Background
Legionella spp. can survive and replicate inside host cells such as protozoa and macrophages. After enough growth, Legionella is released from the host cells as free legionellae or Legionella-filled vesicles. The vesicles support Legionella to survive for a long time in the environment and transmit to a new host. In this study, we identified the differentially expressed genes of Acanthamoeba infected by Legionella (ACA1_114460, ACA1_091500, and ACA1_362260) and examined their roles in the formation of the excreted vesicles and escape of Legionella from the Acanthamoeba.
Methods
After ingestion of Escherichia coli and Legionella pneumophila, expression levels of target genes in Acanthamoeba were measured by real-time polymerase chain reaction (PCR) analysis. The roles of target genes were investigated by transfection of small interfering RNA (siRNA). The formation of Legionella-containing excreted vesicles and the vesicular co-localization with the lysosomes were examined by Giemsa stain and LysoTracker stain.
Results
ACA1_114460, ACA1_091500, and ACA1_362260 were upregulated after ingestion of Legionella in Acanthamoeba. ACA1_114460- and ACA1_091500-silenced Acanthamoeba failed to form the Legionella-containing excreted vesicles. Legionella was released as free legionellae from the Acanthamoeba. When the ACA1_362260 of Acanthamoeba was silenced, Legionella-containing excreted vesicles were fused with the lysosome.
Conclusions
These results indicated that ACA1_114460, ACA1_091500, and ACA1_362260 of Acanthamoeba played important roles in the formation of Legionella-containing excreted vesicles and inhibition of the lysosomal co-localization with the phagosome.
Graphical Abstract
Background and Objectives: Positive airway pressure (PAP) is the treatment of choice for patients with obstructive sleep apnea (OSA). Although PAP is effective in resolving upper airway collapse, a major challenge is maximizing compliance. The purpose of this study was to investigate discontinuing factors in OSA patients with PAP therapy for a long time under the National Health Insurance coverage. Materials and Methods: A total of 229 patients, who were prescribed PAP between July 2018 and July 2020 were recruited and reviewed retrospectively. They were allocated to continue or discontinue group according to duration of PAP usage after 90 days compliance assessment. The discontinuing factors of PAP therapy between two groups were compared. Results: Among 229 patients, 49 failed to PAP therapy during adaptation period. One hundred and eighty patients constituted the final study population and the total good compliance rate at 90 days was 78.6%. The Kaplan-Meier curves showed PAP compliance continues to decrease by 13 months and remains thereafter. PAP-related problem was the major discontinuing factor in less than 13 months and other medical diseases in more than 13 months. Even in the group of continuous use of PAP, patients with low compliance often complained of PAP-related problem. Conclusion: Patients discontinuing PAP therapy within 13 months often have PAP-related problems. Therefore, early identification and rapid troubleshooting of difficulties with PAP use after adaptation period may underlie the higher long-term compliance.
Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis (AK), a vision-threatening parasitic disease whose primary risk factor has been attributed to poor contact lens hygiene. Unfortunately, differential diagnosis of AK is challenging as the clinical manifestations for AK are similar to those of bacterial, fungal, or even viral keratitis. Since delayed AK diagnosis can incur permanent vision impairment, a rapid and sensitive diagnostic method is urgently needed. Here, the diagnostic potential of polyclonal antibodies targeting the chorismate mutase (CM) of Acanthamoeba spp. was evaluated in AK animal models. CM antibody specificity against Acanthamoeba trophozoites and cysts was confirmed by immunocytochemistry after co-culturing Acanthamoeba with Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus, and human corneal epithelial (HCE) cells. Enzyme-linked immunosorbent assay (ELISA) was performed using CM-specific immune sera raised in rabbits, which demonstrated that the antibodies specifically interacted with the Acanthamoeba trophozoites and cysts in a dose-dependent manner. To evaluate the diagnostic potential of the CM antibody, AK animal models were established by incubating contact lenses with an inoculum containing A. castellanii trophozoites and subsequently overlaying these lenses onto the corneas of BALB/c mice for 7 and 21 days. The CM antibody specifically detected Acanthamoeba antigens in the murine lacrimal and eyeball tissue lysates at both time points. Our findings underscore the importance of antibody-based AK diagnosis, which could enable early and differential AK diagnosis in clinical settings.
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