Dataset Information

Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.

ABSTRACT:

Background

Otitis media (OM) is characterized by acute infection progressing to chronic middle ear effusion (MEE). Extracellular secretion of microRNAs (miRNAs) in exosomes is a newly discovered mechanism for cells exerting distant cell genetic regulation. Whether MEE contains exosomes with specific miRNAs is unknown. This study aimed to purify and characterize the exosomal and miRNA content of MEE.

Method

MEEs were subjected to Exoquick exosomal purification and EXOCET exosomal quantification. Extracted vesicles were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM), and immunoblotting of HSP-70. NanoString hybridization was performed to profile miRNAs. Exosomal protein content was profiled by Liquid chromatography tandem mass spectrometry (LC-MS/MS).

Results

EXOCET assays showed presence of exosomes (0-0.5 × 10⁷/ml) in MEEs. DLS confirmed exosomal size between 10 and 200 nm. Western blot analysis showed presence of HSP-70. Twenty-nine miRNAs were found to be unique to MEEs. The most abundant miRNA was miR-223, a miRNA typically secreted by neutrophils. Proteomics demonstrated typical neutrophil markers as well as common innate immune molecules.

Conclusion

To our knowledge, this the first report demonstrating the presence of exosomes transporting miRNAs in MEEs. These findings open a broad and novel area of research in OM pathophysiology as driven by miRNA cell communication.

SUBMITTER: Val S

PROVIDER: S-EPMC8900972 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Publications

Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.

Val Stéphanie S Jeong Stephanie S Poley Marian M Krueger Anna A Nino Gustavo G Brown Kristy K Preciado Diego D

Pediatric research 20170203 6

<h4>Background</h4>Otitis media (OM) is characterized by acute infection progressing to chronic middle ear effusion (MEE). Extracellular secretion of microRNAs (miRNAs) in exosomes is a newly discovered mechanism for cells exerting distant cell genetic regulation. Whether MEE contains exosomes with specific miRNAs is unknown. This study aimed to purify and characterize the exosomal and miRNA content of MEE.<h4>Method</h4>MEEs were subjected to Exoquick exosomal purification and EXOCET exosomal q ...[more]

PMID: 28157838

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Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. There are 8 control samples and 9 samples trans-tympanically injected with H flu 10e9 for 6 hours. Each sample is from a single animal.

Project description:BackgroundOtitis media (OM) is amongst the most common childhood diseases and is associated with multiple microbial pathogens within the middle ear. Global and temporal monitoring of predominant bacterial pathogens is important to inform new treatment strategies, vaccine development and to monitor the impact of vaccine implementation to improve progress toward global OM prevention.MethodsA systematic review of published reports of microbiology of acute otitis media (AOM) and otitis media with effusion (OME) from January, 1970 to August 2014, was performed using PubMed databases.ResultsThis review confirmed that Streptococcus pneumoniae and Haemophilus influenzae, remain the predominant bacterial pathogens, with S. pneumoniae the predominant bacterium in the majority reports from AOM patients. In contrast, H. influenzae was the predominant bacterium for patients experiencing chronic OME, recurrent AOM and AOM with treatment failure. This result was consistent, even where improved detection sensitivity from the use of polymerase chain reaction (PCR) rather than bacterial culture was conducted. On average, PCR analyses increased the frequency of detection of S. pneumoniae and H. influenzae 3.2 fold compared to culture, whilst Moraxella catarrhalis was 4.5 times more frequently identified by PCR. Molecular methods can also improve monitoring of regional changes in the serotypes and identification frequency of S. pneumoniae and H. influenzae over time or after vaccine implementation, such as after introduction of the 7-valent pneumococcal conjugate vaccine.ConclusionsGlobally, S. pneumoniae and H. influenzae remain the predominant otopathogens associated with OM as identified through bacterial culture; however, molecular methods continue to improve the frequency and accuracy of detection of individual serotypes. Ongoing monitoring with appropriate detection methods for OM pathogens can support development of improved vaccines to provide protection from the complex combination of otopathogens within the middle ear, ultimately aiming to reduce the risk of chronic and recurrent OM in vulnerable populations.

Dataset Information

Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.

Background

Method

Results

Conclusion

Publications

Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.

Similar Datasets

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