Name: methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by pseudomonas aeruginosa
Uploaded: 2018-07-12T12:30:00
Description: Watch this Scientific Journal Video about Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa at JoVE.com

Denne forskning blev finansieret i dele af NIH tilskud HL66299 til TS, RB og SL, HL60024 til TS, og HL136869 til MF.

Alle dyr procedurer blev gennemgået og godkendt af den institutionelle og animalsk omhu Udvalget af University of South Alabama og blev udført i overensstemmelse med alle føderale, statslige og lokale bestemmelser. Primære kulturer af rotte pulmonal mikrovaskulære endothelial celler (PMVECs) stammer fra celle kultur Core facilitet på University of South Alabama's Center for lunge biologi. Celler blev udarbejdet ved hjælp af tidligere beskrevne procedurer16.
1. gene...

<ol>
	<li>Brancati, F. L., Chow, J. W., Wagener, M. M., Vacarello, S. J., Yu, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pneumonia+really+the+old+man's+friend?+Two-year+prognosis+after+community-acquired+pneumonia.">Is pneumonia really the old man's friend? Two-year prognosis after community-acquired pneumonia.</a> Lancet. 342 (8862), 30-33 (1993).</li><li>Hedlund, J. U., Ortqvist, A. B., Kalin, M. E., Granath, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+of+importance+for+the+long-term+prognosis+after+hospital+treated+pneumonia.">Factors of importance for the long-term prognosis after hospital treated pneumonia.</a> Thorax. 48 (8), 785-789 (1993).</li><li>Meier, C. R., Jick, S. S., Derby, L. 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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=The+exoenzyme+S+regulon+of+Pseudomonas+aeruginosa.">The exoenzyme S regulon of Pseudomonas aeruginosa.</a> Mol Microbiol. 26 (4), 621-629 (1997).</li><li>Engel, J., Balachandran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Pseudomonas+aeruginosa+type+III+effectors+in+disease.">Role of Pseudomonas aeruginosa type III effectors in disease.</a> Curr Opin Microbiol. 12 (1), 61-66 (2009).</li><li>Ochoa, C. D., Alexeyev, M., Pastukh, V., Balczon, R., Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+exotoxin+Y+is+a+promiscuous+cyclase+that+increases+endothelial+tau+phosphorylation+and+permeability.">Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability.</a> J. Biol. Chem. 287 (30), 25407-25418 (2012).</li><li>Balczon, R., 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=Pseudomonas+aeruginosa+exotoxin+Y-mediated+tau+hyperphosphorylation+impairs+microtubule+assembly+in+pulmonary+microvascular+endothelial+cells.">Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.</a> PLoS One. 8, e74343 (2013).</li><li>Morrow, K. 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Her, er enkel in vitro- metoder beskrevet som give demonstration af generation af cytotoksiske amyloids under infektion med en lungebetændelse forårsager organisme. Disse metoder omfatter celle kultur cytotoksicitet assay, immunoblotting, kvantitering af celle drab ved hjælp af en roman mikroskopimetoden og ThT bindende. Analyser af de cytotoksiske agenter har vist at de amyloid i naturen (figur 2 og figur 4) og udstille Karakteris...

Patienter, der overlever lungebetændelse har forhøjet dødelighed i månederne efter hospitalet decharge1,2,3,4,5,6. I de fleste tilfælde opstår død af en form for ende-orgel bankerot herunder nyre, pulmonal, hjerte, eller lever events samt slagtilfælde5,6. Årsagen til den øgede dødelighed i denne patientgruppe er aldrig blevet etableret.
Lungebetændelse er klassificeret som enten EF-overtagne eller hospitalserhvervede (nosokomielle) og agenter, der kan forårsage lungebetændelse omfatter bakterier, vira, svampe og kemikalier. En af de største årsager til nosokomiel pneumoni er bakterien Pseudomonas aeruginosa. P. aeruginosa er en gram-negative organisme, der bruger en type III sekretion system til at overføre forskellige effektor molekyler, betegnes exoenzymes, direkte til cytoplasma af target celler7,8. Under infektion af pulmonal endotelceller målrette exoenzymes forskellige intracellulære proteiner, herunder en endotel form for mikrotubulus-forbundet protein tau9,10,11,12 , fører til endotel barriere opdeling resulterer i alvorlige lungeødem, nedsat pulmonal funktion og oftentimes, død.
Som nævnt tidligere, har patienter, som overlever de første lungebetændelse forhøjet dødelighed i de første 12 måneder efter hospitalet decharge. En potentiel mekanisme til at forklare dette fænomen er, at nogle type af langlivede toksin genereres under den indledende infektion, der fører til dårlig langsigtede resultat. To bemærkninger støtte denne mulighed. Første, kulturperler pulmonal endotelceller, der er behandlet i første omgang med P. aeruginosa undlader at formere sig op til en uge efter bakterier er dræbt af antibiotika13. Andet, langlivede prioner og agenter med prioner egenskaber er blevet påvist i forskellige menneskers og dyrs sygdomme, navnlig sygdomme forbundet med nervesystemet14,15.
Metoder til at undersøge den potentielle produktion af langlivede cytotoksiske agenter under pulmonal infektion er aldrig blevet beskrevet. Her en række enkle in vitro- assays er skitseret, kan bruges til at undersøge cytotoxin produktion og aktivitet efter infektion ved hjælp af en fælles lungebetændelse forårsager agent, P. aeruginosa. Disse assays bør være let tilpasses til at undersøge mulige cytotoxin induktion efter infektion med andre agenser, der forårsager lungebetændelse, og de supernatanter, der er genereret også bør være nyttige for at undersøge virkningerne af cytotoksiner i hele organer eller dyr. Endelig, de assays, der er skitseret her sandsynligvis vil kunne tilpasses teste dyrs og menneskers biologiske væsker til produktion af cytotoksiner under lungebetændelse.

<table>
	<tbody>
		<tr>
			<td>Rabbit anti beta amyloid</td>
			<td>Thermo</td>
			<td>71-5800</td>
			<td></td>
		</tr>
		<tr>
			<td>A11 amyloid oligomer antibody</td>
			<td>StressMarq</td>
			<td>SPC-506D</td>
			<td></td>
		</tr>
		<tr>
			<td>T22 anti-tau oligomer antibody</td>
			<td>EMD Millipore</td>
			<td>ABN454</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin T</td>
			<td>Sigma Aldrich</td>
			<td>T3516</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 micron syringe filters</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP Goat antirabbit IgG</td>
			<td>Abcam</td>
			<td>ab6721-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Strains PA103 and &#916;PcrV</td>
			<td></td>
			<td></td>
			<td>These strains of P. aeruginosa were obtained from Dr. Dara Frank, University of Wisconsin Medical College, Milwaukee, WI</td>
		</tr>
		<tr>
			<td>FITC Griffonia lectin</td>
			<td>Sigma Aldrich</td>
			<td>L9381</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC Helix pomatia lectin</td>
			<td>Sigma Aldrich</td>
			<td>L1261</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 micron nitrocellulose</td>
			<td>BioRad</td>
			<td>162-0112</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 2 collagenase</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30898.03IH</td>
			<td></td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin Disodium salt</td>
			<td>Sigma&#160;</td>
			<td>C1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcetrifugation concentrators- 10,000 MW cut-off</td>
			<td>Millipore</td>
			<td>UFC801008</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma&#160;</td>
			<td>795496-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium phosphate heptahydrate</td>
			<td>MP Biomedicals</td>
			<td>MP021914221</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>G Biosciences</td>
			<td>50-103-5801</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic heptahydrate</td>
			<td>Sigma</td>
			<td>S9506-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>C10277</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for detecting cytotoxic amyloids following infection of pulmonary endothelial cells by pseudomonas aeruginosa

Patienter, der overlever lungebetændelse har forhøjet dødelighed i månederne efter hospitalet decharge. Det har været en hypotese at infektion af pulmonal væv under lungebetændelse resulterer i produktionen af langlivede cytotoksiner, der kan føre til efterfølgende ende organsvigt. Vi har udviklet in vitro- assays for at teste hypotesen at cytotoksiner produceres under pulmonal infektion. Isolerede rotte pulmonal endotelceller og bakterien Pseudomonas aeruginosa bruges som modelsystemer, og produktionen af cytoxins efter bakterier infektion i endothelial celler påvises ved hjælp af cellekultur efterfulgt af direkte kvantificering ved hjælp af laktat dehydrogenase assays og en roman mikroskopimetoden udnytte ImageJ teknologi. Den amyloid karakter af disse cytotoksiner blev påvist ved thioflavin T bindende assays, immunoblotting og immunodepletion ved hjælp af A11 anti-amyloid antistof. Yderligere analyser ved hjælp af immunoblotting viste at oligomere tau og Aβ blev produceret og udgivet af endothelceller efter P. aeruginosainfektion. Disse metoder bør være let at tilpasse til analyser af humane kliniske prøver.

En simpel i vitro assay er udviklet til at analyse for tilstedeværelse af cytotoksiner i supernatanter af celler inficeret med bakterien P. aeruginosa. Dybest set, næringssubstrat fra inficerede celler er indsamlet 4 h efter bakteriel tilsætning, bakterier er fjernet af filter Sterilisation af supernatanten kultur og derefter sterile supernatanten tilføjes en ny befolkning af celler. Cellerne er derefter observeret 21-24 timer efter tilsætning af supernatanten og ce...

Watch this Scientific Journal Video about Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa at JoVE.com

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

Metoder til påvisning af cytotoksiske Amyloids følgende infektion af pulmonal endotelceller af Pseudomonas aeruginosa

Simple metoder er beskrevet for demonstrerer produktion af cytotoksiske amyloids efter infektion af pulmonal endotel af Pseudomonas aeruginosa.

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

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Immunology and Infection

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

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