חיה המומחה בעלי מסופק על ידי טינה מרטין דייוויד אולברייט מוערכת בהכרת תודה. מחקר זה נתמך על ידי מענקים T32-AI-07285 (ח&quot;נ), NIH R25 2GM064133 (TCL), 1R03-HD061671-01, R24-AI-059830-06 מ-NIH.

<ol>
	<li>Kobel, H. R., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperdiploid+species+hybrids+for+gene+mapping+in+Xenopus.">Hyperdiploid species hybrids for gene mapping in Xenopus.</a> Nature. 279, 157-158 (1979).</li><li>Robert, J., Menoret, A., Basu, S., Cohen, N., Srivastava, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+the+molecular+and+immunological+properties+of+the+chaperones+gp96+and+hsp70.">Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70.</a> Eur J Immunol. 31, 186-195 (2001).</li><li>Robert, J., Gantress, J., Cohen, N., Maniero, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+an+experimental+model+for+studying+evolution+of+hsp--immune+system+interactions.">Xenopus as an experimental model for studying evolution of hsp--immune system interactions.</a> Methods. 32, 42-53 (2004).</li><li>Chardonnens, X., Pasquier, D. u., L, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+skin+allograft+tolerance+during+metamorphosis+of+the+toad+Xenopus+laevis:+a+possible+model+for+studying+generation+of+self+tolerance+to+histocompatibility+antigens.">Induction of skin allograft tolerance during metamorphosis of the toad Xenopus laevis: a possible model for studying generation of self tolerance to histocompatibility antigens.</a> Eur J Immunol. 3, 569-573 (1973).</li><li>Du Pasquier, L., Bernard, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+suppression+of+the+allogeneic+histocompatibility+reactions+during+the+metamorphosis+of+the+clawed+toad+Xenopus.">Active suppression of the allogeneic histocompatibility reactions during the metamorphosis of the clawed toad Xenopus.</a> Differentiation. 16, 1-7 (1980).</li><li>Ramanayake, T., Simon, D. A., Frelinger, J. G., Lord, E. M., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+study+of+T-cell+responses+to+skin+alloantigens+in+Xenopus+using+a+novel+whole-mount+immunohistology+method.">In vivo study of T-cell responses to skin alloantigens in Xenopus using a novel whole-mount immunohistology method.</a> Transplantation. 83, 159-166 (2007).</li><li>Robert, J., Ramanayake, T., Maniero, G. D., Morales, H., Chida, A. S., S, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+glycoprotein+96+ability+to+interact+with+CD91+and+facilitate+antigen+cross-presentation.">Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation.</a> J Immunol. 180, 3176-3182 (2008).</li><li>Robert, J., Gantress, J., Rau, L., Bell, A., Cohen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minor+histocompatibility+antigen-specific+MHC-restricted+CD8+T+cell+responses+elicited+by+heat+shock+proteins.">Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins.</a> J Immunol. 168, 1697-1703 (2002).</li><li>Morales, H., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+techniques+for+comparative+study+of+antiviral+T-cell+responses+in+the+amphibian+Xenopus.">In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus.</a> Biol Proced Online. 10, 1-8 (2008).</li><li>Maniero, G. D., Robert, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+conservation+of+gp96-mediated+antigen-specific+cellular+immunity:+new+evidence+from+adoptive+cell+transfer+in+xenopus.">Phylogenetic conservation of gp96-mediated antigen-specific cellular immunity: new evidence from adoptive cell transfer in xenopus.</a> Transplantation. 78, 1415-1421 (2004).</li></ol>

אין ניגודי אינטרסים הכריז.

Xenopus דוחי הוא מודל ייחודי צדדי שאינם יונקים ללמוד חסינות. השימוש הנרחב שלה מחקר ביו החיסונית הניב רבות כלי מחקר חשוב כמו שיבוטים MHC מוגדר LG-6 ו-LG-15 וכן שורות תאים שונות נוגדנים חד שבטיים. השימוש בכלים אלו הקמנו שונים במבחנה מבחני vivo ללמוד את היכולת של הלם ?...

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<td>Reagents needed:</td>
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<td>Amphibian Phosphate Buffered Saline (APBS): </td>
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<td>NaCl, 1.15 g/L </td>
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<td>Na2HPO4, 0.2 g/L </td>
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<td>KH2PO </td>
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<td>10N NaOH </td>
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<td>Tricaine Methane Sulfonate (TMS, MS-222) </td>
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<td>Crescent Research Chemicals </td>
<td>CAS#886-86-2</td>
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<td>Sodium bicarbonate </td>
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<td>Fisher Scientific </td>
<td>S-233-500</td>
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<td>Histopaque-1077</td>
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<td>Sigma-Aldrich</td>
<td>10771</td>
<td>100ml</td>
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<td>Heparin Sodium Salt </td>
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<td>Sigma-Aldrich </td>
<td>H3149-50KU</td>
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<td>Culture medium for Xenopus 15/0 tumors [see 3 for more details]:</td>
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<td>Iscove DMEM basal medium</td>
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<td>Gibco-Invitrogen </td>
<td>11965</td>
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<td>Insulin</td>
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<td>Non-essential amino acids</td>
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<td>Penicillin-streptomycin </td>
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<td>Kanamycin</td>
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<td>Primatone</td>
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<td>Sheffield Products Division</td>
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<td>&beta;2-mercaptoethanol</td>
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<td>NaHCO3 </td>
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<td>30% double distilled water </td>
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<td>5% featal bovine serum</td>
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<td>20% superantant from a Xenopus kidney cell line A6 </td>
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<td>0.25% of normal Xenopus serum</td>
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<td>Materials and Equipment:</td>
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<td>50 and 15 ml conical centrifuge tubes (sterile)</td>
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<td>25 gauge 5/8 Precision Glide sterile needles</td>
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<td>BD</td>
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<td>18 gauge 1&frac12; Precision Glide sterile needles</td>
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<td>BD</td>
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<td>1 ml Tuberculin Slip Tip Syringe sterile</td>
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<td>BD</td>
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<td>10 ml Slip Tip Syringe sterile</td>
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<td>BD</td>
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<td>60 X 15 mm Polystyrene Petri Dishes sterile</td>
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<td>25 X 75 mm X 1 mm Premium Microscope Slides </td>
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<td>Fisher Scientific</td>
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<td>10 cm glass petri dishes</td>
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<td>9" Pasteur Pipetes Durex Borosilicate Glass Cotton Plugged Disposable</td>
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<td>VWR</td>
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<td>Tygon tubing</td>
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<td>Two # 5 Swiss Jeweler&#x2019;s Forceps</td>
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<td>Miltex Inc.</td>
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<td>Micro Dissecting Spring Scissors McPherson-Vannas straight cutting edge 6 mm</td>
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<td>Roboz</td>
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<td>Helios calipers </td>
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<td>Dissecting microscope</td>
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<td>High intensity illuminator</td>
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<td>Hemacytometer</td>
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<td>Un-bunsen burner</td>
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<td>37&deg;C shaking incubator</td>
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<td>Centrifuge</td>
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comparative in vivo study of gp96 adjuvanticity in the frog xenopus laevis

פיתחנו את Xenopus דוחי laevis מודל ייחודי שאינם יונקים ללמוד את היכולת של חלבונים מסוימים הלם חום (hsps) כגון gp96 כדי להקל צולבות הצגת אנטיגנים מלווה ומעוררים מולד אדפטיבית התגובות תא T. Xenopus דחיית שתל עור מספק פלטפורמה מצוינת ללמוד את היכולת של gp96 לעורר קלאסית MHC בכיתה Ia (Ia בכיתה) מוגבלת התגובות תא T. בנוסף, מערכת מודל Xenopus גם מספק חלופה אטרקטיבית כדי עכברים לחקור את היכולת של gp96 ליצור תגובות נגד גידולים יש למטה מוסדר בכיתה Ia שלהם מולקולות ובכך להימלט מעקב החיסונית. לאחרונה, פיתחנו תא המאמצת להעביר assay ב Xenopus באמצעות שיבוטים leukocytes הצפק כמו תאים הצגת אנטיגן (נגמ&quot;שים), וכן הראו כי gp96 יכולה הממשלה CD8 T התגובות תא in vivo נגד אנטיגנים עור קלים histocompatibility כמו גם נגד הגידול Xenopus הרתי 15 / 0 זה לא להביע Ia מולקולות בכיתה. אנו מתארים כאן את המתודולוגיה המעורבים לבצע את מבחני לרבות העברה, התמחרות פועם המאמצת של לויקוציטים הצפק, וכן השתלת עור מבחני השתלת הגידול. בנוסף אנחנו גם המתאר את קצירת והפרדה של לויקוציטים דם היקפיים המשמשים cytometry זרימה מבחני התפשטות המאפשרים אפיון נוסף של אוכלוסיות מעורבות מפעיל דחייה העור אנטי הגידול התגובות.

Watch this Scientific Journal Video about Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis at JoVE.com

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

הצפרדע<em&gt; Xenopus laevis</em&gt; מספק מודל אטרקטיבי חלופי שאינו לחקר יונקים את היכולת של חלבון הלם חום כגון gp96 לקדם אנטיגן ספציפי CD8 T התגובות התא. אנו מציגים שיטות מחקר<em&gt; In vivo</em&gt; סיוע של המצגת הנגדית של אנטיגנים סרטניים בעור על ידי gp96.

יחסי In vivo לימוד Adjuvanticity gp96 ב הצפרדע Xenopus laevis</em

We have developed in the amphibian Xenopus laevis a unique non-mammalian model to study the ability of certain heat shock proteins (hsps) such as gp96 to ...

comparative-vivo-study-gp96-adjuvanticity-frog-xenopus

Research

JoVE Journal

Immunology and Infection

Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

10.5K Views.  University of Rochester. הצפרדע Xenopus laevis מספק מודל אטרקטיבי חלופי שאינו לחקר יונקים את היכולת של חלבון הלם חום כגון gp96 לקדם אנטיגן ספציפי CD8 T התגובות התא. אנו מציגים שיטות מחקר In vivo סיוע של המצגת הנגדית של אנטיגנים סרטניים בעור על ידי gp96.

Video: Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

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 ...

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 ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Leishmaniasis עורית בעור הגב של אוגרים: מודל שימושי להקרנה של תרופות Antileishmanial

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia culturing ממערכת העצבים המרכזי וילודים למבוגרים

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

במבחנת in vivo המודל ללמוד הידבקות חיידקים לכלי השיט וול תנאי זרימה תחת

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

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 ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retroviral תמרה של Helper T תאים כגישה גנטית לחקר מנגנוני בקרת הבידול והתפקוד שלהם

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

לא פולשנית דגימה של נוזל שורות Mucosal עבור כימות של In Vivo דרכי הנשימה העליונות החיסון-מגשרת רמות

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

לכימות מונוציט האנושי כימוטקסיס במבחנה , לימפוציט מאתר סחר In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.