Pawjai Khampang

Adenoid Reservoir for Pathogenic Biofilm Bacteria

ABSTRACT Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

MUC5AC Expression in Human Middle Ear Epithelium of Patients With Otitis Media

OBJECTIVE To compare levels of middle ear (ME) MUC5AC expression in patients with otitis media (OM) with patients without OM. Mucin gene 5AC has been identified as a major secretory mucin in the ME and is fundamentally important in the development of ME mucoid effusions, hearing loss and also provides ME mucosal protection and bacterial clearance. DESIGN Case control. SETTING Tertiary, academic, pediatric otolaryngology practice. PATIENTS Patients 9 months to 7 years old undergoing routine tympanostomy tube (TT) insertion for recurrent otitis media (RecOM) or chronic otitis media with effusion (COME) were compared with control patients without a history of OM undergoing cochlear implantation. METHODS During routine TT placement or cochlear implantation, a 1-mm biopsy sample of the ME epithelium was obtained. RNA was extracted, and real-time reverse transcriptase-polymerase chain reaction was used to quantify levels of MUC5AC expression. RESULTS Twenty-three patients with OM (12 with RecOM and with 11 COME) were evaluated using 5 controls. Mean age was not different between groups. In the RecOM group, mean expression of MUC5AC was 25.92 times greater than in controls. In the COME group, the mean expression was 155.40 times greater than in controls. CONCLUSIONS Levels of MUC5AC expression in the human ME are significantly increased in patients with RecOM and COME compared with controls. This study demonstrates MUC5AC gene changes in patients with OM and highlights the need for greater understanding of the molecular responses in OM; particularly that of mucin. A thorough exploration of these factors will provide opportunities to develop novel interventions for the extremely common problem of OM.

Mucin gene polymorphisms in otitis media patients.

Mucin genes MUC2, MUC5AC, and MUC5B have been identified as major gel‐forming mucins in the middle ear (ME). This study compared polymorphisms in MUC2, MUC5AC, and MUC5B genes in otitis media (OM) patients and controls.

Mucin gene 19 (MUC19) expression and response to inflammatory cytokines in middle ear epithelium.

Mucin gene 19 (MUC19) has been identified as a major gel-forming mucin in the human middle ear (ME). The objectives of this investigation were to characterize the expression and assess the regulation of MUC19 in the ME cell culture models utilized in the study of otitis media (OM). Findings demonstrate that MUC19 is expressed in both human immortalized cell culture (HMEEC) and chinchilla primary epithelial culture (CMEEC). ME exposure to inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 up-regulate MUC19 transcription, most robustly after exposure to TNF-α. Kinetic experiments suggest a relative early response in MUC19 transcription and a down-regulation after prolonged exposure. Glycoprotein production was increased in response to the increased transcription as well. Similar to other mucin genes in the ME, MUC19 is differentially regulated after exposure to inflammatory cytokines. The large size, gel-forming properties and up-regulation in response to important inflammatory cytokines of MUC19 suggest that it has significant potential to play a role in both physiology and pathophysiology of the ME.

Gene Expression Differences in Infected and Noninfected Middle Ear Complementary DNA Libraries

OBJECTIVES To investigate genetic differences in middle ear mucosa (MEM) with nontypeable Haemophilus influenzae (NTHi) infection. Genetic upregulation and downregulation occurs in MEM during otitis media (OM) pathogenesis. A comprehensive assessment of these genetic differences using the techniques of complementary DNA (cDNA) library creation has not been performed. DESIGN The cDNA libraries were constructed from NTHi-infected and noninfected chinchilla MEM. Random clones were picked, sequenced bidirectionally, and submitted to the National Center for Biotechnology Information (NCBI) Expressed Sequence Tags database, where they were assigned accession numbers. These numbers were used with the basic local alignment search tool (BLAST) to align clones against the nonredundant nucleotide database at NCBI. RESULTS Analysis with the Web-based statistical program FatiGO identified several biological processes with significant differences in numbers of represented genes. Processes involved in immune, stress, and wound responses were more prevalent in the NTHi-infected library. S100 calcium-binding protein A9 (S100A9); secretory leukoprotease inhibitor (SLPI); beta(2)-microglobulin (B2M); ferritin, heavy-chain polypeptide 1 (FTH1); and S100 calcium-binding protein A8 (S100A8) were expressed at significantly higher levels in the NTHi-infected library. Calcium-binding proteins S100A9 and S100A8 serve as markers for inflammation and have antibacterial effects. Secretory leukoprotease inhibitor is an antibacterial protein that inhibits stimuli-induced MUC1, MUC2, and MUC5AC production. CONCLUSIONS A number of genes demonstrate changes during the pathogenesis of OM, including SLPI, which has an impact on mucin gene expression; this expression is known to be an important regulator in OM. The techniques described herein provide a framework for future investigations to more thoroughly understand molecular changes in the middle ear, which will likely be important in developing new therapeutic and intervention strategies.

Differential response of gel-forming mucins to pathogenic middle ear bacteria

OBJECTIVE To assess the differential response of the secretory gel forming mucins (GFM) to the most common bacterial pathogens causing otitis media, Streptococcus pneumoniae (SP), nontypeable Haemophilus influenza (NTHi), and Moraxella catarrhalis (Mcat), in a culture model of human middle ear epithelium (HMEEC). METHODS In vitro cultured HMEEC was exposed to 5 μg/ml of bacterial whole cell lysate (WCL). RNA was extracted to generate cDNA. The expression levels of each of the targeted mucin transcripts, MUC2, MUC5AC, MUC5B and MUC19, were detected by quantitative PCR. RESULTS The submerged HMEEC exposed to NTHi-86028NP WCL demonstrated a significant increase of MUC2, MUC5AC and MUC5B as compared to the control non-treated cells while MUC19 transcript level remained unchanged. WCL of additional major OM pathogens significantly increase the transcription of these three mucin genes as well. A combination of NTHi and SP further synergistically induced MUC2 and MUC5AC gene expression however, not all NTHi strains synergized with SP in the induction. Addition of Mcat WCL to the synergized combination of NTHi and SP did not participate in the synergistic response of mucins. CONCLUSION The specific pathogen combinations were important in determining the degree of synergistic effects to GFM expression. The current data are substantive in guiding future work to extend our understanding of OM pathogens and GFMs.

A novel model of spontaneous otitis media with effusion (OME) in the Oxgr1 knock-out mouse

OBJECTIVE A novel mouse model with a specific genetic mutation in a G protein coupled receptor (GPCR) encoded by the Oxgr1 gene results in a predisposition to spontaneous otitis media with effusion. As a primary component of interest in OME, mucin expression was examined in this model to assess expression as compared to wild type animals and suitability as a murine model of OME. METHOD Mutant (Oxgr1(-/-)) and wild-type (Oxgr1(+/+)) mice between ages of 2 and 5 months were examined by otoscopy and auditory brainstem response (ABR). Histology changes in the middle ear were evaluated. Expression of mucin genes in the middle ear epithelium was determined using RT-PCR and quantitative PCR. RESULT Otoscopic exam showed signs of inflammation in 82% of mutant mice. Significant elevated ABR thresholds were detected in mutant mice indicating hearing loss. Histology analysis of the middle ears demonstrated the presence of inflammatory cells, changes in the mucosal epithelium, and middle ear fluid. RT PCR using universal primers for bacterial 18s rRNA suggested the absence of bacteria in the middle ear. The knockout mice demonstrated expression of Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc9, Muc10, Muc13, Muc15, Muc16, Muc18, Muc19 and Muc20. There was a trend of increase in Muc5B and Muc19 expression in the middle ear of the knockout mice compared to that of wild-type. There was no significant change in the level of Muc2, and Muc5AC was expressed at a level below the detection limit of quantification. CONCLUSION Development of a murine model with genetic defect has several attractive features. The rate of OME in these animals is high at 82%. It is clear that this OME is related to histopathologic changes in the middle ear epithelium of these knock-out mice. Induction of mucus effusion is evident though the viation in dysregulation of GFM does exist in this non-challenge study condition. The underlying cause of these differences between individual animal requires further investigation. Given this, the Oxgr1(-/-) model is likely to be an ideal model to examine mucin regulation in MEE and potentially develop novel GPCR-specific targeted interventions to regulate these processes.

The Chinchilla Research Resource Database: resource for an otolaryngology disease model

The long-tailed chinchilla (Chinchilla lanigera) is an established animal model for diseases of the inner and middle ear, among others. In particular, chinchilla is commonly used to study diseases involving viral and bacterial pathogens and polymicrobial infections of the upper respiratory tract and the ear, such as otitis media. The value of the chinchilla as a model for human diseases prompted the sequencing of its genome in 2012 and the more recent development of the Chinchilla Research Resource Database (http://crrd.mcw.edu) to provide investigators with easy access to relevant datasets and software tools to enhance their research. The Chinchilla Research Resource Database contains a complete catalog of genes for chinchilla and, for comparative purposes, human. Chinchilla genes can be viewed in the context of their genomic scaffold positions using the JBrowse genome browser. In contrast to the corresponding records at NCBI, individual gene reports at CRRD include functional annotations for Disease, Gene Ontology (GO) Biological Process, GO Molecular Function, GO Cellular Component and Pathway assigned to chinchilla genes based on annotations from the corresponding human orthologs. Data can be retrieved via keyword and gene-specific searches. Lists of genes with similar functional attributes can be assembled by leveraging the hierarchical structure of the Disease, GO and Pathway vocabularies through the Ontology Search and Browser tool. Such lists can then be further analyzed for commonalities using the Gene Annotator (GA) Tool. All data in the Chinchilla Research Resource Database is freely accessible and downloadable via the CRRD FTP site or using the download functions available in the search and analysis tools. The Chinchilla Research Resource Database is a rich resource for researchers using, or considering the use of, chinchilla as a model for human disease. Database URL: http://crrd.mcw.edu

Mucin gene expression and mouse middle ear epithelium

OBJECTIVES To investigate the expression of recently identified human mucin genes in an in vitro model of cultured mouse middle ear epithelial cells (MMEEC). METHODS MMEEC were established, RNA was extracted and primers were designed for RT-PCR to assess for expression of mucin genes Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc6, Muc7, Muc8, Muc9, Muc10, Muc11/12, Muc13, Muc15, Muc16, Muc17, Muc18, Muc19 and Muc20 expression. RESULTS Mucin genes Muc1, Muc2, Muc3, Muc4, Muc5AC, Muc5B, Muc9, Muc10, Muc13, Muc15, Muc16, Muc18, Muc19 and Muc20 were identified and expressed in MMEEC. The genes Muc6, Muc7, Muc8, Muc11/12 and Muc17 were not identified. CONCLUSION Many of the mucin genes that have been recently identified in human MEE and chinchilla MEE are also expressed in MMEEC. There are differences in expression, however, which may have implications in utilizing various animal models for study of middle ear physiology and pathogenesis; specifically as it relates to mucin gene expression.

Expression of Mucin (MUC) Genes in Mucoepidermoid Carcinoma

Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland tumor. The presence of mucin (MUC) genes has been correlated with patient prognosis using immunohistochemical techniques. This study was undertaken to 1) investigate the expression of newly discovered MUC genes in MEC specimens, 2) assess the correlation of this expression to prognosis, and 3) further investigate the correlation of previously identified MUC genes to prognosis using molecular techniques.