטרופילים של דגי זברה במהלך תגובות לפצעים

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

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3).
Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy

The mechanism associated with phagocytosis in Leishmania infection remains poorly understood. Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells.