עבודה זו נתמכה על ידי המכון הקנדי למחקר בריאות דרך Catalyst שיתופי זיהומים ועמיתים נלוות גרנט. אריתרוציטים האדם סופקו על ידי הבנק המרכז הרפואי de l&#39;דם מאוניברסיטת לאוול.

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report. , (2005).</li><li>Kublin, J. G., Patnaik, P., Jere, C. S. <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+Plasmodium+falciparum+malaria+on+concentration+of+HIV-1-RNA+in+the+blood+of+adults+in+rural+Malawi:+a+prospective+cohort+study.">Effect of Plasmodium falciparum malaria on concentration of HIV-1-RNA in the blood of adults in rural Malawi: a prospective cohort study.</a> Lancet. 365, 233-240 (2005).</li><li>Cuadros, D. F., Branscum, A. J., Crowley, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-malaria+co-infection:+effects+of+malaria+on+the+prevalence+of+HIV+in+East+sub-Saharan+Africa.">HIV-malaria co-infection: effects of malaria on the prevalence of HIV in East sub-Saharan Africa.</a> Int. J. Epidemiol. 40, 931-939 (2011).</li><li>Xiao, L., Owen, S. M., Rudolph, D. L., Lal, R. B., Lal, 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=Plasmodium+falciparum+antigen-induced+human+immunodeficiency+virus+type+1+replication+is+mediated+through+induction+of+tumor+necrosis+factor-alpha.">Plasmodium falciparum antigen-induced human immunodeficiency virus type 1 replication is mediated through induction of tumor necrosis factor-alpha.</a> J. Infect. Dis. 177, 437-445 (1998).</li><li>Froebel, K., Howard, W., Schafer, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+by+malaria+antigens+renders+mononuclear+cells+susceptible+to+HIV+infection+and+re-activates+replication+of+endogenous+HIV+in+cells+from+HIV-infected+adults.">Activation by malaria antigens renders mononuclear cells susceptible to HIV infection and re-activates replication of endogenous HIV in cells from HIV-infected adults.</a> Parasite Immunol. 26, 213-217 (2004).</li><li>Diou, J., Tardif, M. R., Barat, C., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+derived+from+hemozoin-loaded+monocytes+display+a+partial+maturation+phenotype+that+promotes+HIV-1+trans-infection+of+CD4++T+cells+and+virus+replication.">Dendritic cells derived from hemozoin-loaded monocytes display a partial maturation phenotype that promotes HIV-1 trans-infection of CD4+ T cells and virus replication.</a> J. Immunol. 184, 2899-2907 (2010).</li><li>Diou, J., Tardif, M. R., Barat, C., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+hemozoin+modulates+susceptibility+of+immature+monocyte-derived+dendritic+cells+to+HIV-1+infection+by+inducing+a+mature-like+phenotype.">Malaria hemozoin modulates susceptibility of immature monocyte-derived dendritic cells to HIV-1 infection by inducing a mature-like phenotype.</a> Cell Microbiol. 12, 615-625 (2010).</li><li>Diou, J., Gauthier, S., Tardif, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+the+malaria+pigment+hemozoin+renders+human+macrophages+less+permissive+to+HIV-1+infection.">Ingestion of the malaria pigment hemozoin renders human macrophages less permissive to HIV-1 infection.</a> Virology. 395, 56-66 (2009).</li><li>Davies, J. Q., Gordon, S. <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+culture+of+human+macrophages.">Isolation and culture of human macrophages.</a> Methods Mol. Biol. 290, 105-116 (2005).</li><li>Fortin, J. F., Cantin, R., Lamontagne, G., Tremblay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+ICAM-1+glycoproteins+incorporated+on+human+immunodeficiency+virus+type+1+are+biologically+active+and+enhance+viral+infectivity.">Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity.</a> J. Virol. 71, 3588-3596 (1997).</li><li>Bounou, S., Leclerc, J. E., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J. Virol. 76, 1004-1014 (2002).</li><li>Wei, X., Decker, J. M., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+resistant+human+immunodeficiency+virus+type+1+in+patients+receiving+fusion+inhibitor+(T-20)+monotherapy.">Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.</a> Antimicrob. Agents Chemother. 46, 1896-1905 (2002).</li><li>Trager, W., Jensen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+malaria+parasites+in+continuous+culture.">Human malaria parasites in continuous culture.</a> Science. 193, 673-675 (1976).</li><li>Turrini, F., Ginsburg, H., Bussolino, F., Pescarmona, G. P., Serra, M. V., Arese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+Plasmodium+falciparum-infected+human+red+blood+cells+by+human+monocytes:+involvement+of+immune+and+nonimmune+determinants+and+dependence+on+parasite+developmental+stage.">Phagocytosis of Plasmodium falciparum-infected human red blood cells by human monocytes: involvement of immune and nonimmune determinants and dependence on parasite developmental stage.</a> Blood. 80, 801-808 (1992).</li><li>Akkina, R. K., Walton, R. M., Chen, M. L., Li, Q. X., Planelles, V., Chen, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+CD34++cells+with+a+human+immunodeficiency+virus+type+1-based+retroviral+vector+pseudotyped+with+vesicular+stomatitis+virus+envelope+glycoprotein+G.">High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.</a> J. Virol. 70, 2581-2585 (1996).</li><li>Zhao, C., Papadopoulou, B., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+infantum+enhances+human+immunodeficiency+virus+type-1+replication+in+primary+human+macrophages+through+a+complex+cytokine+network.">Leishmania infantum enhances human immunodeficiency virus type-1 replication in primary human macrophages through a complex cytokine network.</a> Clinical Immunol. 113, 81-88 (2004).</li></ol>

אין ניגוד עניינים הצהיר.

הדגמנו כאן שתי גישות שונות לנתח את ההשפעה של טפיל המלריה על מחזור 1-HIV ויראלי, כלומר על ידי ניתוח או ביטוי גנים ויראלי או וירוס הצאצאים ייצור שכפול מונוציטים הנגזרות מקרופאגים. גישות דומות שימשו במשך שני HIV-1-טפיל שיתוף זיהומים 16. עם זאת, אלה הנתונים החדשים הם צעד קדימה בחקירה של מלריה-HIV-1 עמיתים זיהומים. . אכן, Diou ואח&#39; 8 בחן את ההשפעה של hemozoin, טפילים לא חיים, על שכפול HIV-1, בהסכם עם התוצאות שלנו, הם הבחינו כי hemozoin עצמו היה מספיק כדי לדכא ייצור ויראלי ידי MDM, ולא MDMs. באמצעות פריסת הניסוי המתואר, הבחנו כי פ falciparum מפעיל השפעה מזיקה ברורה על מחזור HIV-1 replicative ב מקרופאגים: עיכוב משמעותי של ייצור ויראלי הוא ציין מקרופאגים מראש שנחשפו טפילים (Figurה 2 א) וכן השפעה מסוימת של הטפיל על שעתוק ויראלי ממחישה 2B איור. עם זאת, אנחנו לא יכולים להשאיר את כל תופעות נוספות של הטפיל על כניסת היתוך או ויראלי (decapsidation) או לאחר שילוב מנגנונים, כגון סינתזה של חלבון או הרכבה החלקיקים ויראלי ניצני. יתר על כן, ייתכן כי השפעה שונה על שכפול HIV-1 היה לקבל אם הטפיל נוספו גם בעת ובעונה אחת או זיהום MDM הבאה עם הנגיף. שלנו במודל שיתוף זיהום חוץ גופית מספק כלי רב עוצמה כדי לבצע חקירות מפורטות של HIV-1 / פ &#39; אינטראקציות falciparum של התא המארח. לדוגמה, שילוב של פריסת זה ניסיוני עם טכניקות אחרות, כגון PCR כמותי זמן אמיתי, שיכול למקד ולכמת צעדים ספציפיים retrotranscription ויראלי ואינטגרציה הגנום הנגיפי אל התא המארח הגנום, הם ריאלי למדי, צריך להניב insi נוסףghts לתוך המנגנונים המעורבים שיתוף זיהומים. יתר על כן, מבחני ספציפיות על מנת להעריך הצעדים הראשונים של מחזור נגיפית (ויראלית היתוך, decapsidation, quantitation הכניסה) ניתן ליישם פרוטוקול זה בסיסי עוד יותר לנתח את ההשפעה על שכפול נגיפי. שינויים אלו מראים כמה גמיש פרוטוקול זה הוא בנוגע HIV-1 quantitation זיהוי: אכן, גם תקן ה-HIV-1 הפוכה quantitation מבחני transcriptase באמצעות טריטיום שכותרתו נוקלאוטידים מתקבל על הדעת להחליף את ELISA ל-HIV-1 p24. בנוסף MDM, אנו מצפים המערכת שלנו להיות להתאמה סוגי תאים אחרים הנוגעים ל-HIV-1-מלריה אינטראקציות, כגון מונוציטים ותאים דנדריטיים. העובדה MDM הם תאים חסיד מאפשר מניפולציה שלהם, ומאפשר טיפול קל הכביסה מתוך iRBC שלא היה נלקח, או נמצאים בקשר עם MDM, ללא ביטול כל מקרופאגים. יתרון כזה לא יחול על תאים דנדריטים ומונוציטים, אשר בתרבית ההשעיה, שמסבך את SEparation של iRBC uRBC וחופשי עם תאים כאלה ואנחנו כרגע מענה לבעיה זו. המערכת שלנו יכולה להיות שימושית גם כדי לחקור אינטראקציות מורכבות יותר, כגון ההשפעה של Plasmodium-שנחשפו מונוציטים הנגזרות תאים על מחזור נגיפי HIV-1 בתאים חיסוניים אחרים כגון מסוג CD4 חיובי בתאי T של שיתוף תרבות ניסויים. לבסוף, פרוטוקול שלנו יכול להיות מתאים לניסוי מחפש את ההשפעה של פ &#39; falciparum iRBCs על הפעלה מחדש ב-HIV-1 PBMC ל-HIV-1 אנשים נגועים. משפט ואתיקה האדם PBMC התקבלו בדם של תורמים בריאים, בהתאם להנחיות ועדת אתיקה של המרכז הרפואי המרכז דה l&#39;אוניברסיטת מחקר לאוול. הסכמה בכתב התקבל כל תורמי הדם.

<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> RPMI 1640 </td><td> Multicell </td><td> 350-000-CL </td><td></td></tr><tr><td> PBS-רעלן פנימי חינם </td><td> סיגמא </td><td> D8662 </td><td></td></tr><tr><td> FBS </td><td> Wisent </td><td> 080-150 </td><td> חום מובטל על 56 מעלות צלזיוס למשך 30 דקות </td></tr><tr><td> Accutase </td><td> eBiosciences </td><td> 00-4555-56 </td><td></td></tr><tr><td> Albumax II </td><td> Gibco </td><td> 11021-037 </td><td> מומס RPMI 1640, פילטר חיטוי </td></tr><tr><td> ההפרדה לימפוציטים בינוני </td><td> Multicell </td><td> 305-010-CL </td><td></td></tr><tr><td> Luminometer צלחות לבנות </td><td> Promega </td><td> Z3291 </td><td></td></tr><tr><td> CS מקינטוש ההפרדה columms </td><td> Miltenyi ביוטק </td><td> 130-041-305 </td><td></td></tr><tr><td> VarioMacs מפריד </td><td> Miltenyi ביוטק </td><td> 130-090-282 </td><td></td></tr><tr><td> פניצילין / סטרפטומיצין פתרון </td><td> Wisent </td><td> 450-201-EL </td><td></td></tr><tr><td> D-סורביטול </td><td> Sigma-Aldrich </td><td> S1876 </td><td></td></tr><tr><td> תא מגרד (25 ס&quot;מ) </td><td> Sarstedt </td><td> 83.1830 </td><td></td></tr><tr><td> 24 ובכן צלחות תרבית תאים </td><td> BD פלקון </td><td> 353047 </td><td></td></tr><tr><td> 150 x 20 מ&quot;מ תרבות צלחת </td><td> Sarstedt </td><td> 83.1803.003 </td><td></td></tr><tr><td> M-CSF </td><td> Genscript </td><td> Z02001 </td><td></td></tr><tr><td> HEPES </td><td> Sigma-Aldrich </td><td> H4034 </td><td></td></tr><tr><td> האדם סרום AB + </td><td> עמק ביו רפואית </td><td> HP1022 </td><td></td></tr><tr><td> HBSS </td><td> Wisent </td><td> 311-505-CL </td><td></td></tr><tr><td> גנטמיצין </td><td> Sigma-Aldrich </td><td> G1397 </td><td></td></tr><tr><td> Hypoxanthine </td><td> Sigma-Aldrich </td><td> H9636 </td><td></td></tr><tr><td> NaHCO 3 </td><td> Sigma-Aldrich </td><td> S5761 </td><td></td></tr><tr><td> Assay בלוציפראז מערכת </td><td> Promega </td><td> E1500 </td><td></td></tr><tr><td> אדם דם תאים אדומים </td><td></td><td></td><td> להשיג מטוהרים מהבנק דם צ&#39;ול. </td></tr></tbody></table>

an in vitro co-infection model to study plasmodium falciparum-hiv-1 interactions in human primary monocyte-derived immune cells

Plasmodium falciparum, הסוכן סיבתי של הטופס הקטלני ביותר של מלריה, נגיף האיידס סוג-1 (HIV-1) הם בין הבעיות הבריאותיות החשובות ביותר ברחבי העולם, להיות אחראי על סך של 4 מיליון מקרי מוות בשנה 1. בשל החפיפה נרחבים שלהם באזורים המתפתחים, בעיקר באפריקה שמדרום לסהרה, עמיתים זיהומים עם מלריה ו-HIV-1 נפוצים, אבל את משחק הגומלין בין שתי המחלות הוא הבין היטב. דיווחים אפידמיולוגיים הראו כי זיהום מלריה זמני משפר HIV-1 שכפול ומגדיל עומס נגיפי HIV-1 ב שיתוף נגועים אנשים 2,3. בגלל viremia זה נשאר גבוה במשך כמה שבועות לאחר טיפול antimalarials, תופעה זו עשויה להיות השפעה על התקדמות המחלה ואת השידור. מנגנוני החיסונית סלולריים מאחורי תצפיות אלו נחקרו רק בקושי. כמה מחקרים במבחנה investigating את ההשפעה של מלריה ב-HIV-1 הראו כי חשיפה אנטיגנים מלריה מסיסים יכולים להגדיל את ה-HIV-1 זיהום מחדש של תאים חיסוניים. עם זאת, מחקרים אלה השתמשו תמציות תאים שלמים של פ &#39; falciparum schizont טפילים בשלב ותאי דם היקפיים (mononuclear PBMC), מה שהופך את זה קשה לפענח איזה מרכיב מלריה (ים) היה אחראי על תופעות שנצפו ומה מארחים את התאים היעד היו 4,5. מחקר שנערך לאחרונה הראה כי חשיפה של מונוציטים הנגזרות תאים דנדריטים בשלים על פיגמנט hemozoin מלריה עלה יכולתם להעביר HIV-1 אל התאים CD4 + טי 6,7, אבל זה ירד זיהום HIV-1 של מקרופאגים 8. כדי לשפוך אור על תהליך מורכב זה, ניתוח שיטתי של אינטראקציות בין טפיל המלריה ו-HIV-1 של אוכלוסיות אנושיות שונות הרלוונטיות תאים ראשוניים יש צורך קריטי. כמה טכניקות לחקור את ההשפעה של HIV-1 על phagocytosis של מיקרואורגניזמים ועל ההשפעה של פתוגנים כאלה על שכפול ה-HIV-1 תוארו. אנחנו כאן להציג שיטה לחקור את ההשפעות של פ &#39; falciparum נגועים אריתרוציטים על שכפול של HIV-1 של האדם הנגזרות מונוציטים מקרופאגים ראשוניים. ההשפעה של החשיפה טפיל על HIV-1 תעתיק / translational אירועים מנוטר באמצעות וירוסים מחזור יחיד pseudotyped שבו הגן כתב בלוציפראז החליף את הגן Env תוך השפעה על כמות של נגיף שפורסמו על ידי מקרופאגים נגועים נקבע על ידי מדידת HIV-1 החלבון p24 capsid ידי ELISA ב supernatants סלולריים.

Watch this Scientific Journal Video about An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells at JoVE.com

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

פיתחנו<em&gt; במבחנה</em&gt; מלריה-HIV-1 שיתוף זיהום מודל ללמוד את ההשפעה של<em&gt; Plasmodium falciparum</em&gt; על מחזור HIV-1 replicative של האדם הנגזרות מונוציטים מקרופאגים ראשוניים. מערכת זו צדדי יכול בקלות להיות מותאם אחרים סוגי התאים העיקריים לסכנת הדבקה ב-HIV-1.

<em&gt; במבחנה</em&gt; שיתוף זיהום דגם ללמוד<em&gt; Plasmodium falciparum</em&gt;-HIV-1 אינטראקציות אדם מונוציטים הנגזרות תאים ראשוניים החיסון

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

an-vitro-co-infection-model-to-study-plasmodium-falciparum-hiv-1

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

13.6K Views.  CHUL (CHUQ), Quebec City, Quebec, Canada. פיתחנו במבחנה מלריה-HIV-1 שיתוף זיהום מודל ללמוד את ההשפעה של Plasmodium falciparum על מחזור HIV-1 replicative של האדם הנגזרות מונוציטים מקרופאגים ראשוניים. מערכת זו צדדי יכול בקלות להיות מותאם אחרים סוגי התאים העיקריים לסכנת הדבקה ב-HIV-1.

Video: An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

שבעה צעדים Stellate תאים

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

שינוי מערכת Transposon piggyBac של תאי T אנושיים ראשוניים

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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 ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

כמותי<em&gt; במבחנה</em&gt; Assay כדי למדוד הידבקות נויטרופילים לתאים מופעלים עיקריים אדם אנדותל כלי הדם בתנאים סטטיים

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

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. ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

דור של תאי דנדריטים אדם נגזר מונוציטים מן הדם מלא

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Monolayers Colonoid האדם ללמוד אינטראקציות בין גורמי מחלה, Commensals אפיתל המעי מארח

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

יעילות גבוהה הדור של העכבר העיקרי של אנטיגן בתאי T ציטוטוקסיים לבדיקות פונקציונליות במודל סוכרת אוטואימוניות

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.