המחברים מודים למכללה האזורית VA-MD ברפואת וטרינרית למתן תמיכת ליבה וטכנית עבור פרויקט זה. עבודה זו נתמכת על פיתוח קריירה פרס NIH (K01DK092355) על ידי.

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המחברים מצהירים שום אינטרסים כלכליים מתחרים.

השלבים הקריטיים ביותר להערכת התגובה החיסונית המארחת בריאות עכבר בהצלחה הוא כדלקמן: 1) לבחור את הזן המתאים עכבר ומין עבור הדגם נבדק; 2) לייעל את משלוח PAMP לריאות; 3) בצורה נכונה לאסוף ולעבד את בלף; ו -4) לתקן כראוי ולהכין של הריאות להערכות histopathological. <p class="jove_content" style=";text-align...

דלקות ריאה הקשורים למיני חיידקים פתוגניים הם גורמים שכיחים לתחלואה ותמותה בעולם. קביעת המנגנונים המניעים את התגובה החיסונית המארחת לפתוגנים אלה יהיו לקדם את הפיתוח של אסטרטגיות חדשניות למניעה וסוכנים טיפוליים שיהיה להחליש את ההשפעה של זיהומים אלה. המטרה הכללית של הפרוטוקול המתואר כאן היא לספק את המשתמש עם שיטה גמישה כדי להעריך את התגובה החיסונית המולדת המארח לזיהום הפתוגן באמצעות דפוס הפתוגן הקשורים מולקולרי (PAMP) כתחליף לחיידקים חיים. רוב המחקרים הקודמים להערכת התגובה החיסונית המולדת לחיידקי המארח התמקדו בדגמי הצפק בשל הקלות היחסית של ביצוע. בעוד מודלים אלה הם שימושיים מאוד והביאו להתקדמות משמעותית בתחום של אינטראקציות בין המאכסן לפתוגן ודלקת מערכתית, הנתונים המופקים ממודלים אלה לא תמיד מתאימים ללימודים involving מערכת הנשימה. הנה, מודל ריאות של דלקת ריאות חריפה מוצע כהרחבה מעשית ורלוונטית קליני של המודלים הזרקה (IP) intraperitoneal הקלאסיים. הטכניקה המוצעת מאפשרת להערכה המקומית של תגובת החיסון המולדת במערכת מודל ספציפית איברים. השיטות שתוארו כאן נועדו לספק טכניקה פשוטה וחזקה כדי לאפשר למשתמשים להעריך את התגובה החיסונית המארחת לLPS, שהוא PAMP משותף. השיטות מבוססות על intratracheal החדרה (את זה) של LPS, אשר גורם לתגובה חיסונית מולדת חזקה לריאות של עכברים ומחקה רבים מהתכונות pathophysiological נצפו בחולים אנושיים הסובלים מזיהומים בדרכי הנשימה ופציעת ריאות חריפות 1. יתרון עיקרי של שיטה זו הוא שהיא מאפשרת למשתמש להעריך את התגובה החיסונית המארחת בלי גורמי בלבול וחששות בטיחות הקשורים לביצוע במחקרי vivoשימוש בחיידקים חיים. כמו כן, יש מסלול ממשלו הלוע התחתון של חשיפה שתוארה בפרוטוקול זה יתרונות משמעותיים על פני טכניקות אחרות מנוצלים בדרך כלל, כוללים אפי (ב) ממשל ומנהלו כירורגית. לדוגמא, ממשלו הלוע התחתון מאפשר dosaging מדויק יחסית ותצהיר ריאות בהשוואה בממשל, אשר בדרך כלל סובל מהשתנות מוגברת של תצהיר ריאות עקב האובדן של סוכנים בחלל האף והסינוסים 2-4. מסלול הממשל עוקף חללים אלו ומאפשר גישה ישירה לקנה הנשימה ודרכי הנשימה. כמו כן, גישתה כירורגית היא שיטת ממשל באופן משמעותי יותר חולנית ודורשת הכשרה מקיפה להורים. הפרוטוקולים המתוארים כאן כוללים גם תיאור טכניקות הנפוצות וסמנים פונדקאיות המשמשים להערכת התקדמות דלקת ומסתיים בפרוטוקול המתאר את הטכניקות המתאימות להכנת luNGS להערכות histopathology. פרוטוקולים אלה התמקדו במזעור מספר העכברים הנדרשים לכל מחקר על ידי למקסם את הנתונים המופקים מבעלי חיים הבודדים. הפרוטוקולים שתוארו הם גמישים ביותר וניתן לשנות בקלות להעריך PAMPs מגוון רחב ודפוסי נזקים הקשורים מולקולריים (damps). יתר על כן, עם כמה שינויים נוספים, יכולים להיות מיושמים גם בפרוטוקולים אלה למחקרים להערכת התקדמות מחלה בדרכי הנשימה אלרגיות או אינטראקציות בין המאכסן לפתוגן עם חיידקים חיים, וירוסים או פטריות 5-10.

<table border="0" cellpadding="0" cellspacing="0" width="596">
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	<tbody>
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			<td style="width:181px;">Ohaus Scale Corporation</td>
			<td style="width:147px;">71142845</td>
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			<td height="20" style="height:20px;width:269px;">Small Animal Rectal Thermometer</td>
			<td style="width:181px;">Braintree Scientific</td>
			<td style="width:147px;">TH 5</td>
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			<td height="20" style="height:20px;width:269px;">Rectal Probe for Rodents</td>
			<td style="width:181px;">Braintree Scientific</td>
			<td style="width:147px;">RET 3</td>
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			<td height="20" style="height:20px;width:269px;">Ear Punch</td>
			<td style="width:181px;">Braintree Scientific</td>
			<td style="width:147px;">EP-S 901</td>
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			<td style="width:181px;">InvivoGen</td>
			<td style="width:147px;">LPS-EB</td>
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			<td style="width:147px;">10010-023</td>
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			<td style="width:181px;">Baxter</td>
			<td style="width:147px;">40032609</td>
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			<td style="width:181px;">ICAP Manufacturing</td>
			<td style="width:147px;">n/a</td>
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			<td style="width:181px;">Braintree Scientific</td>
			<td style="width:147px;">RIS 100</td>
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			<td height="20" style="height:20px;">Scissors (blunt/sharp)</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:147px;">13-806-2</td>
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			<td height="20" style="height:20px;">forceps (straight)</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td>22-327-379</td>
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			<td height="23" style="height:23px;">forceps (45&#186;, curved)</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td>10-275</td>
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			<td height="20" style="height:20px;">Scissors (blunt/blunt)</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td>08-940</td>
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			<td height="20" style="height:20px;width:269px;">Pipette (200 &#181;l Capacity)</td>
			<td style="width:181px;">Gilson</td>
			<td style="width:147px;">F123601</td>
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			<td height="20" style="height:20px;width:269px;">Ethanol</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:147px;">459844</td>
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			<td height="20" style="height:20px;width:269px;">&#160;1 ml Syringe</td>
			<td style="width:181px;">BD Medical</td>
			<td style="width:147px;">301025</td>
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			<td height="20" style="height:20px;width:269px;">10 ml Syringe</td>
			<td style="width:181px;">BD Medical</td>
			<td style="width:147px;">301604</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:269px;">27 G x 0.5 in. needle</td>
			<td style="width:181px;">BD Medical</td>
			<td style="width:147px;">305109</td>
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			<td height="20" style="height:20px;width:269px;">Refrigerated Microcentrifuge</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:147px;">13-100-676</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:269px;">1.2 mm Tracheal Cannulae with Luer-adapter</td>
			<td style="width:181px;">Harvard Apparatus</td>
			<td style="width:147px;">732836</td>
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			<td height="20" style="height:20px;width:269px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:181px;">Life Technologies</td>
			<td style="width:147px;">14025-076</td>
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			<td height="20" style="height:20px;width:269px;">4-0 Silk Braided Surgical Suture</td>
			<td style="width:181px;">Ethicon</td>
			<td style="width:147px;">A183</td>
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			<td style="width:181px;">Harvard Apparatus</td>
			<td style="width:147px;">721406</td>
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			<td style="width:147px;">721664</td>
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			<td style="width:181px;">Sigma</td>
			<td style="width:147px;">R-3603</td>
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			<td height="20" style="height:20px;width:269px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:181px;">Sigma</td>
			<td style="width:147px;">HT501128-4L</td>
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			<td height="20" style="height:20px;width:269px;">Mouse IL-1&#946; OptEIA ELISA Kit</td>
			<td style="width:181px;">BD Biosciences</td>
			<td style="width:147px;">559603</td>
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			<td height="20" style="height:20px;width:269px;">Mouse IL-6 OptEIA ELISA Kit</td>
			<td style="width:181px;">BD Biosciences</td>
			<td style="width:147px;">550950</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Mouse TNF-&#945; OptEIA ELISA Kit</td>
			<td style="width:181px;">BD Biosciences</td>
			<td style="width:147px;">560478</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Hemacytometer</td>
			<td style="width:181px;">Hausser Scientific</td>
			<td style="width:147px;">3520</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Hemacytometer Cover Glasses</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:147px;">22-021-801&#160;</td>
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			<td height="20" style="height:20px;width:269px;">Trypan Blue</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:147px;">SV3008401</td>
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			<td height="20" style="height:20px;">Cytology Funel Clips</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td>10-357</td>
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			<td height="20" style="height:20px;">Cytology Funels</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td>10-354</td>
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			<td height="20" style="height:20px;">Filter Cards</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td>22-030-410</td>
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			<td height="20" style="height:20px;width:269px;">Microscope Slides</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:147px;">12-544-1</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cover Glasses</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:147px;">12-540A</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Cytospin Cytocentrifuge</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:147px;">A78300003&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Diff Quick Staining Kit</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:147px;">47733150</td>
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			<td height="20" style="height:20px;width:269px;">Permount Mounting Medium</td>
			<td style="width:181px;">Fisher Scientific</td>
			<td style="width:147px;">SP15-500</td>
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</table>

the utilization of oropharyngeal intratracheal pamp administration and bronchoalveolar lavage to evaluate the host immune response in mice

התגובה החיסונית המארח לפתוגנים היא תהליך ביולוגי מורכב. רוב במחקרי vivo מועסק קלסי לאפיין אינטראקציות בין המאכסן לפתוגן לנצל את זריקות intraperitoneal של חיידקים בחרו או דפוסי הפתוגן הקשורים מולקולריים (PAMPs) בעכברים. בעוד טכניקות אלה הניבו נתונים עצומים הקשורים pathobiology מחלות זיהומיות, מודלים זריקת intraperitoneal הם לא תמיד מתאימים ללימודי אינטראקציה המאכסן לפתוגן בריאות. ניצול מודל דלקת ריאות חריף בעכברים, ניתן לבצע ניתוח ברזולוציה גבוה של מארח תגובה חיסונית מולדת ניצול lipopolysaccharide (LPS). כאן, אנו מתארים את השיטות לניהול LPS באמצעות ממשל intratracheal הלוע התחתון לא נזקק לניתוח, לעקוב אחר פרמטרים קליניים הקשורים להיווצרות מחלה, ולנצל את נוזל שטיפת רונכואלוואולרית כדי להעריך את התגובה החיסונית המארחת. הטכניקות המתוארותחלים נרחב ללימוד התגובה החיסונית המולדת המארח למגוון רחב של PAMPs ופתוגנים. כמו כן, עם שינויים קלים, יכולות להיות מיושמות גם בטכניקות אלה במחקרים להערכת דלקת בדרכי הנשימה ואלרגיות ביישומים תרופתיים.

דופן התא של חיידקי גרם שלילי מורכבים מLPS, שהוא נרחב ביותר בסביבה. שאיפה של LPS באוכלוסיות אנושיות רגישות מחריפה תגובתיות דרכי הנשימה ומסוגלת להפעיל response11 חיסונית חזק. LPS גם PAMP נפוץ בשימוש במודלים של עכבר כדי לעורר תגובה חיסונית מולדת חזקה. בפרוטוקול מתואר כאן, העכברים ק...

Watch this Scientific Journal Video about The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice at JoVE.com

The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

התגובה החיסונית המארחת לזיהום הפתוגן היא תהליך מוסדר היטב. ניצול מודל חשיפת ריאות lipopolysaccharide בעכברים, ניתן לבצע הערכות ברזולוציה גבוהה של המנגנונים המורכבים הקשורים להיווצרות מחלה.

הניצול של לוע התחתון Intratracheal PAMP מנהל ושטיפה ברונכואלוואולרית כדי להעריך את תגובת מארח חיסון של עכברים

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen ...

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JoVE Journal

Immunology and Infection

16.5K Views.  Virginia Polytechnic Institute and State University.  התגובה החיסונית המארחת לזיהום הפתוגן היא תהליך מוסדר היטב. ניצול מודל חשיפת ריאות lipopolysaccharide בעכברים, ניתן לבצע הערכות ברזולוציה גבוהה של המנגנונים המורכבים הקשורים להיווצרות מחלה. 

Video: The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

מדידת עומס בקטריאלי ותגובות החיסון של עכברים נגועים עם חיידקי ליסטריה</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

מבוא של צרעות טפיליות<em&gt; תסיסנית</em&gt; ואת התגובה החיסונית Antiparasite

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

מדידת כמותית של התגובה החיסונית ובשינה<em&gt; דרוזופילה</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

כימות תגובת Burst נשימה כאינדיקטור של החיסון מולדת בריאות בדג הזברה

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

באמצעות RNA-התערבות לחקר התגובה החיסונית המולדת בהמקרופאגים עכבר

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

בניתוח חוץ גופייה של תיווך Myd88 הסלולרי תגובה החיסונית לוירוס הנילוס המערבי זיהום הזנים Mutant

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data have encouraged pulmonary tuberculosis vaccines as a promising strategy to prevent pulmonary disease, which is responsible for transmission. In this work, we describe the methodology used to demonstrate in the mouse model the benefits of intranasal BCG vaccination when compared to subcutaneous. Our data revealed greater protective efficacy following intranasal BCG administration. In addition, our results indicate that pulmonary vaccination triggers a higher immune response in lungs, including Th1 and Th17 responses, as well as an increase of immunoglobulin A (IgA) concentration in respiratory airways. Our data show correlation between protective efficacy and the presence of IL17-producing cells in lungs post-Mycobacterium tuberculosis challenge, suggesting a role for this cytokine in the protective response conferred by pulmonary vaccination. Finally, we detail the global workflow we have developed to study respiratory vaccination in the mouse model, which could be extrapolated to other tuberculosis vaccines, apart from BCG, targeting the mucosal response or other pulmonary routes of administration such as the intratracheal or aerosol.

We herein detail the methodology followed to compare protective efficacy and lung immune response induced by intranasal and subcutaneous immunization with BCG in mouse model. Our results show the benefits of pulmonary vaccination and suggest a role for IL17-mediated response in vaccine-induced protection.

מגן היעילות ריאתי החיסונית בתגובה תת עורי ואת אפי BCG מינהל עכברים

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is based on the injection of non-pathogenic particles into insects, as the particles will be detected by the immune system and will induce the production of immune effectors. We focus here on the stimulation of the melanization response in the mosquito Anopheles gambiae. The melanization response results in the encapsulation of foreign particles and parasites with a dark layer of melanin. To stimulate this response, mosquitoes are inoculated with beads in the thoracic cavity using microcapillary glass tubes. Then, after 24 hr, the mosquitoes are dissected to retrieve the beads. The degree of melanization of the bead is measured using image analysis software. Beads do not have the pathogenic effects of parasites, or their capacity to evade or suppress the immune response. These injections are a way to measure immune efficacy and the impact of immune stimulations on other life history traits, such as fecundity or longevity. It is not exactly the same as directly studying host-parasite interactions, but it is an interesting tool to study immunity and its evolutionary ecology.

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

ייחסתי<em&gt; Gambiae אנופלס</em&gt; יתושים עם חרוזים כדי לעודד ו למדוד את התגובה החיסונית melanization

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain insight into an ongoing disease state.
Here, a simple and efficient method is described to perform BAL on murine lungs without the need of special tools or equipment. BAL fluid is isolated by inserting a catheter in the trachea of terminally anesthetized mice, through which a saline solution is instilled into the bronchioles. The instilled fluid is gently retracted to maximize BAL fluid retrieval and to minimize shearing forces. This technique allows the viability, function, and structure of cells within the airways and BAL fluid to be preserved.
Numerous techniques may be applied to gain further understanding of the disease state of the lung. Here, a commonly used technique for the identification and enumeration of different types of immune cells is described, where&#160;flow cytometry is combined with a select panel of fluorescently labeled cell surface-specific markers. The BAL procedure presented here can also be used to analyze infectious agents, fluid constituents, or inhaled particles within murine lungs.

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

ברונכאלווולאר של ריאות Murine כדי לנתח דלקת תאים דלקתית

Bronchoalveolar Lavage (BAL) is an experimental procedure that is used to examine the cellular and acellular content of the lung lumen ex vivo to gain ...

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.