תמיכה סופק על ידי בקטון דיקינסון טכנולוגיות. הדעות המובאות הגשת זה הם אלה של הכותבים ואינם מתיימרים לשקף את המדיניות הרשמית של ממשלת ארה&quot;ב. המחקר נערך בהתאם לחוק לרווחת בעלי חיים וכן חוקים ותקנות פדרליים אחרים הקשורים בחיות וניסויים מעורבים בעלי חיים ועקרונות האמורים מדריך לטיפול ושימוש בחיות מעבדה, המועצה הלאומית למחקר, 1996. המתקן שבו המחקר נערך באופן מלא הוא מוכר על ידי האגודה הערכת הסמכה של מעבדה בעלי חיים טיפול הבינלאומי.

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Vaccine Immunol. 18, 667-675 (2011).</li><li>Asanuma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mouse+nasal-associated+lymphoid+tissue.">Isolation and characterization of mouse nasal-associated lymphoid tissue.</a> J. Immunol. Methods. 202, 123-131 (1997).</li><li>Casteleyn, C., Broos, A. M., Simoens, P., Van den Broeck, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NALT+(nasal+cavity-associated+lymphoid+tissue)+in+the+rabbit.">NALT (nasal cavity-associated lymphoid tissue) in the rabbit.</a> Vet. Immunol. Immunopathol. 133, 212-218 (2010).</li><li>Debertin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal-associated+lymphoid+tissue+(NALT):+frequency+and+localization+in+young+children.">Nasal-associated lymphoid tissue (NALT): frequency and localization in young children.</a> Clin. Exp. Immunol. 134, 503-507 (2003).</li><li>Csencsits, K. L., Jutila, M. A., Pascual, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal-associated+lymphoid+tissue:+phenotypic+and+functional+evidence+for+the+primary+role+of+peripheral+node+addressin+in+naïve+lymphocyte+adhesion+to+high+endothelial+venules+in+a+mucosal+site.">Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naïve lymphocyte adhesion to high endothelial venules in a mucosal site.</a> J. Immunol. 163, 1382-1389 (1999).</li><li>Zuercher, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nasal-associated+lymphoid+tissue+is+a+mucosal+inductive+site+for+virus-specific+humoral+and+cellular+immune+responses.">Nasal-associated lymphoid tissue is a mucosal inductive site for virus-specific humoral and cellular immune responses.</a> J. Immunol. 168, 1796-1803 (2002).</li><li>Park, H. S., Francis, K. P., Yu, J., Cleary, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranous+cells+in+nasal-associated+lymphoid+tissue:+a+portal+of+entry+for+the+respiratory+mucosal+pathogen+group+A+streptococcus.">Membranous cells in nasal-associated lymphoid tissue: a portal of entry for the respiratory mucosal pathogen group A streptococcus.</a> J. 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Immunol. 168, 986-990 (2002).</li><li>Rangel-Moreno, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CXC+chemokine+ligand+13,+CC+chemokine+ligand+(CCL)+19,+and+CCL21+in+the+organization+and+function+of+nasal-associated+lymphoid+tissue.">Role of CXC chemokine ligand 13, CC chemokine ligand (CCL) 19, and CCL21 in the organization and function of nasal-associated lymphoid tissue.</a> J. Immunol. 175, 4904-4913 (2005).</li><li>Morefield, G. L., Hawkins, L. D., Ishizaka, S. T., Kissner, T. L., Ulrich, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+Toll-like+receptor+4+agonist+enhances+vaccine+efficacy+in+an+experimental+model+of+toxic+shock+syndrome.">Synthetic Toll-like receptor 4 agonist enhances vaccine efficacy in an experimental model of toxic shock syndrome.</a> Clin. 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אין ניגוד עניינים הצהיר.

הצגנו שיטות קולקטיביות על פיתוח מודל החיה, קבלת דגימות ביולוגיות, מבחני לבחינת NALT הקשורים התגובה החיסונית 1-4. ישנם גורמים נוספים שיש להביא בחשבון בעת ​​ביצוע שיטות אלה. טכניקות סטנדרטיות סטריליים לניתוח ותרבות רקמה יש ללכת. שילוב של סוכנים אנטיבקטריאלי נגד פטריות שימוש במהלך הבידוד ותרבות, כמו גם שמירה על מכשירים מעוקרים, אזור העבודה, ואת חיך מחוטאות תפחית את הסיכון של זיהום. בדיקת הרוק, האף דומים דגימות הפרשת הריריות יש לבדוק על אפשרות של זיהום בדם, כמו נוגדנים בסרום נמצאים בדרך כלל בריכוזים גבוהים יותר. יתר על כן, ההפרשות הריריות צריך רק להיות מדולל במעט לניתוח כי ריכוזים נמוכים יותר של נוגדנים נמצאים דגימות אלה לעומת בסרום. עכברים יש לשמור על חם מיד לאחר הניתוח מראשלפרוק היפותרמיה הנגרמת הפוטנציאל הרדמה. עכברים מנוחה לסירוגין משני שלהם במהלך ההחלמה שלאחר הניתוח כדי למזער נשימה סדירה. ניתוח יעיל יותר עם שלושה אנשים עובדים יחד כדי להשלים את המשימות הבאות: 1 ביצוע הניתוח, 1 כדי לסייע מחזיק את הפה פתוח, ואחד לספק לאחר ניתוח טיפול כמו העכברים להתאושש מן ההרדמה. יש צורך להשתמש ב H &amp; E מכתים של חתכים הגולגולת בסוף כל ההליכים או מחקרים ניסויים כדי לוודא את הצלחת הניתוח על כל עכבר. ניתוח התוצאות האפשריות הן: אבלציה NALT מלא בין שתי המדינות, אבלציה שלם, או NALT ללא פגע. מאחר שלא כל הניתוחים תגרום לאובדן מוחלט של NALT, בעלי חיים עם NALT שיורית או תמים יכול לשמש הבקרה הפנימית. תוצאה נוספת היא כי הפוטנציאל החיך לא מצליח לרפא לחלוטין, משאיר פתח המחבר בין האף לבין הפה עששת. לא מושלםהריפוי של החיך תגרום משקל נמוך וכישלון לשגשג, אלה אנשים יש להסיר מחקרים. בחינת התגובות החיסון הראשון עם מודל העכבר ישמש להקמת תפקיד NALT בתוצאות המחקר המיועד (תגובת נוגדנים, הישרדות וכו &#39;). הסרה בניתוח של NALT מאפשר קביעת תרומות באף חסינות מקומית ומערכתית. הגישה הכירורגית המתוארת כאן היא השיטה הישירה ביותר להשגת במודל עכבר ללא NALT. בחר לדפוק הקצוב מודלים העכבר דווחו NALT חוסר, אבל החיות הללו הם גם חסר ציטוקינים או כמוקינים החיוניים להתפתחות רקמות הלימפה משניים אחרים, ועלול להכיל פגמים נוספים 12,13. יתר על כן, השיטות המתוארות כאן פותחו לבחינת היבטים שונים של התגובה החיסונית שמקורם בתוך מעברי האף. תוצאות הניסוי שלנו מבוססים על מחקרים באמצעות palat העליון כולוהדואר של העכבר על תרביות רקמה, אם כי יתכן כי ניתן יהיה להשתמש בחלקים. לבסוף, המודל NALT תרבותי שימושי לביצוע ניסויים לחלוטין בתרבות רקמות.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> נירוסטה סכין כירורגית סטריליות, מס &#39;11 </td><td> Miltex </td><td> 4-311 </td><td></td></tr><tr><td> ידית הסכין, מס &#39;3 </td><td> Miltex </td><td> 4-7 </td><td></td></tr><tr><td> 48 גם צלחות תרבית תאים </td><td> שחקן המשנה </td><td> 3548 </td><td></td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 11875-093 </td><td></td></tr><tr><td> בסרום שור עוברית, חום מומת </td><td> Gibco / Invitrogen </td><td> 16000-044 </td><td> קונצרט כלשהו הסופי: 10% לפי נפח בתקשורת תרבות </td></tr><tr><td> סטרפטומיצין סולפט </td><td> סיגמא </td><td> S9137 </td><td> קונצרט כלשהו סופי: 100 מיקרוגרם / מ&quot;ל </td></tr><tr><td> פניצילין </td><td> סיגמא </td><td> P7794 </td><td> קונצרט כלשהו סופי: 100 UI / ml </td></tr><tr><td> גנטמיצין </td><td> Sigma-Aldrich </td><td> G1397-10 מ&quot;ל </td><td> קונצרט כלשהו סופי: 50 מיקרוגרם / מ&quot;ל </td></tr><tr><td> Fungizone </td><td> Gibco / Invitrogen </td><td> 15290-018 </td><td> קונצרט כלשהו סופי: 1 מיקרוגרם / מ&quot;ל </td></tr><tr><td> HEPES </td><td> Sigma-Aldrich </td><td> H0887 </td><td> קונצרט כלשהו סופי: 10 מ&quot;מ </td></tr><tr><td> Eppendorf צינורות Microcentrifuge </td><td> Eppendorf </td><td> 022364111 </td><td></td></tr><tr><td> נוטרה ג&#39;ל שרי מזון עכבר בטעמים רטוב </td><td> ביו מגישים </td><td> S4798 מגשים </td><td></td></tr><tr><td> Metacam </td><td> Boehringer Ingelheim </td><td> 601531000 </td><td> טיפה אחת של השעיה מ&quot;ג / מ&quot;ל ​​1.5 שבעל פה </td></tr><tr><td> קטמין </td><td> פייזר </td><td> 00856440301 </td><td> סופי משותףNC: 6.06 מ&quot;ג / מ&quot;ל </td></tr><tr><td> Acepromazine </td><td> Vedco </td><td> VEDC207 </td><td> סופי קונצרט כלשהו: 0.061 מ&quot;ג / מ&quot;ל </td></tr><tr><td> Xylazine </td><td> לויד </td><td> 4811 </td><td> סופי קונצרט כלשהו: 0.667 מ&quot;ג / מ&quot;ל </td></tr><tr><td> Puralube משחה וטרינר </td><td> Pharmaderm לבריאות בעלי חיים </td><td> 1621 </td><td></td></tr><tr><td> 0.9% נתרן כלורי להזרקה USP </td><td> בקסטר </td><td> 2B1302 </td><td></td></tr><tr><td> 0.5 מ&quot;מ Microcurette </td><td> Roboz </td><td> RS-6350 </td><td></td></tr><tr><td> מצרב Thermal Unit </td><td> גייגר </td><td> 150-S/150A-S </td><td></td></tr><tr><td> עצה החלפת TCU, לופ פיין סטרייט </td><td> גייגר </td><td> 214 </td><td></td></tr><tr><td> כירורגי מספריים </td><td></td><td></td><td></td></tr><tr><td> 10% ניטרלי שנאגרו פורמלין </td><td> סיגמא </td><td> HT501128 </td><td></td></tr><tr><td> חומצה פורמית </td><td> דיג </td><td> A119P-4 </td><td> מערבבים 426 מ&quot;ל חומצה פורמית למים מהברז 1047 מ&quot;ל, ואז מוסיפים 45 מ&quot;ל Rexyn 101 (ג) </td></tr><tr><td> Rexyn 101 (ג) </td><td> דיג </td><td> R231-500 </td><td></td></tr><tr><td> רקמת-Tek VIP E150/E300 </td><td> סאקורה </td><td></td><td></td></tr><tr><td> רוטרי Microtome </td><td> Leica </td><td> RM2255 </td><td></td></tr><tr><td> מאיר חברה Hematoxylin </td><td> סיגמא </td><td> MHS16-500ml </td><td></td></tr><tr><td> גיל מס &#39;1 Hematoxylin </td><td> סיגמא </td><td> GHS116 </td><td></td></tr><tr><td> Eosin B </td><td> סיגמא </td><td> 2853 </td><td></td></tr><tr><td> Microtainer מפריד סרום </td><td> BD Medical </td><td> 365956 </td><td></td></tr><tr><td> פוספט שנאגרו מלוחים </td><td></td><td></td><td> 100 מ&quot;מ לאא 2 PO 4, 140 מ&quot;מ NaCl, pH 7.4 </td></tr><tr><td> מעכבי פרוטאז קוקטייל, EDTA ללא </td><td> Thermo Scientific </td><td> 78415 </td><td></td></tr></tbody></table>

examining the role of nasopharyngeal-associated lymphoreticular tissue (nalt) in mouse responses to vaccines

את האף והלוע הקשורים רקמות lymphoreticular (NALT) נמצאו אצל בני אדם, מכרסמים, ויונקים אחרים, לתרום חסינות הסינוסים באף 1-3. NALT שני מקבילים פעמון מבנים הממוקמים את מעברי האף מעל החיך הקשה, והם נחשבים בדרך כלל להיות מרכיבים משניים של מערכת הרירית הקשורים הלימפה 4-6. ממוקם בתוך NALT הם תאים נפרדים של לימפוציטים מסוג B ו-T-וביניהם אנטיגן הצגת תאים דנדריטים 4,7,8. תאים אלו מוקפים בשכבת תאים אפיתל intercalated עם M-התאים אשר אחראים אחזור אנטיגן מן המשטחים הריריים של מעברי האוויר 9,10. לימפוציטים נאיביים במחזור דרך NALT עומדים להגיב על המפגשים הראשונים עם גורמי מחלה בדרכי הנשימה 7. בעוד NALT נעלמים אצל בני אדם עד גיל שנתיים, טבעת ומובנית דומה של Waldeyer הלימפה לאיברים להמשיך להתמיד הroughout חיים 6. בניגוד לבני אדם, עכברים לשמור NALT לאורך כל החיים, ובכך לספק מודל חיה נוח לחקר המערכת החיסונית שמקורם בתוך הסינוסים האף 11. תרבויות של תא יחיד המתלים של NALT לא מעשית בשל תשואות נמוכות של תאים mononuclear. עם זאת, ביולוגיה NALT ניתן לבדוק על ידי culturing vivo לשעבר של איבר שלם, בשיטה זו יש יתרון נוסף של שמירה על הרקמה הטבעית. עבור במחקרים vivo, מודלים בנוקאאוט הצגת פגמים גנטיים מוגבל NALT לא זמין כרגע בשל הבנה לקויה של מסלול ההתפתחות. לדוגמה, בעוד lymphotoxin למוצרי אלפא עכברים בנוקאאוט התנוון NALT, תיקונים של Peyer, בלוטות לימפה הפריפריה, דנדריטים ותאי זקיקי הלימפה ורקמות אחרות שינו גם אלה מניפולציות גנטיות בעכברים 12,13. כחלופה עכברים בנוקאאוט גנים, עמ &#39;אבלציה כירורגיתermanently מבטלת NALT מן המעבר באף מבלי לפגוע ברקמות אחרות. מודל העכבר וכתוצאה מכך נעשה שימוש כדי ליצור קשרים בין התגובות NALT ו החיסונית לחיסונים 1,3. אוסף סדרתי של נסיוב, רוק, שטיפות האף הפרשות הנרתיק הכרחי להקמת בסיס של תגובות מארחים את החיסון, תוך כדי התגובה החיסונית שמקורם ישירות NALT שניתן לאשר על ידי תרביות רקמה. ההליכים הבאים מתארים את הניתוחים, תרבות רקמות ואיסוף המדגם יש לבחון מקומיים מערכתית התגובה החיסונית לחיסון הלחות (IN) אפי.

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Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

שיטות לבחון את תרומתם של האף והלוע הקשורים רקמות lymphoreticular (NALT) על המערכת החיסונית האף מערכתית של עכברים לחיסונים אפי מתוארים. אנחנו מדגימים ניתוח לבסס מודל NALT תלוי העכבר<em&gt; לשעבר vivo</em&gt; תרבויות NALT חילוץ.

לבחון את תפקידה של רקמות האף והלוע הקשורים Lymphoreticular (NALT) בתגובות עכבר לחיסונים

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

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Research

JoVE Journal

Immunology and Infection

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

25.5K Views.  U.S. Army Medical Research Institute of Infectious Diseases. שיטות לבחון את תרומתם של האף והלוע הקשורים רקמות lymphoreticular (NALT) על המערכת החיסונית האף מערכתית של עכברים לחיסונים אפי מתוארים. אנחנו מדגימים ניתוח לבסס מודל NALT תלוי העכבר לשעבר vivo תרבויות NALT חילוץ.

Video: Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

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 ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Intranasal המינהל של החיסונים שפעת רקומביננטי במודלי כימרי עכבר ללמוד חסינות רירית

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

<em&gt; במבחנה</em&gt; מודל למדידה של מערכת חיסונית למלריה בהקשר של HIV Co-זיהום

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility provides X. fastidiosa a means for long-distance intra-plant movement and colonization, contributing toward pathogenicity in X. fastidiosa. The twitching motility of X. fastidiosa is operated by type IV pili. Type IV pili of Xylella fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon encoding proteins that are involved with signal transduction pathways. To elucidate the roles of pilG in the twitching motility of X. fastidiosa, a pilG-deficient mutant Xf&#916;pilG and its complementary strain Xf&#916;pilG-C containing native pilG were developed. A microfluidic chambers integrated with a time-lapse image recording system was used to observe twitching motility in Xf&#916;pilG, Xf&#916;pilG-C and its wild type strain. Using this recording system, it permits long-term spatial and temporal observations of aggregation, migration of individual cells and populations of bacteria via twitching motility. X. fastidiosa wild type and complementary Xf&#916;pilG-C strain showed typical twitching motility characteristics directly observed in the microfluidic flow chambers, whereas mutant Xf&#916;pliG exhibited the twitching deficient phenotype. This study demonstrates that pilG contributes to the twitching motility of X. fastidiosa. The microfluidic flow chamber is used as a means for observing twitching motility.

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

In this study, a nano-microfluidic flow chamber was employed to visualize and functionally characterize the twitching motility of Xylella fastidiosa, a bacterium that causes Pierce&#39;s disease in grapevine.

ויזואליזציה של תנועתיות מפרכסים אפיון תפקידו של<em&gt; PilG</em&gt; ב<em&gt; Fastidiosa Xylella</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

בידוד נויטרופילים וניתוח כדי לקבוע את תפקידם רגישות תאים לימפומה לסוכנים טיפוליים

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

דגם IL-8 עכבר transgenized חולף עבור<em&gt; בויבו</emמעקב ארוך טווח אחר תגובות דלקתיות

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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

Overexpression וטיהור האדם<em&gt; Cis</em&gt; פרנליטרונפרפרז ב<em&gt; Escherichia coli</em

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

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.

בידוד של סלמונלה typhimurium-המכיל Phagosomes של מקרופאגים

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

חיסונים של דג זברה בוגרת להקרנה פרה של חיסונים מבוסס DNA

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

הערכה של פתוגן-פונדקאי תגובות והיעילות החיסון בעכברים

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...