Otitis Media Impacts Hundreds of Mouse Middle and Inner Ear Genes

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.     

A Mouse Model of Otitis Media Identifies HB-EGF as a Mediator of Inflammation-Induced Mucosal Proliferation

Objective

Otitis media is one of the most common pediatric infections. While it is usually treated without difficulty, up to 20% of children may progress to long-term complications that include hearing loss, impaired speech and language development, academic underachievement, and irreversible disease. Hyperplasia of middle ear mucosa contributes to the sequelae of acute otitis media and is of important clinical significance. Understanding the role of growth factors in the mediation of mucosal hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae.

Methods

From a whole genome gene array analysis of mRNA expression during acute otitis media, we identified growth factors with expression kinetics temporally related to hyperplasia. We then tested these factors for their ability to stimulate mucosal epithelial growth in vitro, and determined protein levels and histological distribution in vivo for active factors.

Results

From the gene array, we identified seven candidate growth factors with upregulation of mRNA expression kinetics related to mucosal hyperplasia. Of the seven, only HB-EGF (heparin-binding-epidermal growth factor) induced significant mucosal epithelial hyperplasia in vitro. Subsequent quantification of HB-EGF protein expression in vivo via Western blot analysis confirmed that the protein is highly expressed from 6 hours to 24 hours after bacterial inoculation, while immunohistochemistry revealed production by middle ear epithelial cells and infiltrating lymphocytes.

Conclusion

Our data suggest an active role for HB-EGF in the hyperplasia of the middle ear mucosal epithelium during otitis media. These results imply that therapies targeting HB-EGF could ameliorate mucosal growth during otitis media, and thereby reduce detrimental sequelae of this childhood disease.     

MRI Findings of Otic and Sinus Barotrauma in Patients with Carbon Monoxide Poisoning during Hyperbaric Oxygen Therapy

Background and Purpose

To study the MRI findings of otic and sinus barotrauma in patients with carbon monoxide(CO) poisoning during hyperbaric oxygen (HBO) therapy and examine the discrepancies of otic and sinus abnormalities on MRI between barotrauma and acute otitis media with effusion.

Materials and Methods

Eighty patients with CO-poisoning diagnosed with otic and sinus barotrauma after HBO therapy were recruited. Brain MRI was performed to predict delayed encephalopathy. Over the same period, 88 patients with acute otitis media with effusion on MRI served as control. The abnormalities of the middle ear and paranasal sinuses on MRI were noted and were compared between groups. Nine patients with barotrauma were followed up by MRI.

Results

In the barotrauma group, 92.5% of patients had bilateral middle ear abnormalities on MRI, and 60% of patients had both middle ear cavity and mastoid cavity abnormalities on MRI in both ears. Both rates were higher than those in the control group (p = 0.000). In the two groups, most abnormalities on MRI were observed in the mastoid cavity. The rate of sinus abnormalities of barotrauma was 66.3%, which was higher than the 50% in the control group (p = 0.033). In the nine patients with barotrauma followed up by MRI, the otic barotrauma and sinus abnormalities had worsened in 2 patients and 5 patients, respectively.

Conclusion

MRI is able to depict the abnormalities of otic and sinus barotrauma in patients with CO-poisoning during HBO therapy and to differentiate these from acute otitis media with effusion.     

Genetic requirement for pneumococcal ear infection

Background

Ear infection or otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the major OM pathogens. However, little is known about the genetic basis of bacterial OM largely due to practical difficulties in conducting research in ear infection models and genetically manipulating clinical isolates. Here, we report the first genome-scale in vivo screen for bacterial genes required for ear infection in a chinchilla model by signature tagged mutagenesis (STM), a high throughput mutant screen technique.

Methodology/Principal Findings

STM strains were constructed with a multi-drug resistant OM isolate ST556 (serotype 19F) and screened in a chinchilla OM model. Out of 5,280 mutants tested, 248 mutants were substantially underrepresented in the mutant pools recovered from the middle ear fluids of the infected chinchillas, indicating the impaired ability to survive and replicate in the middle ears due to genetic disruptions in the chromosome of strain ST556. Further DNA sequencing analysis mapped the mutations to 169 pneumococcal genes. Surprisingly, only 52 of these genes were required for pneumococcal nasopharyngeal colonization in a murine model. This infection site-specific gene requirement was verified by targeted mutagenesis in the selected genes.

Conclusions/Significance

These findings suggest that there are a subset of pneumococcal genes required for ear infection and that these may be distinct from those required for nasal colonization. Our data thus provide comprehensive gene targets for mechanistic understanding of pneumococcal ear infection. Finally, this study has also developed a model for future genome-scale search for virulence determinants in other pathogens associated with ear infections.     

Neutrophil Extracellular Traps and Bacterial Biofilms in Middle Ear Effusion of Children with Recurrent Acute Otitis Media – A Potential Treatment Target

Background

Bacteria persist within biofilms on the middle ear mucosa of children with recurrent and chronic otitis media however the mechanisms by which these develop remain to be elucidated. Biopsies can be difficult to obtain from children and their small size limits analysis.

Methods

In this study we aimed to investigate biofilm presence in middle ear effusion (MEE) from children with recurrent acute otitis media (rAOM) and to determine if these may represent infectious reservoirs similarly to those on the mucosa. We examined this through culture, viability staining and fluorescent in situ hybridisation (FISH) to determine bacterial species present. Most MEEs had live bacteria present using viability staining (32/36) and all effusions had bacteria present using the universal FISH probe (26/26). Of these, 70% contained 2 or more otopathogenic species. Extensive DNA stranding was also present. This DNA was largely host derived, representing neutrophil extracellular traps (NETs) within which live bacteria in biofilm formations were present. When treated with the recombinant human deoxyribonuclease 1, Dornase alfa, these strands were observed to fragment.

Conclusions

Bacterial biofilms, composed of multiple live otopathogenic species can be demonstrated in the MEEs of children with rAOM and that these contain extensive DNA stranding from NETs. The NETs contribute to the viscosity of the effusion, potentially contributing to its failure to clear as well as biofilm development. Our data indicates that Dornase alfa can fragment these strands and may play a role in future chronic OM treatment.     

Parent-Reported Otorrhea in Children with Tympanostomy Tubes: Incidence and Predictors

Purpose

Although common in children with tympanostomy tubes, the current incidence of tympanostomy tube otorrhea (TTO) is uncertain. TTO is generally a sign of otitis media, when middle ear fluid drains through the tube. Predictors for otitis media are therefore suggested to have predictive value for the occurrence of TTO.

Objective

To determine the incidence of TTO and its predictors.

Methods

We performed a cohort study, using a parental web-based questionnaire to retrospectively collect data on TTO episodes and its potential predictors from children younger than 10 years of age with tympanostomy tubes.

Results

Of the 1,184 children included in analyses (total duration of time since tube placement was 768 person years with a mean of 7.8 months per child), 616 children (52%) experienced one or more episodes of TTO. 137 children (12%) had TTO within the calendar month of tube placement. 597 (50%) children had one or more acute TTO episodes (duration <4 weeks) and 46 children (4%) one or more chronic TTO episodes (duration ≥4 weeks). 146 children (12%) experienced recurrent TTO episodes. Accounting for time since tube placement, 67% of children developed one or more TTO episodes in the year following tube placement. Young age, recurrent acute otitis media being the indication for tube placement, a recent history of recurrent upper respiratory tract infections and the presence of older siblings were independently associated with the future occurrence of TTO, and can therefore be seen as predictors for TTO.

Conclusions

Our survey confirms that otorrhea is a common sequela in children with tympanostomy tubes, which occurrence can be predicted by age, medical history and presence of older siblings.     

IgG Responses to Pneumococcal and Haemophilus Influenzae Protein Antigens Are Not Impaired in Children with a History of Recurrent Acute Otitis Media

Background

Vaccines including conserved antigens from Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) have the potential to reduce the burden of acute otitis media. Little is known about the antibody response to such antigens in young children with recurrent acute otitis media, however, it has been suggested antibody production may be impaired in these children.

Methods

We measured serum IgG levels against 4 pneumococcal (PspA1, PspA 2, CbpA and Ply) and 3 NTHi (P4, P6 and PD) proteins in a cross-sectional study of 172 children under 3 years of age with a history of recurrent acute otitis media (median 7 episodes, requiring ventilation tube insertion) and 63 healthy age-matched controls, using a newly developed multiplex bead assay.

Results

Children with a history of recurrent acute otitis media had significantly higher geometric mean serum IgG levels against NTHi proteins P4, P6 and PD compared with healthy controls, whereas there was no difference in antibody levels against pneumococcal protein antigens. In both children with and without a history of acute otitis media, antibody levels increased with age and were significantly higher in children colonised with S. pneumoniae or NTHi compared with children that were not colonised.

Conclusions

Proteins from S. pneumoniae and NTHi induce serum IgG in children with a history of acute otitis media. The mechanisms in which proteins induce immunity and potential protection requires further investigation but the dogma of impaired antibody responses in children with recurrent acute otitis media should be reconsidered.     

Gene Expression Analysis of Peripheral Cells for Subclassification of Pediatric Inflammatory Bowel Disease in Remission

Objective

In current clinical practice, optimal treatment of inflammatory bowel disease (IBD) aims at the induction and maintenance of clinical remission. Clinical remission is apparent when laboratory markers of inflammation are normal and clinical symptoms are absent. However, sub-clinical inflammation can still be present. A detailed analysis of the immune status during this inactive state of disease may provide a useful tool to categorize patients with clinical remission into subsets with variable states of immune activation.

Design

By using Affymetrix GeneChips, we analysed RNA gene expression profiles of peripheral blood leukocytes from pediatric IBD patients in clinical remission and controls. We performed (un)supervised clustering analysis of IBD-associated genes and applied Ingenuity® pathway software to identify specific molecular profiles between patients.

Results

Pediatric IBD patients with disease in clinical remission display heterogeneously distributed gene expression profiles that are significantly distinct from controls. We identified three clusters of IBD patients, each displaying specific expression profiles of IBD-associated genes.

Conclusion

The expression of immune- and IBD-associated genes in peripheral blood leukocytes from pediatric IBD patients in clinical remission was different from healthy controls, indicating that sub-clinical immune mechanisms are still active during remission. As such, RNA profiling of peripheral blood may allow for non-invasive patient subclassification and new perspectives in treatment regimes of IBD patients in the future.     

Mutational Analysis of EYA1, SIX1 and SIX5 Genes and Strategies for Management of Hearing Loss in Patients with BOR/BO Syndrome

Background

Branchio-oto-renal (BOR) or branchio-otic (BO) syndrome is one of the most common forms of autosomal dominant syndromic hearing loss. Mutations in EYA1, SIX1 and SIX5 genes have been associated with BOR syndrome. In this study, clinical and genetic analyses were performed in patients with BOR/BO syndrome focusing on auditory manifestations and rehabilitation.

Methods

The audiologic manifestations were reviewed in 10 patients with BOR/BO syndrome. The operative findings and hearing outcome were analyzed in patients who underwent middle ear surgeries. The modality and outcome of auditory rehabilitation were evaluated. Genetic analysis was performed for EYA1, SIX1, and SIX5 genes.

Results

All patients presented with mixed hearing loss. Five patients underwent middle ear surgeries without successful hearing gain. Cochlear implantation performed in two patients resulted in significant hearing improvement. Genetic analysis revealed four novel EYA1 mutations and a large deletion encompassing the EYA1 gene.

Conclusions

Auditory rehabilitation in BOR/BO syndrome should be individually tailored keeping in mind the high failure rate after middle ear surgeries. Successful outcome can be expected with cochlear implantations in patients with BOR/BO syndrome who cannot benefit from hearing aids. The novel EYA1 mutations may add to the genotypic and phenotypic spectrum of BOR syndrome in the East Asian population.     

Identification and distribution of the NBS-LRR gene family in the Cassava genome

Background

Plant resistance genes (R genes) exist in large families and usually contain both a nucleotide-binding site domain and a leucine-rich repeat domain, denoted NBS-LRR. The genome sequence of cassava (Manihot esculenta) is a valuable resource for analysing the genomic organization of resistance genes in this crop.

Results

With searches for Pfam domains and manual curation of the cassava gene annotations, we identified 228 NBS-LRR type genes and 99 partial NBS genes. These represent almost 1% of the total predicted genes and show high sequence similarity to proteins from other plant species. Furthermore, 34 contained an N-terminal toll/interleukin (TIR)-like domain, and 128 contained an N-terminal coiled-coil (CC) domain. 63% of the 327 R genes occurred in 39 clusters on the chromosomes. These clusters are mostly homogeneous, containing NBS-LRRs derived from a recent common ancestor.

Conclusions

This study provides insight into the evolution of NBS-LRR genes in the cassava genome; the phylogenetic and mapping information may aid efforts to further characterize the function of these predicted R genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1554-9) contains supplementary material, which is available to authorized users.     

Duplicated paralogous genes subject to positive selection in the genome of Trypanosoma brucei

Background

Whole genome studies have highlighted duplicated genes as important substrates for adaptive evolution. We have investigated adaptive evolution in this class of genes in the human parasite Trypanosoma brucei, as indicated by the ratio of non-synonymous (amino-acid changing) to synonymous (amino acid retaining) nucleotide substitution rates.

Methodology/Principal Findings

We have identified duplicated genes that are most rapidly evolving in this important human parasite. This is the first attempt to investigate adaptive evolution in this species at the codon level. We identify 109 genes within 23 clusters of paralogous gene expansions to be subject to positive selection.

Conclusions/Significance

Genes identified include surface antigens in both the mammalian and insect host life cycle stage suggesting that competitive interaction is not solely with the adaptive immune system of the mammalian host. Also surface transporters related to drug resistance and genes related to developmental progression are detected. We discuss how adaptive evolution of these genes may highlight lineage specific processes essential for parasite survival. We also discuss the implications of adaptive evolution of these targets for parasite biology and control.     

Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach

Background

The master transactivator CIITA is essential to the regulation of Major Histocompatibility Complex (MHC) class II genes and an effective immune response. CIITA is known to modulate a small number of non-MHC genes involved in antigen presentation such as CD74 and B2M but its broader genome-wide function and relationship with underlying genetic diversity has not been resolved.

Results

We report the first genome-wide ChIP-seq map for CIITA and complement this by mapping inter-individual variation in CIITA expression as a quantitative trait. We analyse CIITA recruitment for pathophysiologically relevant primary human B cells and monocytes, resting and treated with interferon-gamma, in the context of the epigenomic regulatory landscape and DNA-binding proteins associated with the CIITA enhanceosome including RFX, CREB1/ATF1 and NFY. We confirm recruitment to proximal promoter sequences in MHC class II genes and more distally involving the canonical CIITA enhanceosome. Overall, we map 843 CIITA binding intervals involving 442 genes and find 95% of intervals are located outside the MHC and 60% not associated with RFX5 binding. Binding intervals are enriched for genes involved in immune function and infectious disease with novel loci including major histone gene clusters. We resolve differentially expressed genes associated in trans with a CIITA intronic sequence variant, integrate with CIITA recruitment and show how this is mediated by allele-specific recruitment of NF-kB.

Conclusions

Our results indicate a broader role for CIITA beyond the MHC involving immune-related genes. We provide new insights into allele-specific regulation of CIITA informative for understanding gene function and disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0494-z) contains supplementary material, which is available to authorized users.     

Network analysis identifies protein clusters of functional importance in juvenile idiopathic arthritis

Introduction

Our objective was to utilise network analysis to identify protein clusters of greatest potential functional relevance in the pathogenesis of oligoarticular and rheumatoid factor negative (RF-ve) polyarticular juvenile idiopathic arthritis (JIA).

Methods

JIA genetic association data were used to build an interactome network model in BioGRID 3.2.99. The top 10% of this protein:protein JIA Interactome was used to generate a minimal essential network (MEN). Reactome FI Cytoscape 2.83 Plugin and the Disease Association Protein-Protein Link Evaluator (Dapple) algorithm were used to assess the functionality of the biological pathways within the MEN and to statistically rank the proteins. JIA gene expression data were integrated with the MEN and clusters of functionally important proteins derived using MCODE.

Results

A JIA interactome of 2,479 proteins was built from 348 JIA associated genes. The MEN, representing the most functionally related components of the network, comprised of seven clusters, with distinct functional characteristics. Four gene expression datasets from peripheral blood mononuclear cells (PBMC), neutrophils and synovial fluid monocytes, were mapped onto the MEN and a list of genes enriched for functional significance identified. This analysis revealed the genes of greatest potential functional importance to be PTPN2 and STAT1 for oligoarticular JIA and KSR1 for RF-ve polyarticular JIA. Clusters of 23 and 14 related proteins were derived for oligoarticular and RF-ve polyarticular JIA respectively.

Conclusions

This first report of the application of network biology to JIA, integrating genetic association findings and gene expression data, has prioritised protein clusters for functional validation and identified new pathways for targeted pharmacological intervention.