עבודה זו נתמכה על ידי מקס פלאנק החברה.

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9 (7), 671-675 (2012).</li><li>Sliusarenko, O., Heinritz, J., Emonet, T., Jacobs-Wagner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput,+subpixel+precision+analysis+of+bacterial+morphogenesis+and+intracellular+spatio-temporal+dynamics.">High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.</a> Mol. Microbiol. 80 (3), 612-627 (2011).</li><li>Ducret, A., Quardokus, E. M., Brun, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicrobeJ,+a+tool+for+high+throughput+bacterial+cell+detection+and+quantitative+analysis.">MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis.</a> Nat. Microbiol. 1, 16077 (2016).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7 (10), R100 (2006).</li><li>Morales-Soto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+Imaging,+and+Quantification+of+Bacterial+Surface+Motility+Assays.">Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays.</a> J. Vis. Exp. (98), e52338 (2015).</li><li>Paintdakhi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oufti:+An+integrated+software+package+for+high-accuracy,+high-throughput+quantitative+microscopy+analysis.">Oufti: An integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.</a> Mol. Microbiol. 99 (4), 767-777 (2016).</li><li>de Jong, I. G., Beilharz, K., Kuipers, O. P., Veening, J. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell+Imaging+of+Bacillus+subtilis+and+Streptococcus+pneumoniae+using+Automated+Time-lapse+Microscopy.">Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy.</a> J. Vis. Exp. (53), e1-e6 (2011).</li></ol>

שום ניגוד אינטרסים לא הכריז.

מאמר זה מדווח על שיטת הדמיה מיקרוסקופית של תאים V. parahaemolyticus swarmer , ואחריו מנתח במורד המיועד לפתור לכמת את לוקליזציה subcellular ותכונות אחרות של חלבונים שנחקרו fluorescently שכותרתו. למרות מספר כלים קיימים לעיבוד תמונה מיקרוסקופית וניתוח 14 , 15...

חיידקים כל הזמן חווים שינויים בסביבה החיצונית שלהם פיתחו מספר טכניקות לשנות ולהתאים את התנהגותם בהתאם. מנגנון אחד כזה כולל את ההבחנה לסוגים שונים של תאים המשלימים טוב יותר את הסביבה המשתנה. בידול לעיתים קרובות כרוך שינויים גדולים הרגולציה של מחזור התא, מורפולוגיה התא, ואת הארגון spatiotemporal של התאים. אורגניזם אחד שיכול לעבור בידול הוא Vibrio parahaemolyticus . V. parahaemolyticus שייך Vibrionaceae , אשר משפחה של proteobacteria כי בדרך כלל מים טריים או מלוחים. Vibrionaceae מופצים באופן נרחב בסביבה וכוללים מספר מינים הגורמים זיהומים בדרכי העיכול בבני אדם, כולל גם cholerae.V Vibrio. Parahaemolyticus הוא אורגניזם dimorphic והוא מסוגל להבדיל לשני סוגי תאים נפרדים כתגובה כדי להתאים את השינויים שלהסביבה חיצונית. בסביבות מימיות, הוא קיים כמו תא שוחה קצר בצורת מוט עם דגלום קוטב יחיד ממוקם על עמוד התא הישן. על פני השטח מגע, בידול לתא swarmer מופעלת. הבדל תאים swarmer כוללת שני שינויים עיקריים: morphogenesis תא swarmer באמצעות עיכוב חלוקת התא, ואת אינדוקציה של מערכת השני flagella. התוצאה היא היווצרותו של תא נחיל, בעל צורה מוטורית ואורך מוארך, אשר יכול להמשיך את סגנון החיים של נחיל, שבו אירועים של חלוקה מביאים לתאי נחילים, או לחילופין להבדיל חזרה לתאי שחיינים. מספר גורמים דווחו כדי להשפיע או להשפיע על בידול swarmer. הגירוי העיקרי נראה מונע על ידי mechanosensing שבו V. parahaemolyticus משתמש flagell הקוטב כחיישן מישוש המזהה עיכוב של סיבוב על פני השטח קשר, אבל גורמים נוספים מעורבים כמוטוב 1 . במעבדה, סיבוב של דגלום הקוטב יכול להיות מעוכבת באופן מלאכותי על ידי תוספת של phenamil, אשר חוסם את ערוץ הנתרן מונע סיבוב flagellar, ובכך לגרום swarmer differiation 2 . יתר על כן, במחקר מוקדם יותר, Vibrio alginolyticus היה המושרה נחיל על מדיה מוצקה כאשר תאים היו מופצות על המדיום צמיחה בצלחת פטרי חתום עם קלטת פלסטיק ברורה. נייר סינון רווי אלקלי מונע התנפחות תחת תנאים אלה, ומכאן טוען כי אחד או יותר חומצות נדיפות עשוי להיות מעורב אינדוקציה של נחיל. לפיכך, איטום צלחת פטרי עם קלטת פלסטיק מותר ככל הנראה על הצטברות של חומצות נדיפות, נוצר כמו תוצרי לוואי של חילוף החומרים הסלולרי, בתוך שטח הראש של הצלחת. אותו אפקט הושג כאשר H 2 O 2 נוספה בינוני הצמיחה במנות אטום אטום על מנת לייצר באופן מלאכותי חומצות נדיפות ידי hydrolysing רכיבי מדיה <supClass = &quot;xref&quot;&gt; 3 , 4 , 5 . עם זאת, הזהות של חומצות נדיפות כאלה עדיין לא ידוע. יתר על כן, הוכח כי זמינות עודפת של סידן 6 ו-הגבלה ברזל 7 הן לשפר את הבדל swarmer והתפשטות על פני משטחים מוצקים. תאים ניתן לרעב ברזל על ידי הוספת המתחם 2,2&#39;-Bipyridyl למדיום הצמיחה, אשר הוכח להשפיע על בידול swarmer 8 . הגורמים ידועים לוויסות בידול מיושמים עכשיו בעיצוב של פרוטוקול זה reproducibly גורם V. parahaemolyticus בידול לתוך תאים swarmer ואת התפשטות שלהם על משטחים אגר מוצק. V. parahaemolyticus בידול כוללת שינויים משמעותיים ויסות חלוקת התא, מורפולוגיה הסלולר, ואת המיקום של מכונות macromolecular כגון flagellA ו chemotaxis מנגנוני - תהליכים שדורשים כל לוקליזציה ספציפית של חלבונים בהתאם מחזור התא. לכן, היכולת ללמוד את לוקליזציה תאיים של חלבונים כאלה חיונית להבנת התהליכים הסלולר הנ&quot;ל. על מנת לבצע מחקרים כאלה מיקרוסקופ פלואורסצנטי על תאים בודדים נדרשת. זה יכול להיות מאתגר במיוחד כאשר תאים הדמיה הקיימות בתוך אוכלוסיות חיידקים צפופים, כמו במקרה של תאים swarmer. היו ניסיונות לגרום להבדל swarmer בתקשורת נוזלית, אשר יכול ליצור תאים בודדים למחקרים מיקרוסקופיים. ואף על ברמת תעתיק תאים אלה גרמו חלקית את התוכנית swarmer-differentiation, הם לא עוברים את אותם שינויים מורפולוגיים ברורים כמו תאים נבדל מובחן לחלוטין גדל על בינוני מוצק 8 . מאמר זה מציע פרוטוקול חזק לשחזור של שיטה כדי לגרום swarmer CELl הבחנה על פני אגר. הפרוטוקול מייצר אוכלוסייה של תאים swarmer נגיש בקלות זמין עבור מיקרוסקופ תא בודד וניתוח לאחר מכן. יתר על כן, הפרוטוקול מאפשר מחקרים לוקליזציה של חלבונים שכותרתו fluorescently בשחיין סוגי התאים swarmer (סעיפים 1, 2, 3, 4). בנוסף, פרוטוקול מתאר את זרימת העבודה הבאים על איך לעבד ולנתח את הנתונים שנוצר ניסויים מיקרוסקופ פלואורסצנטי, המאפשר ניתוח דמוגרפי של חברות חיידקים (סעיף 5).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB-medium</td>
			<td>Roth</td>
			<td>X968.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Agar, granulated</td>
			<td>BD</td>
			<td>214510</td>
			<td>For preparation of LB agar with LB-medium</td>
		</tr>
		<tr>
			<td>Difco Heart Infusion Agar</td>
			<td>BD</td>
			<td>244400</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Agarose NEEO, ultra quality</td>
			<td>Roth</td>
			<td>2267.3</td>
			<td>For preparation of microscopy agarose pads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Additives</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-arabinose</td>
			<td>Roth</td>
			<td>5118.3</td>
			<td>Used to induce pBAD derivatives</td>
		</tr>
		<tr>
			<td>2,2`-Bipyridyl</td>
			<td>Sigma Aldrich</td>
			<td>D216305-25G</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Roth</td>
			<td>5239.1</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Roth</td>
			<td>K029.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Roth</td>
			<td>3886.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>-</td>
			<td>-</td>
			<td>Standard recipe for Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>D(+) Sucrose</td>
			<td>Roth</td>
			<td>4621.1</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Roth</td>
			<td>3783.5</td>
			<td>For preparation of electrocompetent cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Hardware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 150x20mm with cams</td>
			<td>Sarstedt</td>
			<td>82.1184.500</td>
			<td>For preparation of HI agar swarming plates</td>
		</tr>
		<tr>
			<td>kelvitron t (B 6420)</td>
			<td>Heraeus</td>
			<td>-</td>
			<td>Used for drying HI agar swarming plates</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Microbial System</td>
			<td>BioRad</td>
			<td>1652662</td>
			<td>Used for elctroporation of plasmid DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MetaMorph Offline (version 7.7.5.0)</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Used for microscopy image analysis</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>-</td>
			<td>Used for microscopy data analsis</td>
		</tr>
	</tbody>
</table>

induction of cellular differentiation and single cell imaging of vibrio parahaemolyticus swimmer and swarmer cells

היכולת ללמוד את לוקליזציה תאיים של חלבונים חיונית להבנה של תהליכים סלולריים רבים. בתורו, זה דורש את היכולת להשיג תאים בודדים עבור מיקרוסקופ פלואורסצנטי, אשר יכול להיות מאתגר במיוחד כאשר תאים הדמיה הקיימות בקהילות חיידקים. לדוגמה, האדם הפתוגן Vibrio parahaemolyticus קיים כמו קצר בצורת מוט שחין בצורת בתנאים נוזליים כי על פני השטח קשר להבדיל subpopulation של תאים swarmer מוארך מאוד התמחה לצמיחה על משטחים מוצקים. מאמר זה מציג שיטה לבצע יחיד תא פלואורסצנטי ניתוח מיקרוסקופ של V. parahaemolyticus בשני מצבי ההפרש שלה. פרוטוקול זה reproducibly מאוד גורם הפרדה של V. parahaemolyticus לתוך מחזור חיי תא מחזורי ומקל על התפשטות שלהם על פני משטחים מוצקים. השיטה מייצרת התפרצויות של תאים swarmer מובחנת המשתרעת מ tהוא קצה של מושבה נחיל. יש לציין, בקצה מאוד של התנפחות- swarm, תאים swarmer קיים בשכבה אחת של תאים, אשר מאפשר העברת קל שלהם שקופיות מיקרוסקופ ולאחר מכן מיקרוסקופ פלואורסצנטי הדמיה של תאים בודדים. בנוסף, זרימת העבודה של ניתוח תמונות עבור ייצוג דמוגרפי של חברות חיידקים מוצג. כהוכחה לעיקרון, ניתוח של לוקליזציה תאיים של chemotaxis איתות מערכים בשחיינים ותאי swarmer של V. parahaemolyticus מתואר.

אינדוקציה של בידול ודור של מושבות נחפזות איור 1 מספק סכימה של השלבים החשובים מעורב בהפקת מושבות נחיל של V. parahaemolyticus ( איור 1A-C ). תרבות שחיינים זוהה על נחיל אגר ו מודגרות על 24 <...

Watch this Scientific Journal Video about Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells at JoVE.com

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

פרוטוקול זה מאפשר מיקרוסקופית תא בודד של מובחן Vibrio parahaemolyticus שחיין ותאי swarmer . השיטה מייצרת אוכלוסייה של תאים swarmer זמין בקלות עבור ניתוח תא בודד ומכסה הכנה של תרבויות תאים, אינדוקציה של בידול swarmer, הכנת המדגם, ניתוח תמונה.

אינדוקציה של דיפרנציאציה נייד תא הדמיה אחת של<em&gt; Vibrio parahaemolyticus</em&gt; שחיין ותאי נחיל

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

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Research

JoVE Journal

Immunology and Infection

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

9.3K Views.  Max Planck Institute for Terrestrial Microbiology.  פרוטוקול זה מאפשר מיקרוסקופית תא בודד של מובחן Vibrio parahaemolyticus שחיין ותאי swarmer . השיטה מייצרת אוכלוסייה של תאים swarmer זמין בקלות עבור ניתוח תא בודד ומכסה הכנה של תרבויות תאים, אינדוקציה של בידול swarmer, הכנת המדגם, ניתוח תמונה. 

Video: Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

Visualisation וכימות של אינטראקציות תאיים של Neisseria meningitidis ואת האדם α-actinin על ידי הדמיה confocal

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

קולוניזציה של Euprymna scolopes קלמארי על ידי Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Assay כמותית תפוקה גבוהה מתא בודד Cytotoxicity לתאי T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

יישום קליני של<em&gt; יפהפה נרדם</em&gt; ומלאכותי תאי מציגי אנטיגן גנטי לשינוי תאי T מדם טבורים היקפי וטבורים

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

בידוד ובידול של Th17 הנאיביים T מסוג CD4 לימפוציטים

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

קליפת המוח אקטין זרימה בתאי T לכמת ידי מתאם תמונת ספקטרוסקופיה מרחב ובזמן של תאורה מיקרוסקופית נתונים מובנים

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

פיתוח בינוני אגר chromogenic רגיש וספציפי יותר לצורך זיהוי של<em&gt; Parahaemolyticus Vibrio</em&gt; ו אחר<em&gt; Vibrio</em&gt; מינים

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

מאוד מרבב, סופר ברזולוציה הדמיה של תאים T באמצעות madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

הערכת מתח סלולארית, דלקת דיסקרטית מפריש אוקסיטוצין המוח גרעינים בחולדה Neonatal לפני ואחרי האכלה קולוסטרום הראשון

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Functionalization of Atomic Force Microscope Cantilevers with Single-T Cells or Single-Particle for Immunological Single-Cell Force Spectroscopy

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This technique allows for probing interaction strengths and dynamics on a live cell membrane, including those between cells, receptor and ligands, and alongside many other variations. It also works as a mechanism to deliver a physical or biochemical stimulus on single cells in a spatiotemporally controlled manner, thus allowing specific cell activation and subsequent cellular events to be monitored in real-time when combined with live-cell fluorescence imaging. The key step in those AFM-SCFS measurements is AFM-cantilever functionalization, or in other words, attaching a subject of interest to the cantilever. Here, we present methods to modify AFM cantilevers with a single T cell and a single polystyrene bead respectively for immunological studies. The former involves a biocompatible glue that couples single T cells to the tip of a flat cantilever in a solution, while the latter relies on an epoxy glue for single bead adhesion in the air environment. Two immunological applications associated with each cantilever modification are provided as well. The methods described here can be easily adapted to different cell types and solid particles.

We present a protocol to functionalize atomic force microscope (AFM) cantilevers with a single T cell and bead particle for immunological studies. Procedures to probe single-pair T cell-dendritic cell binding by AFM and to monitor the real-time cellular response of macrophages to a single solid particle by AFM with fluorescence imaging are shown.

הפונקציונליזציה של מיקרוסקופ כוח אטומי מנופים עם תאים חד-טי או חלקיק יחיד לספקטרוסקופיית של כוח בודד אימונולוגיים

Atomic force microscopy based single cell force spectroscopy (AFM-SCFS) is a powerful tool for studying biophysical properties of living cells. This ...