המחברים מודים Therwa חמזה, John E. Tidwell, נינה קלוביס, וסוזן סמית לסיוע ניסיוני וסוזן סמית להגהה. המחברים גם להודות לג&#39;ון תומאס, PhD למתן הקליני מבודדי החיידקים וג&#39;ון ב &#39;ברנט, דוקטורט על תמיכתו ואת השימוש במעבדת הבטיחות הביולוגית במחלקה למיקרוביולוגיה, אימונולוגיה וביולוגיה של תא באוניברסיטת מערב וירג&#39;יניה. החוקרים מכירים בתמיכה כספית מOsteosynthesis וטראומת טיפול הקרן והקרן לאומית למדע (# 1003907). ניסויי מיקרוסקופ וניתוח תמונה בוצעו גם במתקן אוניברסיטת מערב וירג&#39;יניה ההדמיה, אשר נתמך בחלקו על ידי מרי אב רנדולף מרכז הסרטן והמענק NIH P20 RR016440. שימוש בבעלי חיים לדם מושך אושרו על ידי הטיפול באוניברסיטת מערב וירג&#39;יניה המוסדי ובעלי חי ועדת שימוש. כל הניסויים בוצעו בעמידה בכל guidelin הרלוונטיes, תקנות, וגופים רגולטוריים.

1. הכנה והפעלה של PRP להקיז דם 1.1 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרדם ארנב על ידי שאיפה של isoflurane (2% בO 2 לזירוז ו1% לתחזוקה). </li><li style=";text-align:right;direction:rtl"> צייר 2 0.129 ציטראט המ&quot;ל M תלת נתרן (פתרון anticoagulent) לתוך מזרק מ&quot;ל 20. פתרון ציטראט תלת נתרן הוא הוכן על ידי המסת 1.897 ציטראט תלת נתרן גרם ב 50 המ&quot;ל המזוקק H 2 O וסינון עם מסנן 0.22 מיקרומטר סטרילי. </li><li style=";text-align:right;direction:rtl"> לחטא את אוזן הארנב באמצעות אתנול 70%. </li><li style=";text-align:right;direction:rtl"> להקיז דם (5 מ&quot;ל לדוגמה) מוריד אוזן הארנב באמצעות מחט פרפר (25-G) מחובר למזרק. </li><li style=";text-align:right;direction:rtl"> מערבב את הדם עם פתרון ציטראט תלת נתרן על ידי תסיסה עדינה. יחס הנפח של תמיסת ציטרט תלת נתרן בדם והוא 09:01. </li></ol> 1.2 הכנת PRP (איור 1) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> העברת דם anticoagulated לצינור פלסטיק צנטריפוגה מ&quot;ל 50. קח aliquot של 10 μlדם כדי לקבוע את ספירת הטסיות הבסיסית באמצעות hemocytometry. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה הדם ב 300 XG למשך 10 דקות בטמפרטורת חדר (RT) בצנטריפוגה עם הרוטור נדנדה החוצה. הגדר את התאוצה ומהירות בלם לנמוך (איור 1). </li><li style=";text-align:right;direction:rtl"> לאחר צנטריפוגה, הדם מתחלק לשלוש שכבות. השכבה התחתונה היא תאי דם אדומים בעיקר, השכבה האמצעית (המכונה גם &quot;מעייל באפים&quot;) מורכבת מטסיות דם ותאי דם לבנים מרוכזים, והשכבה העליונה היא בעיקר פלזמה, המהווה את המרכיב הנוזלי של דם, וטסיות דם ( איור 1). בזהירות להעביר את צינור צנטריפוגה למכסת מנוע תרבית תאים; לא להפריע את השכבות. להעביר את כל הפלזמה, מעייל באפים, ושכבת 2-3 מ&quot;מ עובי תאי דם אדומה לתוך צינור פלסטיק 15 מ&quot;ל באמצעות pipettor פלסטיק 1 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה המדגם הועבר בפעם שנייה ב3000 XG במשך 15 דקות בRT. השכבה העליונה (supernatant) נחשבת עניה פלזמת טסיות דם (PPP)ד מועבר לצינור חדש. </li><li style=";text-align:right;direction:rtl"> השג PRP ידי התאמת ריכוז טסיות הדם בדגימת הדם שנותרה באמצעות PPP להשיג 10 x 2.0 6 טסיות / μl (נקבע על ידי hemocytometry). </li></ol> 1.3 הפעלת PRP <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את פתרון הפעלת PRP ידי המסת תרומבין שור 5000 יחב&quot;ל עם מ&quot;ל 10% כלוריד 5 סידן לריכוז העבודה של 1,000 יחב&quot;ל / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הוסף את הפתרון להפעלה וPRP-PPP, ומערבב את הפתרון על ידי pipetting שוב ושוב כדי ליצור PRP-PPP ו- ג. יחס הנפח של הפתרון להפעלת PRP או PPP הוא 01:04. </li></ol> 2. במבחן Antimicrobial מבחנה של PRP השימוש Assay Curve Kill (איור 2) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> באמצעות לולאת inoculating סטרילי, להוסיף כמה מושבות של ס &#39; סטפילוקוקוס מתרבות צלחת הלילה שלה לתוך 5 מ&quot;ל של מרק מילר הינטון (MHB) בצינור פלסטיק. מערבולת קצרה ואז דגירת המדגם עבור 2 שעות ב 37 ° C. הבא, הצפיפות האופטית של תקשורת החיידקים נקבעה באמצעות ספקטרופוטומטר והותאמה לצפיפות שווה ל ~ 1 אופטית x 10 8 CFU / מ&quot;ל על בסיס העקום הסטנדרטיות שנקבע מראש. </li><li style=";text-align:right;direction:rtl"> הפוך דילול 100x באמצעות PBS להשיג 1 x 10 6 CFU / מ&quot;ל ולמקם את inoculums על קרח. </li><li style=";text-align:right;direction:rtl"> להגדיר ולתייג 5 שפופרות סטריליות, חד פעמיות מיליליטר ישבן עגול קלקר, ולהכין את הקבוצות לדוגמא הבאות, שהוזכרו בטבלת המס &#39;1 לנפח סופי של 2 מ&quot;ל בכל מבחנה. </li><li style=";text-align:right;direction:rtl"> הוסף PRP, PPP, או PBS ראשון שצינורות הקלקר, ואחריו פתרון תרומבין לצורך ההפעלה (היווצרות קריש). בשלב בא, ולאחר מכן להוסיף MHB ס inoculums aureus (1 x 10 6 CFU / מ&quot;ל) להשיג ריכוז הסופי של 1 x 10 5 CFU / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> דגירת הצינורות על 37 מעלות צלזיוס עם תסיסת הקפה ב 150 סל&quot;ד. </li><li style=";text-align:right;direction:rtl"> בנקודתי זמן שנקבעו מראש (לדוגמה 0, 1, ו2 שעות), לערבב את הפתרונים בכל צינור באמצעות pipetting החוזר (זההצעד חשוב שכן עשויים להיות לכודי חיידקים בתוך הג&#39;ל PRP). קחו 10 μl של מדגם, לדלל סדרתי עם מי מלח 0.9% סטרילית, ופיפטה aliquot 100 μl של כל דילול על אגר סויה Tryptic (TSA, עם 5% דם כבשים) צלחת לספירת CFU. </li><li style=";text-align:right;direction:rtl"> תרבות את הצלחות אגרו הלילה על 37 מעלות צלזיוס, ואז תספורנה ולהקליט מושבות הצלחת. נתוני עלילה בקנה מידת logarithimic עם זמן (שעות) על ציר ה-X וCFU / מ&quot;ל ​​על ציר y. </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+improving+the+handling+of+particulate+cancellous+bone+and+marrow+grafts+using+platelet+gel.">A technique for improving the handling of particulate cancellous bone and marrow grafts using platelet gel.</a> J. Oral. Maxillofac. Surg. 56, 1217-1218 (1998).</li><li>Currie, L. J., Sharpe, J. R., Martin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fibrin+glue+in+skin+grafts+and+tissue-engineered+skin+replacements:+a+review.">The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review.</a> Plast. Reconstr. Surg. 108, 1713-1726 (2001).</li><li>Nikulin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+calcium,+thrombin+and+nucleotides+(ADP,+cAMP,+cGMP)+on+blood+platelet+glycolysis+and+energy+metabolism.">Effect of calcium, thrombin and nucleotides (ADP, cAMP, cGMP) on blood platelet glycolysis and energy metabolism.</a> Farmakol. Toksikol. 43, 585-590 (1980).</li><li>Hantgan, R. R., Taylor, R. G., Lewis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets+interact+with+fibrin+only+after+activation.">Platelets interact with fibrin only after activation.</a> Blood. 65, 1299-1311 (1985).</li><li>Hantgan, R., Fowler, W., Erickson, H., Hermans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+assembly:+a+comparison+of+electron+microscopic+and+light+scattering+results.">Fibrin assembly: a comparison of electron microscopic and light scattering results.</a> Thromb. Haemost. 44, 119-124 (1980).</li><li>Li, B., Jiang, B., Boyce, B. M., Lindsey, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+polypeptide+nanoscale+coatings+incorporating+IL-12+for+the+prevention+of+biomedical+device-associated+infections.">Multilayer polypeptide nanoscale coatings incorporating IL-12 for the prevention of biomedical device-associated infections.</a> Biomaterials. 30, 2552-2558 (2009).</li><li>Li, B., Jiang, B., Dietz, M. J., Smith, E. S., Clovis, N. B., Rao, K. M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+local+MCP-1+and+IL-12+nanocoatings+for+infection+prevention+in+open+fractures.">Evaluation of local MCP-1 and IL-12 nanocoatings for infection prevention in open fractures.</a> J. Orthop. Res. 28, 48-54 (2010).</li><li>Boyce, B. M., Lindsey, B. A., Clovis, N. B., Smith, E. S., Hobbs, G. R., Hubbard, D. F., Emery, S. E., Barnett, J. B., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+effects+of+exogenous+IL-12+supplementation+and+antibiotic+treatment+in+infection+prophylaxis.">Additive effects of exogenous IL-12 supplementation and antibiotic treatment in infection prophylaxis.</a> J. Orthop. Res. 30 (2), 196-202 (2012).</li></ol>

החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

פלזמה טסיות דם עשיר כבר בשימוש יותר ויותר ליישומים קליניים בשל מאפייניה ריפוי המקדם 15-17. במחקר הנוכחי, PRP הוצג כגישה חדשה למניעת זיהום. PRP נמצא יש מאפיינים מיקרוביאלית חזקים נגד MRSA, MSSA, הקבוצה סטרפטוקוקוס, וNeisseria gonorrhoeae. היתרונות העיקריים של PRP, בהשוואה לטיפול...

זיהום שתל הקשורים הוא סיבוך קליני משמעותי. סטפילוקוקוס אוראוס (ס aureus) הוא אחד ממיקרואורגניזמים הנפוצים ביותר שבודדו מזיהומים הקשורים שתל. הוא מסוגל לייצר biofilm המכסה את פני השטח של שתלים ועלולים להוביל לזיהום 1,2 עמיד לאנטיביוטיקה. טיפול בזיהום נלווה שתל-לעתים קרובות דורש אשפוז ארוך טווח לdebridements החוזרים ונשנים וטיפול אנטיביוטי ממושך Parenteral. במקרים עמידים לאנטיביוטיקה, הסרת השתל עשויה להיות נחוצה. ההתנגדות הגוברת של חיידקים לאנטיביוטיקה יש גם מכונה על ידי המרכז לבקרת מחלות ומניעתן (CDC), &quot;אחד מבעיות הבריאות הקשות ביותר בעולם.&quot; עם זמן, ללא התפתחות של טיפולים חדשים ויעילים מיקרוביאלית, זה אפשרי כי פתוגנים עמידים לשילוב תרופות יהיו חשוכים עם אנטיביוטיקה קונבנציונלית. מניעת שתל הקשוריםזיהום לכן חשוב וסוכנים מניעתי רומן או גישות יש צורך למניעת זיהומים כאלה. פלזמה טסיות דם עשיר (PRP) היא ריכוז של דם אוטולוגית המכיל מעל 30 גורמי גדילה אשר יכול לסייע בריפוי עצם ושתל עצם 3-5. היישום של PRP כדי לשפר את התחדשות עצם ורקמות רכות התבגרות דווח יותר ויותר במרפאות בגלל הריכוז הגבוה של גורמי גדילה שונים שפורסמו על ידי טסיות. כמה מאפיינים של PRP PRP עולים כי יכול להיות גם מאפיינים מיקרוביאלית 6-9. PRP מכיל מספר גדול של טסיות דם, ריכוז גבוה של תאי דם לבנים (שעשוי להחזיק פעולות מארח הגנה נגד חיידקים ופטריות), ופפטידים מיקרוביאלית מרובים 7,8,10. במחקר שנערך לאחרונה בעוקבה גדולה של חולים ניתוחיים לב, התגלה כי שימוש intraoperative של PRP ג&#39;ל במהלך סגירת הפצע signifירידת icantly השכיחות של זיהום עצם חזה שטחי ועמוק 11. המסיבות והתצפיות הללו, שערו כי PRP, מלבד תכונותיו, למדו היטב ריפוי המקדם, יש מאפיינים מיקרוביאלית. היתרונות הפוטנציאליים של שימוש PRP כדי למנוע זיהום יכולים לכלול: (i) PRP הוא פחות סביר לגרום להתנגדות בהשוואה לטיפולים אנטיביוטיים קונבנציונליים. (Ii) PRP יש גם תכונות שמקדמות ריפוי אשר עשוי להיות השפעה סינרגיסטית על מניעת זיהום; המאפיינים של PRP ריפוי המקדם יכולים לספק חותם כדי למנוע עיקול חיידקים ובכך להפחית את הסיכויים לזיהום כפתוגנים ותאי אדם הם מירוץ למשטחי שתל 12 , 13. (Iii) PRP הוא מטבעו ביולוגי, ובטוח וחופשי מהסיכון למחל העברה. המטרה שלנו לטווח הארוך היא להשתמש PRP כגישה חדשה למניעת שתל הקשורים infectiתוספות. מטרת מחקר זה היה להכין PRP באמצעות גישת צנטריפוגה פעמים, כדי לבחון PRP של נכסים במבחנת מיקרוביאלית, וכדי לתאר את הפרוטוקולים להערכת מאפיינים מיקרוביאלית כאלה.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine thrombin</td>
 <td>King Pharmaceuticals, Inc </td>
 <td>60793-215-05</td>
 <td>Thrombin (bovine origin)</td>
 </tr>
 <tr>
 <td>Calcium chloride</td>
 <td>King Pharmaceuticals, Inc</td>
 <td>60793-215-05</td>
 <td>10% calcium chloride</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td>Baxter</td>
 <td>1001936060</td>
 <td></td>
 </tr>
 <tr>
 <td>Mueller Hinton broth</td>
 <td>Becton, Dickinson and Company</td>
 <td>275710</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate-buffered saline</td>
 <td>Sigma-Aldrich</td>
 <td>D8662</td>
 <td></td>
 </tr>
 <tr>
 <td>Tri-sodium citrate</td>
 <td>Sigma-Aldrich</td>
 <td>W302600</td>
 <td></td>
 </tr>
 <tr>
 <td>Tryptic soy agar</td>
 <td>Fisher Scientific</td>
 <td>R01202</td>
 <td></td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Kendro Laboratory Products</td>
 <td>750043077</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Millipore</td>
 <td>SLGP033RS</td>
 <td></td>
 </tr>
 </tbody>
</table>

prp as a new approach to prevent infection: preparation and in vitro antimicrobial properties of prp

זיהום שתל הקשורים הופך להיות יותר ויותר מאתגר לתעשיית הבריאות עולמי בשל הגדלת עמידות לאנטיביוטיקה, העברה של חיידקים עמידים לאנטיביוטיקה בין חיות ובני אדם, ועלות הגבוהה של טיפול בזיהומים. במחקר זה, אנו חושפים את אסטרטגיה חדשה שעשויה להיות יעילים במניעת שתל הקשורים זיהום על בסיס התכונות מיקרוביאלית הפוטנציאליות של פלזמה עשירה בטסיות (PRP). בשל תכונותיו, למדו היטב לקידום ריפוי, PRP (מוצר ביולוגי) כבר משמש יותר ויותר ליישומים קליניים כוללים ניתוחים אורתופדיים, ניתוחי חניכיים ופה, ניתוחי לסת, ניתוחים פלסטיים, רפואת ספורט, וכו &#39; PRP יכול להיות חלופה מתקדמת לטיפולים אנטיביוטיים קונבנציונליים במניעת זיהומים הקשורים שתל. השימוש בPRP עשוי להיות יתרון בהשוואה לטיפולים אנטיביוטיים קונבנציונליים siNCE PRP הוא פחות סביר לגרום לעמידות לאנטיביוטיקה וPRP של מיקרוביאלית ותכונות ריפוי מקדם עשויים להיות השפעה סינרגיסטית על מניעת זיהום. זה ידוע היטב כי פתוגנים ותאי אדם הם מירוץ למשטחי שתל, והמאפיינים של PRP ריפוי קידום יכולים לשפר מצורף תא אנושי ובכך להפחית את הסיכויים לזיהום. בנוסף, PRP הוא מטבעו ביולוגי, ובטוח וחופשי מהסיכון למחל העברה. למחקר שלנו, יש לנו נבחרו מספר זני חיידקים קליניים שמצויים לרוב בזיהומים אורתופדיים ובדקו האם יש בPRP מאפיינים מיקרוביאלית מבחנה נגד חיידקים אלה. הכנו PRP באמצעות גישת צנטריפוגה פעמים אשר מאפשרת את אותו ריכוז טסיות כדי להתקבל לכל המדגמים. השגנו ממצאים מיקרוביאלית עקביים ומצאנו שיש לו חזק בPRP מאפיינים מיקרוביאלית מבחנה נגד חיידקים כמו methiaureus רגיש-cillin והעמיד methicillin סטפילוקוקוס, קבוצת סטרפטוקוקוס, וNeisseria gonorrhoeae. לכן, השימוש בPRP יכול להיות פוטנציאל למניעת הזיהום ולהפחית את הצורך בטיפול יקר שלאחר ניתוח שתל של זיהומים הקשורים.

PRP מוכן reproducibly באמצעות גישת צנטריפוגה פעמים (איור 1). PRP נמצא להציג חזק (ירידה של עד 100-לקפל בCFUs) במאפיינים מיקרוביאלית מבחנה נגד methicillin העמיד ס סטפילוקוקוס (MRSA) (איור 3), הנמצא בדרך כלל בבתי חולים ברחבי עולם 14. כמו כן, PRP יש מאפיינים מי?...

Watch this Scientific Journal Video about PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP at JoVE.com

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

זיהום שתל הקשורים הוא סיבוך קליני משמעותי. מחקר זה מתאר גישה באמצעות פלזמה עשירה בטסיות (PRP) כדי למנוע זיהומים הקשורים שתל, מציג את הפרוטוקול להכנת PRP עם ריכוז טסיות דם קבוע, ומדווח על נכסים שזוהו לאחרונה מיקרוביאלית של PRP ופרוטוקולים הקשורים לבחינת מאפיינים מיקרוביאלית כאלה<em&gt; במבחנה.</em

PRP כגישה חדשה למניעת זיהום: הכנה ו<em&gt; במבחנה</em&gt; מאפיינים מיקרוביאלית של PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

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Immunology and Infection

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

21.2K Views.  West Virginia University. זיהום שתל הקשורים הוא סיבוך קליני משמעותי. מחקר זה מתאר גישה באמצעות פלזמה עשירה בטסיות (PRP) כדי למנוע זיהומים הקשורים שתל, מציג את הפרוטוקול להכנת PRP עם ריכוז טסיות דם קבוע, ומדווח על נכסים שזוהו לאחרונה מיקרוביאלית של PRP ופרוטוקולים הקשורים לבחינת מאפיינים מיקר?...

Video: PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

התמרה retroviral של T-cell קולטנים עכבר מתאי T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

היכרות עם מתח שאר בחקר הדבקה חיידקית

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

פשוט וחזק In vivo ו במבחנה גישה ללימוד הרכבה וירוס

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

שימוש בינוני כיח מלאכותית לבדיקת יעילות אנטיביוטי נגד<em&gt; Pseudomonas aeruginosa</em&gt; בתנאי רלוונטיים יותר ריאות סיסטיק פיברוזיס

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

<em&gt; במבחנה</em&gt; מודל ללמוד ההטרוגניות של בידול Macrophage אדם וקיטוב

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

שימוש<em&gt; Mellonella Galleria</em&gt; כאורגניזם מודל לחקר<em&gt; הלגיונלה pneumophila</em&gt; זיהום

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

ניטור שינויים בממברנה קוטביות, ממברנה יושרה, וריכוזי יון תאיים ב<em&gt; Streptococcus pneumoniae</em&gt; שימוש צבעי ניאון

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

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 ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

גישה פשוטה ויעילה לבנות וקטורי וירוס Vaccinia Mutant

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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 ...