This research was funded in parts by NIH grants HL66299 to TS, RB, and SL, HL60024 to TS, and HL136869 to MF.

All animal procedures were reviewed and approved by the Institutional and Animal Care Committee of the University of South Alabama and were performed in accordance with all federal, state, and local regulations. Primary cultures of rat pulmonary microvascular endothelial cells (PMVECs) were obtained from the Cell Culture Core Facility at the University of South Alabama&#8217;s Center for Lung Biology. Cells were prepared using previously described procedures16.
1. Generat...

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F., 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=Association+between+hospitalization+for+pneumonia+and+subsequent+risk+of+cardiovascular+disease.">Association between hospitalization for pneumonia and subsequent risk of cardiovascular disease.</a> J Am Med Assoc. 313 (3), 264-274 (2015).</li><li>Frank, D. 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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. 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=Pseudomonas+aeruginosa+exoenzymes+U+and+Y+induce+a+transmissible+endothelial+proteinopathy.">Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy.</a> Am J Physiol Lung Cell Molec Physiol. 310 (4), L337-L353 (2016).</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+infection+liberates+transmissible,+cytotoxic+prion+amyloids.">Pseudomonas aeruginosa infection liberates transmissible, cytotoxic prion amyloids.</a> FASEB Journal. 31 (7), 2785-2796 (2017).</li><li>Stevens, T. 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Here, simple in vitro methods are outlined which allow demonstration of the generation of cytotoxic amyloids during infection with a pneumonia causing organism. These methods include a cell culture cytotoxicity assay, immunoblotting, quantitation of cell killing using a novel microscopic method, and ThT binding. Analyses of the cytotoxic agents have demonstrated that they are amyloid in nature (Figure 2 and Figure 4) and exhibit charact...

Patients who survive pneumonia have elevated death rates in the months following hospital discharge1,2,3,4,5,6. In most cases, death occurs by some type of end-organ failure including renal, pulmonary, cardiac, or liver events, as well as stroke5,6. The reason for the elevated death rate in this patient population has never been established.
Pneumonia is classified as being either community-acquired or hospital-acquired (nosocomial), and agents that can cause pneumonia include bacteria, viruses, fungi, and chemicals. One of the major causes of nosocomial pneumonia is the bacterium Pseudomonas aeruginosa. P. aeruginosa is a gram-negative organism that uses a type III secretion system to transfer various effector molecules, termed exoenzymes, directly to the cytoplasm of target cells7,8. During infection of pulmonary endothelial cells, the exoenzymes target various intracellular proteins, including an endothelial form of the microtubule-associated protein tau9,10,11,12, leading to endothelial barrier breakdown resulting in severe pulmonary edema, decreased pulmonary function and, oftentimes, death.
As stated previously, patients who survive the initial pneumonia have elevated death rates in the first 12 months following hospital discharge. A potential mechanism for explaining this phenomenon is that some type of long-lived toxin is generated during the initial infection that leads to poor long-term outcome. Two observations support this possibility. First, cultured pulmonary endothelial cells that are treated initially with P. aeruginosa fail to proliferate for up to a week after the bacteria are killed by antibiotics13. Second, long-lived prions and agents with prion characteristics have been demonstrated in various human and animal diseases, particularly diseases associated with the nervous system14,15.
Methods for examining the potential production of long-lived cytotoxic agents during pulmonary infection have never been described. Here a series of simple in vitro assays are outlined that can be used for investigating cytotoxin production and activity following infection using a common pneumonia causing agent, P. aeruginosa. These assays should be readily adaptable to investigate possible cytotoxin induction following infection using other agents that cause pneumonia, and the supernatants that are generated also should be useful for investigating effects of the cytotoxins in whole organs or animals. Finally, the assays that are outlined here most likely will be adaptable to test animal and human biological fluids for the production of cytotoxins during pneumonia.

<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

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.

A simple in vitro assay has been developed to assay for the presence of cytotoxins in supernatants of cells infected with the bacterium P. aeruginosa. Basically, culture medium from infected cells is collected 4 h after bacterial addition, the bacteria are removed by filter sterilization of the culture supernatant, and then the sterile supernatant is added to a new population of cells. The cells are then observed 21 - 24 h after the addition of supernatant and cell killi...

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

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

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 ...

This method can help answer key questions in the pulmonary care field, such as why do patients who survive hospital acquired pneumonia have such poor long term outcomes? The advantage of this method is that it's simple and reliable, which means that anybody who comes in the lab can be taught it and the next day they're generating useful and repeatable data. Though these analytical methods can be applied to provide insight into the infection of cultured cells in vitro, they can also be applied to animal models and human patients.To produce cytotoxic supernatant, wash a 150 centimeter dish of endothelial cells with HBSS, and dilute a P.aeruginosa colony to an absorbance of 0.25 at 540 nanometers. Further dilute the bacterial cells`to allow a multiplicity of infection of 20 to one in 20 milliliters of HBSS and seed the bacteria onto the plate of endothelial cells. Then place the infected cells in a 37 degrees Celsius and 5%CO2 incubator for four to five hours.It is essential that the incubation of the bacteria and the endothelial cells occur for an appropriate length of time. If the incubation is too short, the amyloids will not be released into the supernatant. If the incubation is too long, the cells will actually lyse and release all of their contents into the supernatant.The indicator that we use for an appropriate duration of incubation is the formation of gaps in the endothelial monolayer. When gaps can be observed in the cell monolayer by light microscopy harvest the supernatant for centrifugation to remove any cellular debris. Decant the supernatant into a syringe that is equipped with a 0.2 micron filter and pass the supernatant through the filter to remove any contaminating bacteria.Then set aside 1.5 milliliters of sterile supernatant for cytotoxicity testing and freeze the rest of the sample at minus 80 degrees Celsius. To assess the cytotoxicity of the harvested supernatant, add the 1.5 milliliter aliquot of filter sterilized supernatant to a single well of a six well plate containing a confluent pulmonary microvascular endothelial cell culture. Place the plate in a CO2 incubator for 21 to 24 hours.Then obtain images of regions of the treated and control cultures. Import the images into a custom imagej macro and adjust the contrast to 15%saturated pixels. Duplicate the contrast adjusted images and use subtract background to obtain high contrast images of both the intact cells and the gap area within the field of view.Subtract this high contrast image from the original image and use the image calculator and function to combine the resulting image with the original image. Use the threshold function to convert the combined image to a mask with the gaps in black and the intact cells in white. And use the binary erode function to remove any noise from the image.Then use the area fraction to measure the ratio of black to white pixels within the resultant image. And plot and express the fractional areas for each treatment time point, as the percent of the maximal gap area. To quantify the amyloids within the supernatant, add 20 microliters of freshly prepared and filtered 50X thioflavin T stock solution to one milliliter of PBS in a one milliliter spectrophotometer cuvette, and load the diluted sample onto a spectrofluorometer.Measure the baseline fluorescence emission using a 425 nanometer excitation. Scanning the fluorescence emission from 450 to 575 nanometers in two nanometer increments. Then set the instrument to perform a time lapse scan using a 425 nanometer excitation and 482 nanometer emission with data acquisition every 0.2 seconds for 60 seconds.Pause the scan after 20 seconds and 10 microliters of filter sterilized supernatant to the cuvette. After mixing by inversion, reload the cuvette onto the spectrofluorometer and resume the time based scan for the final 40 seconds. Upon completion of the time lapse, perform a final fluorescence emissions spectrum scan using the original scan settings.The addition P.aeruginosa to confluent layers of pulmonary microvascular endothelial cells induces gap formation between the cells. Using imagej to assess supernatant cytotoxicity as demonstrated during the first 12 hours of treatment, still healthy confluent cell monolayers can be visualized as all white regions within the microscopic field. However, by 18 hours after the addition of supernatant collected from PA 103 infected cultures, gaps in the monolayer can be detected with the area of the culture dish devoid of cells increasing in a linear manner until 36 hours of treatment, at which point almost no intact cells can be observed.Similar results can be obtained using lactate dehydrogenase release as a marker of cytotoxicity. With lactate dehydrogenase first detected at 18 hours after the addition of the cytotoxic supernatant and increasing in a linear fashion until maximal cell killing is measured at 36 hours. The supernatants can also be assessed by immunoblot analysis or by measuring the change in thioflavin T fluorescence intensity due to confirmational changes induced by amyloid binding to determine the presence of cytotoxic amyloids in the supernatants.After watching this video you should have a good understanding how to generate cytotoxic supernatants following the infection of endothelial cells with citomonsis aeruginosa. In addition, you should be well versed in the types of assays needed to character and analyze the cytotoxins that are present in those supernatants.

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5.4K Views.  University of South Alabama. This method can help answer key questions in the pulmonary care field, such as why do patients who survive hospital acquired pneumonia have such poor long term outcomes? The advantage of this method is that it's simple and reliable, which means that anybody who comes in the lab can be taught it and the next day they're generating useful and repeatable data. Though these analytical methods can be applied to provide insight into the infection of cultured cells in vitro, they can also be applied...