ברצוננו להודות ג O Toole הדרכה והצעות בפיתוח המודלים האלה. עבודה זו נתמכה על ידי קרן סיסטיק פיברוזיס (ANDERS06F0 כדי GGA, STANTO07RO ו STANTO08GA כדי BAS), המכונים הלאומיים לבריאות (T32A107363 כדי GGA ו-R01 HL074175 כדי BAS), לבין המרכז הלאומי למחקר משאבים מרכזי מצוינות למחקר ביו (COBRE P20-RR018787 כדי BAS).

<ol>
	<li>Anderson, G. G., Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+analysis+of+tobramycin-treated+Pseudomonas+aeruginosa+biofilms+on+cystic+fibrosis-derived+airway+epithelial+cells.">In vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on cystic fibrosis-derived airway epithelial cells.</a> Infect Immun. 76, 1423-1433 (2008).</li><li>Bruscia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+CF+cell+lines+corrected+at+DeltaF508-CFTR+locus+by+SFHR-mediated+targeting.">Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting.</a> Gene Ther. 9, 683-685 (2002).</li><li>Cozens, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFTR+expression+and+chloride+secretion+in+polarized+immortal+human+bronchial+epithelial+cells.">CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.</a> Am J Respir Cell Mol Biol. 10, 38-47 (1994).</li><li>Hentchel-Franks, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+airway+cl-+secretion+in+human+subjects+by+adenosine.">Activation of airway cl- secretion in human subjects by adenosine.</a> Am J Respir Cell Mol Biol. 31, 140-146 (2004).</li><li>Moreau-Marquis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DeltaF508-CFTR+mutation+results+in+increased+biofilm+formation+by+Pseudomonas+aeruginosa+by+increasing+iron+availability.">The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability.</a> Am J Physiol Lung Cell Mol Physiol. 295, 25-37 (2008).</li><li>Kuchma, S. L., Connolly, J. P., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-component+regulatory+system+regulates+biofilm+maturation+and+type+III+secretion+in+Pseudomonas+aeruginosa.">A three-component regulatory system regulates biofilm maturation and type III secretion in Pseudomonas aeruginosa.</a> J Bacteriol. 187, (2005).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+reproducibility+in+flow-chamber+biofilms.">Experimental reproducibility in flow-chamber biofilms.</a> Microbiology. 146 (Pt 10), 2409-2415 (2000).</li><li>Heydorn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Anderson, G. G., Yahr, T. L., Lovewell, R. R., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Pseudomonas+aeruginosa+magnesium+transporter+MgtE+inhibits+transcription+of+the+type+III+secretion+system.">The Pseudomonas aeruginosa magnesium transporter MgtE inhibits transcription of the type III secretion system.</a> Infect Immun. 78, (2010).</li><li>Costerton, 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=Overview+of+microbial+biofilms.">Overview of microbial biofilms.</a> J Ind Microbiol. 15, 137-140 (1995).</li><li>Hoiby, N., Frederiksen, B., Pressler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eradication+of+early+Pseudomonas+aeruginosa+infection.">Eradication of early Pseudomonas aeruginosa infection.</a> J Cyst Fibros. 4, 49-54 (2005).</li><li>Ko, Y. H., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis:+a+brief+look+at+some+highlights+of+a+decade+of+research+focused+on+elucidating+and+correcting+the+molecular+basis+of+the+disease.">Cystic fibrosis: a brief look at some highlights of a decade of research focused on elucidating and correcting the molecular basis of the disease.</a> J Bioenerg Biomembr. 33, 513-521 (2001).</li><li>Gibson, R. L., Burns, J. L., Ramsey, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+management+of+pulmonary+infections+in+cystic+fibrosis.">Pathophysiology and management of pulmonary infections in cystic fibrosis.</a> Am J Respir Crit Care Med. 168, 918-951 (2003).</li><li>Furukawa, S., Kuchma, S. L., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+their+options+open:+acute+versus+persistent+infections.">Keeping their options open: acute versus persistent infections.</a> J Bacteriol. 188, 1211-1217 (2006).</li><li>Merritt, J. H., Kadouri, D. E., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+and+analyzing+static+biofilms.">Growing and analyzing static biofilms.</a> Curr Protoc Microbiol. Chapter 1, Unit 1B 1-Unit 1B 1 (2005).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signalling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis.</a> Mol Microbiol. 28, 449-461 (1998).</li><li>Gomez, M. I., Prince, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunistic+infections+in+lung+disease:+Pseudomonas+infections+in+cystic+fibrosis.">Opportunistic infections in lung disease: Pseudomonas infections in cystic fibrosis.</a> Curr Opin Pharmacol. 7, 244-251 (2007).</li></ol>

אין ניגודי אינטרסים הכריז.

Biofilms הן הקהילות של חיידקים היוצרים בתגובה לגירויים סביבתיים. אלה אותות סביבתיים להוביל שינויים רגולטוריים הגלובלית בתוך כל חיידק, וכתוצאה מכך מחייב משטח, צבירה, ייצור exopolysaccharides ו פנוטיפים אחרים כגון עמידות לאנטיביוטיקה גדל 10. במהלך השנתיים האחרונות של עשרות ש...

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		<th>Material Name</th>
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		<th>Comment</th>
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<td>FCS2 (Focht Live-Cell) chamber</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319131616</td>
<td>&nbsp;</td>
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<td>FCS2 chamber controller</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>060319-2-0303</td>
<td>&nbsp;</td>
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<td>40 mm glass coverslips</td>
<td>&nbsp;</td>
<td>Bioptechs, Butler, PA</td>
<td>PH 40-1313-0319</td>
<td>&nbsp;</td>
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<td>MEM</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>#10-010-CV</td>
<td>&nbsp;</td>
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<td>MEM without phenol red</td>
<td>&nbsp;</td>
<td>Mediatech, Manassass, VA</td>
<td>Mediatech, Manassass, VA</td>
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co-culture models of pseudomonas aeruginosa biofilms grown on live human airway cells

Biofilms חיידקי נקשרו עם מספר מחלות אנושיות שונות, אבל פיתוח biofilm יש בדרך כלל נחקרה על משטחים שאינם חיים. במאמר זה, אנו מתארים פרוטוקולים biofilms להרכיב Pseudomonas aeruginosa על תאים אנושיים אפיתל דרכי הנשימה (תאים CFBE) גדל בתרבות. בשיטה הראשונה (כינה את המודל הסטטי שותף תרבות biofilm), עמ &#39; aeruginosa הוא מודגרות עם תאים CFBE גדל כמו monolayers ומחוברות על צלחות תרבות תקן רקמות. למרות החיידק הוא רעיל למדי לתאי האפיתל, תוספת של עיכובים ארגינין הרס monolayer מספיק זמן בשביל ליצור biofilms על תאים CFBE. השיטה השנייה (המכונה תא זרימה משותפת תרבות דגם biofilm), כולל התאמה של מנגנון הזרימה תא biofilm, המשמש לעתים קרובות biofilm מחקר, כדי להתאים coverslip זכוכית תמיכה monolayer ומחוברות של תאים CFBE. Monolayer זה מחוסן עם פ aeruginosa ומשאבה peristaltic אז זורם בינוני טרי על פני התאים. שתי המערכות, biofilms חיידקי טופס בתוך 6-8 שעות לאחר חיסון. ויזואליזציה של biofilm מוגברת על ידי שימוש P. זנים aeruginosa constitutively לבטא חלבון פלואורסצנטי ירוק (GFP). סטטית תזרים Co-Cell תרבות מבחני biofilm הן מערכות מודל פ מוקדם aeruginosa זיהום של הריאות סיסטיק פיברוזיס (CF), ואת הטכניקות הללו מאפשרים היבטים שונים של פ biofilm aeruginosa היווצרות ארסיות להיחקר, כולל cytotoxicity biofilm, מדידת biofilm CFU, והכתים באופן חזותי biofilm.

Watch this Scientific Journal Video about Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells at JoVE.com

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

מאמר זה מתאר שיטות שונות של גידול<em&gt; Pseudomonas aeruginosa</em&gt; Biofilms על בתרבית תאים אפיתל דרכי הנשימה האנושית. פרוטוקולים אלה ניתן להתאים לימוד היבטים שונים של היווצרות biofilm, כולל הדמיה של biofilm, מכתים של biofilm, מדידת המושבה להרכיב יחידות (CFU) של biofilm, ולומדים cytotoxicity biofilm.

Co-מודלים של תרבות Pseudomonas aeruginosa Biofilms גדל על תאים חיים Airway האדם

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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Immunology and Infection

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

22.1K Views.  Dartmouth College. מאמר זה מתאר שיטות שונות של גידול Pseudomonas aeruginosa Biofilms על בתרבית תאים אפיתל דרכי הנשימה האנושית. פרוטוקולים אלה ניתן להתאים לימוד היבטים שונים של היווצרות biofilm, כולל הדמיה של biofilm, מכתים של biofilm, מדידת המושבה להרכיב יחידות (CFU) של biofilm, ולומדים cytotoxicity biofilm.

Video: Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa ו biofilm Saccharomyces cerevisiae בתוך תאים Flow

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA בידוד Pseudomonas aeruginosa מיישבים את מערכת העיכול Murine

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

כרוני לטווח ארוך<em&gt; Pseudomonas aeruginosa</em&gt; זיהום דרכי הנשימה בעכברים

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

הערכת חישה המולדת של CD4 הנגוע ב- HIV-1<sup&gt; +</sup&gt; T תאים על ידי תאים דנדריטים Plasmacytoid שימוש<em&gt; Ex vivo</em&gt; עמית תרבות מערכת.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa מושרה ריאות פגיעה דגם

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

שיטות זיהוי ציטוטוקסיות Amyloids הבאים זיהום של אנדותל תאי הריאות על ידי Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

תרבות של וריאציה של המושבה הקטנה של הפסאודומונס וככמת של האלגיאט

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

מדבקת החיסון נגד הידבקות כרונית בנגיף הדלקת בפצע

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

פ. ארוגינוזה נגוע 3D שיתוף תרבות של תאים אפיתל הסיפונות מקרופאגים בממשק אוויר נוזלי להערכה פרה-סרטנית של אנטי זיהומים

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.