Acute Otitis Media Model Research Articles

A Pseudo-Surfactant Chemical Permeation Enhancer to Treat Otitis Media via Sustained Transtympanic Delivery of Antibiotics.

Chemical permeation enhancers (CPEs) represent a prevalent and safe strategy to enable noninvasive drug delivery across skin-like biological barriers such as the tympanic membrane (TM). While most existing CPEs interact strongly with the lipid bilayers in the stratum corneum to create defects as diffusion paths, their interactions with the delivery system, such as polymers forming a hydrogel, can compromise gelation, formulation stability, and drug diffusion. To overcome thischallenge, differing interactions between CPEs and the hydrogel system are explored, especially those with sodium dodecyl sulfate (SDS), an ionic surfactant and a common CPE, and those with methyl laurate (ML), a nonionic counterpart with a similar length alkyl chain. Notably, the use of ML effectively decouples permeation enhancement from gelation, enabling sustained delivery across TMs to treat acute otitis media (AOM),which is not possible with the use of SDS. Ciprofloxacin and ML are shown to form a pseudo-surfactant that significantly boosts transtympanic permeation. The middle ear ciprofloxacin concentration is increased by 70-fold in vivo in a chinchilla AOM model, yielding superior efficacy and biocompatibility than the previous highest-performing formulation. Beyond improved efficacy and biocompatibility, this single-CPE formulation significantly accelerates its progression toward clinical deployment.

Pathogenesis of Streptococcus pneumoniae serotype 3 during natural colonization and infections among children and its IgG correlate of protection in a mouse model

Current licensed pneumococcal conjugate vaccines (PCVs) are effective against pneumococcal diseases caused by the serotypes contained in the PCvs However; several studies evaluating pneumococcal colonization and acute otitis-media (AOM) prevention in young children vaccinated with PCV13, observed less effectiveness against serotype-3. One possible reason for less effectiveness may be release of the capsular polysaccharide (CPS) of serotype-3 (CPS-3) as an immune evasion mechanism. Here we evaluated free CPS-3 levels released from 6 clinical isolates from young children compared to WU2 strain and to serotype-19A CPS (CPS-19A) released in vitro when interacting with nasopharyngeal, middle-ear and lung cell-lines. Clinical serotype-3 strains showed greater release of CPS than WU2 with the interaction to 2 cell-lines and all 6 clinical serotype-19A strains. We next evaluated CPS-3 vs CPS-19A levels in middle-ear fluid (MEF) and the nasopharynx (NP) of young children and found higher levels of CPS-3 compared to CPS-19A in MEF during AOM but not in NP secretions during colonization. With anti-CPS-3 IgG in MEF and NP secretions at time of health and onset of AOM, a significant negative correlation (r = -0.75, p < 0.05) between unbound anti-CPS-3 IgG levels and free- anti-CPS-3 in MEF were found, and a significant lower detection of unbound anti-CPS-3 IgG in NP at the time of health with serotype-3 SPN (p < 0.05) compared to irrelevant SPN serotypes were found. In a mouse model of AOM and pneumonia, we sought a correlate of protection against serotype-3 infection using human serum-derived anti-CPS-3 IgG. We conclude that serotype-3 clinical isolates from children release more capsule than WU2 strains or 19A strains during in vitro testing; release more capsule in the MEF of children during AOM than serotype 19A; unbound anti-CPS-3 IgG levels negatively correlate with free-anti-CPS-3; and a level of 2.8 µg/ml anti-CPS-3 antibody protects mice from AOM and pneumonia but not colonization.

Effect of Angiogenesis and Lymphangiogenesis in Diesel Exhaust Particles Inhalation in Mouse Model of LPS Induced Acute Otitis Media.

Lymphangiogenesis and angiogenesis might have significant involvement in the pathogenesis of otitis media with effusion. This study investigated the effect of diesel exhaust particles (DEP) on inflammation and lymphangiogenesis in a mouse model of acute otitis media (AOM). BALB/c mice were injected with LPS and exposed to 100 µg/m3 DEP. The mice were divided into four groups: control (no stimulation), AOM, AOM + DEP, and DEP + AOM.The effects of DEP inhalation pre- and post-DEP induction were estimated based on measurements of the auditory brainstem response, mRNA levels of lymphangiogenesis-related genes and cytokines, and histology of the middle ear. Cell viability of human middle ear epithelial cells decreased in a dose-response manner at 24 and 48 hours post-DEP exposure. DEP alone did not induce AOM. AOM-induced mice with pre- or post-DEP exposure showed thickened middle ear mucosa and increased expression of TNF-α and IL1-β mRNA levels compared to the control group, but increased serum IL-1β levels were not found in the AOM + Post DEP. The mRNA expression of TLR4, VEGFA, VEGFAC, and VEGFR3 was increased by pre-AOM DEP exposure.The expression of VEFGA protein was stronger in the AOM + Post DEP group than in any other group. The expression of CD31 and CD45 markers in the mouse middle ear tissue was higher in the Pre DEP + AOM group than in the AOM group. This result implies that pre-exposure to DEP more strongly increases inflammation and lymphangiogenesis in a mouse model of acute otitis media.

Single-Cell Transcriptome Profiling Identifies Phagocytosis-Related Dual-Feature Cells in A Model of Acute Otitis Media in Rats.

BackgroundThe molecular mechanisms of acute otitis media (AOM) development, and the intercellular crosstalk within the multicellular ecosystem of AOM, are not clear.MethodsWe established a model of AOM in rats (with normal rats as controls) and undertook single-cell RNA sequencing (scRNA-seq) for the middle-ear mucosa (MEM). Cell clustering and trajectory analyses were undertaken using Seurat and Monocle 2 packages in R software. Pathway analyses were done by gene set enrichment analysis (GSEA). Cell–cell interactions were inferred by CellChat. Cell scores were calculated to identify cells with dual-feature.ResultsA total of 7023 cells from three samples of inflamed MEM and 5258 cells from three samples of healthy MEM underwent scRNA-seq, which identified 20 cell clusters belonging to eight major cell types. After exposure to lipopolysaccharide, the MEM underwent significant conversion of cell types characterized by rapid infiltration of macrophages and neutrophils. M2 macrophages seemed to play a key part in inflammatory intercellular crosstalk, which facilitated the maintenance and proliferation of macrophages, cell chemotaxis, and regulation of the proinflammatory activities of cytokines. Three rare cell clusters with phagocytosis-related dual-feature were also identified. They coexisted with professional phagocytes in the MEM, and displayed distinct immunoregulatory functions by maintaining a normal immune microenvironment or influencing inflammation progression.ConclusionsMacrophages might be the “master” initiators and regulators of the inflammatory response of the MEM to external stimuli. And their functions are fulfilled by a specific polarization status (M2) and sophisticated intercellular crosstalk via certain signaling pathways. Besides, the coexistence of professional phagocytes and non-professional phagocytes as well as their interplay in the MEM provides new clues for deciphering the underlying pathogenic mechanisms of AOM.

From Evidence to Clinical Guidelines in Antibiotic Treatment in Acute Otitis Media in Children.

Acute otitis media (AOM) in children represents a public health concern, being one of the leading causes of health care visits and antibiotic prescriptions worldwide. The overall aim of this paper is to unravel the major current insights into the antibiotic treatment of AOM in children. Our approach is three-fold: 1. a preclinical evaluation of antibiotics in animal models of AOM stressing on the advantages of different species when testing for different schemes of antibiotics; 2. an overview on the new antimicrobial agents whose efficacy has been demonstrated in refractory cases of AOM in children; and 3. an analysis of the different guidelines stressing on the differences and similarities between the various schemes of antibiotic treatment. The preferred therapeutic agents remain amoxicillin and the amoxicillin-clavulanate combination for AOM caused by Streptococcus pneumoniae, whereas oral cephalosporin is preferred in AOM due to Moraxella catarrhalis and Haemophilus influenzae. As for the second and third line antimicrobial treatments, there is a wide variety of suggested antibiotic classes with variations in duration and posology. The decision to prescribe antimicrobial treatment as a first-line choice is based on the severity of the symptoms in 16 of the guidelines included in this review.

Macrophage Depletion in CCR2-/- Mice Delays Bacterial Clearance and Enhances Neutrophil Infiltration in an Acute Otitis Media Model.

Otitis media (OM) is a common and potentially serious disease of childhood. Although OM is multifactorial on origin, bacterial infection is a unifying component. Many studies have established a critical role for innate immunity in bacterial clearance and OM resolution. A key component of innate immunity is the recruitment of immune and inflammatory cells, including macrophages. To explore the role of macrophages in OM, we evaluated the expression of genes related to macrophage function during a complete episode of acute OM in the mouse caused by middle ear (ME) inoculation with Haemophilus influenzae. We also combined CCR2 deficiency with chlodronate liposome toxicity to deplete macrophages during OM. Macrophage genes were robustly regulated during OM. Moreover, macrophage depletion enhanced and prolonged the infiltration of neutrophils into the infected ME and increased the persistence of bacterial infection. The results illustrate the critical role played by macrophages in OM resolution.

TLR4 regulates ROS and autophagy to control neutrophil extracellular traps formation against Streptococcus pneumoniae in acute otitis media.

Otitis media (OM), a prevalent pediatric infectious disease, is mainly caused by Streptococcus pneumoniae (S.pn). Neutrophil extracellular traps (NETs), a novel antimicrobial strategy, were reported in 2004. We found that NETs formed in the middle ear with acute otitis media (AOM) induced by S.pn. However, the mechanisms of NETs formation are not entirely clear. We stimulated neutrophils isolated from mouse bone marrow with S.pn clinical stain 19F in vitro, and established mouse model of AOM via transbullar injection with S.pn. NETs formation, reactive oxygen species (ROS) production, autophagy activation and bacterial load were analyzed in TLR4-/- and wild-type neutrophils stimulated in vitro with S.pn and in vivo during AOM. We found that autophagy and ROS were required for S.pn-induced NETs formation. Moreover, TLR4 partly mediated NETs formation in response to S.pn in vitro and in vivo during AOM. We also showed that attenuated NETs formation in TLR4-/- neutrophils correlated with an impaired ROS production and autophagy activation in vitro and in vivo. In addition, both the in vivo and in vitro-produced NETs were able to engulf and kill S.pn. TLR4 regulates ROS and autophagy to control NETs formation against S.pn in the course of AOM. S.pn can induce NETs formation in vitro and in vivo; TLR4 regulates NETs formation by ROS and autophagy; NETs contribute to the clearance of bacteria in acute otitis media. In this study, we firstly found that autophagy and ROS were required for S.pn-induced NETs formation in the model of acute otitis media (AOM). And to some extent, TLR4 mediated NETs formation during AOM. Our research might provide a potential strategy for the treatment of otitis media.

What is the effectiveness of beta-glucan for treatment of acute otitis media?

IntroductionAs a supplement, beta–glucan has various therapeutic healing effects generated by the immune cells. It has been scientifically approved and proven to be a biological defense modifier. The aim of this study was to investigate the effects of beta–glucan on treatments administered in an acute otitis media model ObjectivesThis study investigated the effect of beta–glucan on the treatment of acute otitis media in an acute otitis media -induced animal model. Efficacy was evaluated both immunologically and histologically. MethodsThe study sample comprised 35 adult rats, randomly separated into 5 groups of 7: Group 1 (control), Group 2 (acute otitis media, no treatment), Group 3 (acute otitis media+antibiotic), Group 4 (acute otitis media+beta–glucan) and Group 5 (acute otitis media+beta–glucan+antibiotic). Analyses were made of the histopathology and immunology examination results in respect of thickening of the tympanic membrane, epithelium damage, inflammation, and sclerosis. In all groups the serum levels of TNF-α, IL-4, IL-6 and IL-1β were evaluated. ResultsAll serum cytokine levels were significantly lower in the beta–glucan and antibiotic-treated groups compared to the acute otitis media Group. Significant differences in tympanic membrane thickness, inflammation, epithelium damage, and sclerosis values were observed between the acute otitis media+antibiotic and acute otitis media+beta–glucan Groups. According to these parameters, the values in aute otitis media+antibiotic+beta–glucan Group were markedly lower than those of the other groups. There was a significant difference in the acute otitis media+antibiotic+beta–glucan Groups compared to acute otitis media Group (p < 0.001). ConclusionsBoth antibiotic and beta–glucan treatment reduced acute otitis media signs of inflammations in an acute otitis media-induced rat model, decreasing histological damage and cytokine levels. Co-administration of antibiotic and beta–glucan led to a significant reduction in tympanic membrane thickness, inflammation, and epithelium damage. Antibiotic+beta–glucan treatment resulted in a greater decrease in tympanic membrane thickness, inflammation, and epithelium damage than in the other groups. From these results, it can be suggested that beta–glucan, in combination with antibiotics may provide an alternative for the treatment of acute otitis media.

Innate Anti-microbial and Anti-chemotaxis Properties of Progranulin in an Acute Otitis Media Mouse Model.

Acute otitis media (AOM) is one of the most common infectious diseases primarily caused by Streptococcus pneumoniae (S.pn) among children. Progranulin (PGRN) is a multifunctional growth factor widely expressed in various tissues and cells. Studies have confirmed that PGRN is involved in the development of a variety of inflammatory diseases. We found that the expression of PGRN increased significantly in the middle ear of wild mice with AOM. However, its physiological functions in AOM still remain unknown. To examine the role of PGRN during AOM, we established an acute otitis media model in both C57BL/6 wild mice and PGRN-deficient (PGRN−/−) mice via transbullar injection with S.pn clinical strain serotype 19F. Interestingly, we observed dual results: on one hand, macrophage recruitment notably increased in PGRN−/− mice compared with WT mice; on the other hand, the overall bacterial clearance was surprisingly dampened in PGRN−/− mice. The enhanced recruitment of macrophages was associated with increased production of chemokine (C-C motif) ligand 2 (CCL2), while the decreased bacterial clearance was associated with impaired endocytosis capacity of macrophages. The scavenging ability of bacteria in PGRN−/− mice was recovered with administration of recombinant PGRN. These results suggested a novel dual role of PGRN in affecting the activities of macrophages.

Apigetrin treatment attenuates LPS-induced acute otitis media though suppressing inflammation and oxidative stress

The natural course of otitis media in children is acute and self-limiting. Nevertheless, about 10–20% children could experience recurrent or persistent otitis media. Thus, finding effective candidate to prevent acute otitis media is urgently required. In our study, mouse acute otitis media model was constructed by lipopolysaccharide (LPS) injection into the middle ear of mice via the tympanic membrane. Apigetrin (APT) is a flavonoid isolated from various herbal medicines, possessing anti-inflammatory and anti-oxidative bioactivities. However, if APT could attenuate acute otitis media in LPS-induced animal models, little is to be known. Hematoxylin and eosin (H&E) staining suggested that APT treatment reduced LPS-induced higher mucosa thickness. LPS-triggered inflammatory response was also inhibited by APT, as evidenced by the down-regulated neutrophils and macrophages. Additionally, the reduced inflammatory factors, including interleukin-1β (IL-lβ), tumor necrosis factor α (TNF-α), IL-6 and vascular endothelial growth factor (VEGF) were observed in APT-treated mice with acute otitis media. The process was associated with the inhibition of toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) pathway, which was proved by the blockage of TLR4, MyD88, p-IKKα, p-IκBα, and p-NF-κB using western blot analysis. Moreover, the production of reactive oxygen species (ROS) caused by LPS was also reduced by APT through promoting anti-oxidants, involving superoxide dismutase (SOD) activity, heme oxygenase-1 (HO-1), NADP(H) quinone oxidoreductase 1 (NQO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) expressions. In contrast, high levels of MDA and kelch-like ECH-associated protein 1 (Keap 1) in LPS-treated mice were down-regulated by APT, which might be associated with the inactivation of NF-κB. In vitro, APT exhibited anti-inflammatory and anti-oxidant effects with little cytotoxicity in LPS-stimulated cells. Together, the data above indicated that APT could ameliorate acute otitis media through inhibiting inflammation and oxidative stress.

Surface Motion of Tympanic Membrane in a Chinchilla Model of Acute Otitis Media.

The conductive hearing loss caused by acute otitis media (AOM) is commonly related to a reduction of the tympanic membrane (TM) mobility in response to sound stimuli. However, spatial alterations of the TM surface motion associated with AOM have rarely been addressed. In this study, the TM surface motion was determined using scanning laser Doppler vibrometry (SLDV) in a chinchilla model of AOM. The AOM was established by transbullar injection of nontypeable Haemophilus influenzae. The TM surface vibration was measured in control (uninfected) animals and two AOM groups of animals: 4days (4D) and 8days (8D) post inoculation. To quantify the effect of middle ear pressure in those infected ears, the SLDV measurement was first conducted in unopened AOM ears and then in middle ear pressure released ears. Results showed that middle ear infection generally reduced the TM displacement across the entire surface, but the reduction in the umbo displacement over the time course, from 4 to 8days post inoculation, was less than the reduction in the displacement at the center of each quadrant. The presence of middle ear fluid shifted the occurrence of traveling-wave-like motion on the TM surface to lower frequencies. The observation of the spatial variations of TM surface motion from this study will help refine our understanding of the middle ear sound transmission characteristics in relation to AOM.

Intranasal coinfection model allows for assessment of protein vaccines against nontypeable Haemophilus influenzae in mice.

Nontypeable Haemophilus influenzae (NTHi) is a commensal in the human nasopharynx and the cause of pneumonia, meningitis, sinusitis, acute exacerbations of chronic obstructive pulmonary disease and acute otitis media (AOM). AOM is the most common ailment for which antibiotics are prescribed in the United States. With the emergence of new strains of antibiotic-resistant bacteria, finding an effective and broad coverage vaccine to protect against AOM-causing pathogens has become a priority. Mouse models are a cost-effective and efficient way to help determine vaccine efficacy. Here, we describe an NTHi AOM model in C57BL/6J mice, which also utilizes a mouse-adapted H1N1 influenza virus to mimic human coinfection. We tested our coinfection model using a protein vaccine formulation containing protein D, a well-studied NTHi vaccine candidate that can be found in the 10-valent Streptococcus pneumoniae conjugate vaccine. We verified the usefulness of our mouse model by comparing bacterial loads in the nose and ear between protein D-vaccinated and control mice. While there was no measurable difference in nasal bacterial loads, we did detect significant differences in the bacterial loads of ear washes and ear bullae between vaccinated and control mice. The results from this study suggest that our NTHi AOM coinfection model is useful for assessing protein vaccines.

Animal models of acute otitis media – A review with practical implications for laboratory research

Considerable animal research has focused on developing new strategies for the prevention and treatment of acute otitis media (AOM). Several experimental models of AOM have thus been developed. A PubMed search of the English literature was conducted from 1975 to July 2016 using the search terms “animal model” and “otitis media” from which 91 published studies were included for analysis, yielding 123 animal models. The rat, mouse and chinchilla are the preferred animals for experimental AOM models with their individual advantages and disadvantages. The most common pathogens used to create AOM are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Streptococcus pneumoniae (types 3, 23 and 6A) and non-typeable Haemophilus influenzae (NTHi) are best options for inoculation into rat and mouse models. Adding viral pathogens such as RSV and Influenza A virus, along with creating ET dysfunction, are useful adjuncts in animal models of AOM. Antibiotic prophylaxis may interfere with the inflammatory response without a significant reduction in animal mortality.

Factors affecting sound energy absorbance in acute otitis media model of chinchilla

Acute otitis media (AOM) is a rapid-onset infection of the middle ear which results in middle ear pressure (MEP), middle ear effusion (MEE), and structural changes in middle ear tissues. Previous studies from our laboratory have identified that MEP, MEE, and middle ear structural changes are three factors affecting tympanic membrane (TM) mobility and hearing levels (Guan et al., 2014, 2013). Sound energy reflectance or absorbance (EA) is a diagnostic tool increasingly used in clinical settings for the identification of middle ear diseases. However, it is unclear whether EA can differentiate these three factors in an AOM ear. Here we report wideband EA measurements in the AOM model of chinchilla at three experimental stages: unopened, pressure released, and effusion removed. These correspond to the combined and individual effects of the three factors on sound energy transmission. AOM was produced by transbullar injection of Haemophilus influenzae in two treatment groups: 4 days (4D) and 8 days (8D) post inoculation. These time points represent the relatively early and later phase of AOM. In each group of chinchillas, EA at 250–8000 Hz was measured using a wideband tympanometer at three experimental stages. Results show that the effects of MEP, MEE, and tissue structural changes over the frequency range varied with the disease time course. MEP was the primary contributor to reduction of EA in 4D AOM ears and had a smaller effect in 8D ears. MEE reduced the EA at 6–8 kHz in 4D ears and 2–8 kHz in 8D ears and was responsible for the EA peak in both 4D and 8D ears. The residual EA loss due to structural changes was observed over the frequency range in 8D ears and only at high frequencies in 4D ears. The EA measurements were also compared with the published TM mobility loss in chinchilla AOM ears.

The critical role of myeloperoxidase in Streptococcus pneumoniae clearance and tissue damage during mouse acute otitis media.

We have recently reported that neutrophils play a pivotal role in innate defense against Streptococcus pneumoniae ( Spn) during mouse acute otitis media (AOM). However, the underlying mechanism remains unclear. By constructing models of pneumococcal AOM in C57BL/6 mice and using a specific inhibitor invivo, we investigated the role of myeloperoxidase (MPO), one of the most important protein components of neutrophils. Experiment results showed a significant increase in MPO production of the recruited neutrophils in Spn-infected mice. Neutrophils killed Spn in a MPO-dependent manner. MPO facilitated the generation of reactive oxygen species (ROS), and consequently promoted Spn clearance at an early stage and exacerbated tissue damage. Moreover, MPO induced neutrophil apoptosis and necrosis, which, in turn, worsened tissue damage. In summary, our study demonstrates that neutrophil MPO plays a paradoxical role in bacterial clearance and tissue damage in pneumococcal AOM.

Dynamic property changes in stapedial annular ligament associated with acute otitis media in the chinchilla

Located at the end of the ossicular chain, the stapedial annular ligament (SAL) serves as a closed yet mobile boundary between the cochlear fluid and stapes footplate. It is unclear how SAL properties change with acute otitis media (AOM). This paper reports the measurements of SAL dynamic properties in chinchilla AOM model using dynamic mechanical analyzer (DMA) and frequency-temperature superposition (FTS) principle. AOM was analyzed in two infection groups: 4 days (4D) and 8 days (8D) post induction. SAL specimens were measured using DMA at three temperatures: 5, 25, and 37°C. To extend the testing frequencies to higher levels, FTS principle was employed. Then generalized Maxwell model was utilized to define the constitutive equations of the SAL. The complex shear moduli were obtained from seven samples of control, 4D, and 8D groups. Results show that the storage and loss shear moduli of SALs decreased due to AOM. The storage moduli for 4D and 8D ears were similar below 100Hz, and the loss modulus for 4D was significantly larger than 8D across the entire frequency range. This study reports data that contributes to ear biomechanics and improves understanding on the effects of AOM in middle ear tissues.

Morphological changes in the tympanic membrane associated with Haemophilus influenzae-induced acute otitis media in the chinchilla

ObjectiveThe tympanic membrane (TM) couples sound waves entering the outer ear canal to mechanical vibrations of the ossicular chain in the middle ear. During acute otitis media (AOM), dynamic structural changes in the TM can occur, which potentially affect sound transmission. It has remained unclear whether TM changes contribute significantly to the conductive hearing loss associated with human AOM. Studies that systematically and quantitatively assess the impact of morphological and mechanical characteristics of the TM on hearing in animal models of AOM have been few in number and lack detail. Our current study focused on the identification of quantitative morphological changes in the TM of the adult chinchilla. MethodAOM was produced by transbullar injection of the nontypeable (acapsular) Haemophilus influenzae strain 86-028NP into two treatment groups of chinchillas: one 4 days (4D) post bacterial challenge, and a second treatment group after 8 days (8D) post challenge. Structure and thickness were examined histologically at nine locations over the TM in untreated controls and in animals from both AOM treatment groups. ResultsTM thickness was found to have increased significantly (110–150%) at all measured locations of H. influenzae-infected ears when compared with uninfected (normal) TMs at 4D post bacterial challenge. Cellular proliferation and infiltration in the outer epithelial layer were primary contributors to this thickening. In ears infected for 8D, the TM was substantially thicker, a 200–300% increase from uninfected control values, due to edema and cell proliferation in both the outer and inner epithelial layers. In both 4D and 8D ears, thickening of the TM was more prominent in the superior-anterior quadrant. ConclusionThis study provides unequivocal structural evidence that significant TM thickness increases are associated with AOM induced by a well characterized H. influenzae human clinical isolate of low passage number. These and additional thickness data from early and later stages in middle ear infection will be used to derive the mechanical properties of the TM in a future study from our laboratory.

Effect of pH and oxygen on biofilm formation in acute otitis media associated NTHi clinical isolates.

Biofilms occur in animal models of acute otitis media (AOM) and in children with recurrent AOM (rAOM) and chronic otitis media with effusion (OME). We therefore studied the ability of nontypeable Haemophilus influenzae (NTHi) strains from children to form biofilms in vitro under conditions we presumed occurred in the middle ear during AOM, rAOM, and OME. Evaluate NTHi isolates for biofilm formation across a pH range under aerobic, microaerophilic, and anaerobic conditions. Using a crystal violet biofilm assay we studied 12 NTHi pediatric clinical isolates to investigate biofilm formation over a pH range of 4.5 to 10 under aerobic, microaerophilic, and anaerobic conditions. Our findings included: 1) not all clinical NTHi strains form biofilms (75% did); 2) the pH of middle ear fluid collected from AOM (n = 170; age range, 4-36 months), rAOM (n = 54; age range, 7-36 months), and OME (n = 30; age range, 9-60 months) subjects tested immediately after withdrawal was similar (mean = 8.0;range 7.0-9.0); 3) biofilms formed optimally at pH 8.0, a finding that is consistent with previous studies by other investigators; 4) biofilms did not form under aerobic conditions as likely occurs in AOM, whereas under microaerophilic and anaerobic conditions biofilm formation was observed as likely occurs during rAOM and OME. We concluded that biofilm formation by NTHi does not occur in all strains, occurs best where the pH = 8.0 and in anaerobic conditions as likely occurs in children during rAOM and OME. However, biofilm formation is limited or absent under aerobic conditions as likely occurs during AOM. NA.

Blocking macrophage migration inhibitory factor activity alleviates mouse acute otitis media in vivo

This study was to investigate the role of macrophage migration inhibitory factor (MIF) in mouse acute otitis media (AOM), we hypothesize that blocking MIF activity will relieve mouse AOM. A mouse AOM model was constructed by injecting lipopolysaccharide (LPS) into the middle ear of C57BL/6 mice through the tympanic membrane (TM). MIF levels were measured by real-time PCR (RT-PCR) and ELISA after LPS application. Normal or AOM mice were given PBS or ISO-1 (MIF antagonist) every day for 10 days and the hearing levels were determined by measuring auditory brainstem response (ABR) threshold. After the ABR test finished, H&E staining was conducted and the inflammation was also measured by detecting interleukin (IL)-1β, tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) levels with RT-PCR and ELISA. TLR-4 expression was determined by western blotting and NF-κB activation was determined by electrophoretic mobility shift assays. Compared with the normal control, MIF levels in the middle ear of LPS-induced AOM mice were significant increased. The ABR results showed that mean ABR thresholds in ISO-1 treated AOM mice were significantly reduced compared with PBS treated AOM mice since day 7, indicating that ISO-1 treatment potentially improved the hearing levels of AOM mice. H&E staining showed that ISO-1 treatment could reduce the mucosal thickness of AOM mice. In ISO-1 treated mice, TLR-4 expression and levels of IL-1β, TNF-α and VEGF were significantly lower compared with PBS treated AOM mice. ISO-1 treatment also significantly inhibited NF-κB activation in AOM mice compared with PBS treated AOM mice. These results suggested that blocking the activity of MIF by ISO-1 could reduce the inflammation in AOM mice in which process TLR-4 and NF-κB were involved. The reduction in MIF activity is conducive to alleviate mouse AOM, which may serve as a potential therapeutic target for the treatment of AOM.

Nonencapsulated Streptococcus pneumoniae Cause Acute Otitis Media in the Chinchilla That Is Enhanced by Pneumococcal Surface Protein K.

Use of the pneumococcal conjugate vaccine has led to serotype replacement of carriage and acute otitis media (AOM) pneumococcal isolates. Increases in nonencapsulated Streptococcus pneumoniae (NESp) isolates have also occurred, and there are increasing reports of NESp-associated disease. Disease prevalence and virulence factors of NESp isolates have not been studied. A chinchilla model of pneumococcal AOM was utilized, and disease was assessed through bacterial enumeration along with scoring visible signs of pathology. An adhesion-invasion assay using a human epithelial cell line was performed. Nonencapsulated Streptococcus pneumoniae strains containing pneumococcal surface protein K (PspK) were more likely to cause AOM and pathology upon infection. Deletion of PspK from an isolate significantly reduced bacterial loads. Increased epithelial cell adhesion correlated with increased virulence of NESp isolates naturally lacking PspK. Furthermore, expression of PspK by an avirulent NESp resulted in virulence. The presence of PspK increased the disease potential of NESp. Pneumococcal surface protein K is not the only virulence factor of NESp in AOM. Expression of PspK in an avirulent NESp mediated the progression to pneumococcal disease. Genetic exchange between pneumococci may allow dissemination of PspK, increasing the potential of NESp disease. The current study is the first report of a NESp-specific virulence factor.