{'fraction_non_alphanumeric': 0.02072538860103627, 'fraction_numerical': 0.003700962250185048, 'mean_word_length': 6.208, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Development of a Novel Point of Care Test for Toxic Amyloids in Patients Recovering from Hospital Acquired Pneumonia

Amyloids have autofluorescence properties that may be exploited for their detection in patients recovering from hospital acquired pneumonia (HAP). Recovering HAP patients have cytotoxic amyloids in their bronchoalveolar lavage fluid, blood, and cerebrospinal fluid, and these amyloids may be the cause of the cognitive decline and secondary organ failure patients encounter after HAP. Infection of pulmonary microvascular endothelial cells (PMVECs) produces bacterial strain‐dependent cytotoxic or antimicrobial amyloids. Currently, there are no detection methods for pulmonary amyloids at the bedside. Here, we hypothesize that cytotoxic and antimicrobial amyloids can be detected and distinguished by their autofluorescence properties. PMVECS were infected with mutants of gram‐negative bacterium Pseudomonas aeruginosa, including virulent ExoY+ and avirulent ∆PcrV, to generate amyloids with distinctive phenotypes. Infection with ExoY+ for 7 hours produced cytotoxic amyloids, while infection with ∆PcrV for 4 hours yielded antimicrobial amyloids. To resolve whether the autofluorescence properties are concentration‐dependent, protein concentrations of the supernatants were standardized to 50 μg/mL. To assess whether fibrillar or oligomeric amyloids are responsible for the autofluorescent signature of ExoY+ and ∆PcrV supernatants, the supernatants were immunodepleted with the amyloid fibril‐specific OC or amyloid oligomer‐specific A11 antibody. Fluorescence spectroscopy was performed to obtain the fluorescence spectra of the unstandardized, standardized, and immunodepleted ExoY+ and ΔPcrV supernatants. The unstandardized fluorescence spectra of ExoY+ and ΔPcrV were indistinguishable; however, when standardized to 50 μg/mL, the fluorescence intensity of ΔPcrV supernatant containing antimicrobial amyloids was significantly greater than ExoY+ supernatant containing cytotoxic amyloids. Immunodepleting the supernatant with the OC antibody eliminated the fluorescence intensity of ΔPcrV supernatant but did not diminish the fluorescence intensity of the ExoY+ supernatant. However, fluorescence of the ExoY+ supernatant was eradicated after immunodepletion with the A11 antibody. Our work suggests that the intrinsic fluorescence of amyloids can be developed as a point‐of‐care diagnostic tool in HAP. Antimicrobial amyloids may be detected by the immunodepletion of amyloid fibrils whereas cytotoxic amyloids can be distinguished by immunodepleting oligomeric amyloids. In the future we will apply this method to patient samples to assess its use as a diagnostic tool.