עבודה זו נתמכה על ידי מענק מטעם משרד המדע והטכנולוגיה (MOST 103-2633-B-002-002; 104-2320-B-002-048). אנו מודים בני זוג. בטי וו-צ&#39;יי Chien-קואו לי בבית NTU, ליי זרבוני באוניברסיטת סטנפורד וד&quot;ר פיטר הופמן באוניברסיטת הוואי לקריאת כתב היד, יון Chien ב NTU לקבלת סיוע טכני, וד&quot;ר וון-צ&#39;י וויי ב ביוטכנולוגיה החקלאות מרכז המחקר בבית Academia Sinica לקבלת ייעוץ טכני.

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L., 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=An+alternatively+spliced+IL-15+isoform+modulates+abrasion-induced+keratinocyte+activation.">An alternatively spliced IL-15 isoform modulates abrasion-induced keratinocyte activation.</a> J Invest Dermatol. 135, 1329-1337 (2015).</li><li>Rudy, S. 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אין המחברים אינטרסים פיננסיים לחשוף.

השלב הקריטי ביותר להבטיח את הביטוי של פלסמיד דנ&quot;א acufected הוא להתנדנד באופן שווה לשחרר את שכבת חרמן העור. בעוד דוחף מחטים בעדינות בלי לחתוך את העור, הכוח צריך להיות מספיק חזק כדי לדכא את השטח. כדי להקל על קליטת ה- DNA בתמיסה 10 μL על פני שטח 1 ס&quot;מ x 1 ס&quot;מ, מחטים צריך להתנד?...

העור הוא קו ההגנה הראשון המארח. קרטינוציטים (KCS) הם סוג התאים העיקרי בעור של בני אדם ועכברים. בתגובה לגירויים סביבתיים (למשל השמש, חמצן, כימיקלים הפלישה פתוגניים), KCS מופעלים לייצר מגוון רחב של ציטוקינים מעודדי דלקת וכמוקינים כגון IL-8, IL-6, IL-1α, IL-1β, TNF- α ו- GM-CSF 1. יחד הם מפעילים את הגיוס של תאי מערכת החיסון של העור. דלקת עורית מחמירות קשורה לעיתים קרובות עם מחלות אנושיות רבות כולל דלקת עור ממגע אקטופי חריפה ודלקת בתיווך התא T כרונית (דלקת עור אלרגית ממגע למשל ופסוריאזיס) 2. ויסות תגובות מעודדות דלקת בעור על ידי עיכוב הפעלת KC היא גישה סבירה לטיפול בדלקת עורית. פרוטוקול זה מתאר גישה חדשה לבטא את גן ציטוקינים זמני האפידרמיס על מנת ללמוד מיל חיסוניponse תוצאתי כדי טיפול כזה בעור. האפידרמיס מלחין השכבה השטחית ביותר של העור. הוא משמש כמחסום פיזי שמירת חומרים חיצוניים כגון חומצות גרעין ופתוגנים מלהיכנס לשכבות העמוקות של העור. כמה טכניקות מחט ללא הוקמו DNA אפידרמיס העברת 3, 4. דנ&quot;א בתמיסה או הקשורים ליפוזומים קטיוני או וקטורים אדנווירוס יושם ישירות האפידרמיס שונה עם טכניקות כגון הסרת השכבה הקרנית או הטיפול עם depilating מגיב. הסרת האפיתל cornified מקל DNA חציית מחסום האפידרמיס ואינטראקציה עם קרטינוציטים ותאי לנגרהנס לגרום תגובה חיסונית 5. בעוד שטח פנים גדול זמין עבור העברת דנ&quot;א גישה זו אינה כרוכה מחטים, ישנם חסרונות לרבות הדרישהעבור הכמויות הגדולות של DNA (10 - 100 מיקרוגרם), יחס קשה על העור על ידי הפשטה, ואת התוצאות לא העקביות בזירוז תגובה חיסונית של חיות המחוסנות בלי מאיץ משלוח DNA 6. Microparticle בתיווך משלוח תאי של DNA ערום אל העור הוכח להיות יעיל מאוד לשחזור גרימת תגובה חיסונית 7, 8. אקדח גני כף יד נעשה שימוש כדי להפציץ את אתר יעד עור עם חלקיקי זהב מצופה פלסמיד דנ&quot;א 2 מיקרומטר בקוטר בגודל באמצעות זרימת אוויר הליום בלחץ 9. ה- DNA פלסמיד כנראה הוא נלקח על ידי תאים דנדריטים KCsand (DCS) בעור ואת החלבון מתבטא באופן מקומי או מועברים ניקוז בלוטות הלימפה ידי DCs 10. למרות שמערכת אקדח גנים היא פשוטה ודורשת ניסיון טכני מוגבל, העלות להכנה ושולח את &quot;bul DNAתן &quot;(כלומר אבקות זהב, גז הליום) ועבור אקדח הגן עצמו הגביל היישום הכללי שלה. בנוסף, הדרישה עבור מערכת הולכת גז הליום מגביל את הקלות של תחבורה אקדח גנים כאשר הניסויים נערכים במקומות שונים. Microseeding הודגם להשיג יעילות גבוהה יותר של העברת גנים מ זריקה אחת חלקיקי הפגזה 11. Microseeding משתמשת אקדח קעקוע להעביר דנ&quot;א לעור ללא שימוש חרוזי חלקיקים 11. מתנודד microneedles על העור באמצעות גישה זו עשוי ישירות לגרד את קרום התא ובכך להעביר DNA לתאים מרובים בו זמנית. עם זאת, הכאב הקשור למספר רב של זריקות עם מחטים 0.254 מ&quot;מ בקוטר הוא חיסרון. כאן אנו מספקים גישה חלופית למסירת DNA in vivo. את &quot;acufection&quot; </strאונג&gt; פרוטוקול שתיארנו כאן מספק דרך חסכונית ויעילה כדי לספק פלסמיד דנ&quot;א בעור העכבר. בקצרה, עשר מחטי הדיקור (0.2 מ&quot;מ קוטר x 13 מ&quot;מ אורך) היו כבולים צרור ידי דבק. נפח של 10 μL של PBS עם 10 מיקרוגרם של ה- DNA פלסמיד המכיל את הגן של העניין הושם על עור אגף המגולח, מקריח שטופל בקרם. באמצעות חבילה מחט דיקור להתנדנד המשטח (1 ס&quot;מ x 1 ס&quot;מ) של העור, keratins על שכבת חרמן התרופפו ואת פלסמיד דנ&quot;א מיושם על פני השטח נטמע האפידרמיס. ניזק לעור היה פיקוח ונמצא מינימאלי. ביטוי של גן טרנספקציה בעור acufected אושר על ידי שני כמוני בזמן האמת PCR (qRT-PCR) ו- ELISA. השפעות modulatory החיסוניות של-נמסר acufection IL-15ΔE7 על דלקת עורית הודגמו באמצעות מודל עכבר מטופל-IMQ 12. בעוד acufection מוכיח להיות Dema קל ופחותnding שיטה למסירה DNA in vivo, את הכמות האופטימלית של DNA עבור גנים שונים בזמנים לדקור את העור צריך להיות מותאם בקפידה כדי להשיג תוצאות לשחזור.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accu Handy Needle</td>
			<td>Accu</td>
			<td>36Gx0.5</td>
			<td>Medical device</td>
		</tr>
		<tr>
			<td>NairTM Lotion</td>
			<td>Church &#38; Dwight</td>
			<td>N/A</td>
			<td>Use to remove hair</td>
		</tr>
		<tr>
			<td>Shandon Xyline substitute</td>
			<td>Thermo Fisher Scientific</td>
			<td>6764506</td>
			<td>Deparaffinization</td>
		</tr>
		<tr>
			<td>Avertin (2,2,2-Tribromethanol)</td>
			<td>Sigma-Aldrich</td>
			<td>T4,840-2</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>2-methyl-2-butanol</td>
			<td>Sigma-Aldrich</td>
			<td>152463</td>
			<td>Solvent for the dissolution of avertin</td>
		</tr>
		<tr>
			<td>DifcoTM LB broth, Miller</td>
			<td>BD</td>
			<td>244620</td>
			<td>Propagation and maintenance of E. coli for molecular biology</td>
		</tr>
		<tr>
			<td>QIAGEN&#9415; Plasmid Midi Kit</td>
			<td>QIAGEN</td>
			<td>12143</td>
			<td>Purification of plasmid DNA from E. coli</td>
		</tr>
		<tr>
			<td>Mouse IL-15/IL-15R Complex ELISA Ready-SET-Go kit</td>
			<td>eBioscience</td>
			<td>88-7215</td>
			<td>Measure IL-15 protein</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6G</td>
			<td>BioLegend</td>
			<td>127601</td>
			<td>Antibody used for immunohistochemical stain</td>
		</tr>
		<tr>
			<td>anti-mouse Ki-67</td>
			<td>eBioscience</td>
			<td>14-5698-80</td>
			<td>Antibody used for immunohistochemical stain</td>
		</tr>
		<tr>
			<td>Simple Stain Mouse MAXPO (Rat)</td>
			<td>Nichirei Biosciences</td>
			<td>414341F</td>
			<td>Reagent to block background signal in mouse-on-mouse stain</td>
		</tr>
		<tr>
			<td>DAB Peroxidase (HRP) Substrate Kit</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Peroxidse substrate for immunohistochemical stain</td>
		</tr>
		<tr>
			<td>Ultra V block</td>
			<td>Thermo Fisher Scientific</td>
			<td>TA-060-UB</td>
			<td>Reduce nonspecific background staining</td>
		</tr>
		<tr>
			<td>Methyl green</td>
			<td>Sigma-Aldrich</td>
			<td>M8884</td>
			<td>Nuclear staining</td>
		</tr>
		<tr>
			<td>Vecta MountTM</td>
			<td>Vector Laboratories</td>
			<td>M-5000</td>
			<td>Permanently preserving histochemical stains</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>04-693-116-001</td>
			<td>Inhibit protease activity in cell lysate</td>
		</tr>
		<tr>
			<td>Aldara cream</td>
			<td>3M Parmaceuticals</td>
			<td>N/A</td>
			<td>5% imiquimod cream</td>
		</tr>
		<tr>
			<td>Bessman Tissue Pulverizer</td>
			<td>Spectrum Labs</td>
			<td>189475</td>
			<td>Use to pulverize skin tissue</td>
		</tr>
		<tr>
			<td>ABI7900HT cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>quantitative real-time PCR assay</td>
		</tr>
		<tr>
			<td>Camcorder</td>
			<td>Panasonic</td>
			<td>HDC-SD60</td>
			<td>Image documentation</td>
		</tr>
		<tr>
			<td>Axio Scope.A1</td>
			<td>ZEISS</td>
			<td>N/A</td>
			<td>Bright-field microscopy</td>
		</tr>
		<tr>
			<td>ScanScope&#9415; XT</td>
			<td>Aperio</td>
			<td>N/A</td>
			<td>Whole-field image scanner</td>
		</tr>
		<tr>
			<td>MetaMorph&#9415; Research Imaging</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Quantitative analysis of skin thickness and immune-reactive cells</td>
		</tr>
	</tbody>
</table>

development of an economical dna delivery system by "acufection" and its application to skin research

חוסר ויסות תגובה חיסונית עור קשור מחלות עור אדם רבות. העברה ישירה של גנים חיסוניים הקשורות לתוך רקמת עור היא גישה מרתקת לחקור אפנון חיסון של דלקת עורית בעכברי מודל של מחלות אנושיות. כאן אנו מציגים פרוטוקול חסכוני כי נמסר DNA עירומה עור עכבר ומוביל ביטוי transgene. השיטה שטבע &quot;acufection&quot;, המציין fection טרנס בתיווך לנקב ACU DNA. כדי לבצע acufection, עכבר עור היה חדור ראשון עם ה- DNA פוספט שנאגר מלוח (PBS) ולאחר מכן דקר בקלילות עם חבילה של מחטי דיקור כדי להקל על קליטת ה- DNA ואת transfection לתוך תאים. ה- DNA פלסמיד כנראה נלקח על ידי keratinocyte ותאים דנדריטיים (DCS) בעור והביע לתוך חלבון. שמוק מכני עם מחטים כשלעצמה לא לגרום נזק לעור או לגרום ההפעלה keratinocyte. הביטוישל גנים טרנספקציה אותרה בעור ברמות תעתיק ו translational הן הבאות acufection עבור 2 ימים ומתוחזק עד 7 ימים. המטרה העיקרית לפיתוח שיטה acufection זה היה כדי לחקור איזופורם מוגדר בעבר של IL-15. באמצעות שיטה זו, איזופורם שחבור חלופי IL-15 עם 7 אקסון נמחק חלקית (IL-15ΔE7) הובעה בעור ובהמשך מטופלים עם קולטן אגרה דמוי 7 (TLR7) אגוניסט, imiquimod (IMQ), כדי לגרום לדלקת. Acufection-נמסר IL-15ΔE7 בעור מודחקים התפשטות keratinocyte, עובי אפידרמיס גיוס נויטרופילים דלקת עורית הנגרמת IMQ. עם הגדלת ריבית בזיהוי מנגנוני הוויסות של דלקת עורית, הפרוטוקול המתואר כאן מספק אלטרנטיבה יעילה תכליתית עלות למערכת אקדח הגנים או microseeding למסירת DNA in vivo. זה עלול פוטנציאלי לאפשר גילוי הפונקציה של רומןהגן בעור או לחקירת הטיפול החדש למחלות עורית.

בעוד כמה אדמומיות בלטה במהלך השעה הראשונה לאחר לדקור עם מחטי דיקור, העור מנוקה ביום 2 ואין תגובה של עור לוואי עד יום 7 נצפתה לאחר לדקור (איור 2 א). דוקר מחט המושרה ברמה נמוכה מאוד של orthokeratosis אפידרמיס הסתננות עורי מינימלית על ידי ניתוח H &a...

Watch this Scientific Journal Video about Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research at JoVE.com

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

פרוטוקול זה מהווה חלופה חסכונית להיבעת פלסמיד דנ&quot;א עירומה עור עכבר. המטרה הכללית של הפרוטוקול היא לספק גנים חיסוניים הקשורות לתוך רקמות עור כדי להתוות את התפקיד הפונקציונלי של גן ספציפי דלקת עורית.

פיתוח של מערכת מסירת DNA החסכוני ידי &quot;Acufection&quot; ויישומו מחקר עור

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

development-an-economical-dna-delivery-system-acufection-its

Research

JoVE Journal

Immunology and Infection

7.3K Views.  National Taiwan University College of Medicine.  פרוטוקול זה מהווה חלופה חסכונית להיבעת פלסמיד דנ"א עירומה עור עכבר. המטרה הכללית של הפרוטוקול היא לספק גנים חיסוניים הקשורות לתוך רקמות עור כדי להתוות את התפקיד הפונקציונלי של גן ספציפי דלקת עורית. 

Video: Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

פיתוח מרקר לבחירה שלילי Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

פיתוח נייד מסוג ספציפי נגד HIV gp120 aptamers למסירה siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

יישום של עכבר Ligated מעיים תיקון של Peyer Assay Loop להערכת ספיגת חיידקים על ידי תאים M

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

טיהור ויזואליזציה של Lipopolysaccharide של חיידקים גראם שליליים, על ידי הפקת מימית, פנול חם

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

הקמתה של<em&gt; במבחנה</em&gt; מערכת ללמוד את התנהגות תאית של<em&gt; קנדידה glabrata</em&gt; בהמקרופאגים THP-1 אדם

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

יישום של פלורסנט חלקיקים לחקר שיפוץ של מערכת Endo-lysosomal על ידי חיידקים תאיים

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

פיתוח<em&gt; במבחנה</em&gt; מערכת מודל לחקר האינטראקציה של<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE עם קולטן גבוה הזיקה שלה FcεRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

לימפה Duct cannulated עכברוש דגם mesenteric: בקשה להערכה של המעיים הלימפה תרופות תחבורה

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

טיפוח<em&gt; Heligmosomoides Polygyrus:</em&gt; נמטודות טפיל מערכת חיסונית ומוצרים המופרשים

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

פיתוח מודל קוליטיס מונחה Antigen ללמוד מצגת של אנטיגנים על ידי אנטיגן הצגת תאים לתאי T

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...