Animal Model Of Otitis Media Research Articles

Otitis media: recent advances in otitis media vaccine development and model systems.

Otitis media is an inflammatory disorder of the middle ear caused by airways-associated bacterial or viral infections. It is one of the most common childhood infections as globally more than 80% of children are diagnosed with acute otitis media by 3 years of age and it is a common reason for doctor's visits, antibiotics prescriptions, and surgery among children. Otitis media is a multifactorial disease with various genetic, immunologic, infectious, and environmental factors predisposing children to develop ear infections. Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most common culprits responsible for acute otitis media. Despite the massive global disease burden, the pathogenesis of otitis media is still unclear and requires extensive future research. Antibiotics are the preferred treatment to cure middle ear infections, however, the antimicrobial resistance rate of common middle ear pathogens has increased considerably over the years. At present, pneumococcal and influenza vaccines are administered as a preventive measure against otitis media, nevertheless, these vaccines are only beneficial in preventing carriage and/or disease caused by vaccine serotypes. Otitis media caused by non-vaccine serotype pneumococci, non-typeable H. influenza, and M. catarrhalis remain an important healthcare burden. The development of multi-species vaccines is an arduous process but is required to reduce the global burden of this disease. Many novel vaccines against S. pneumoniae, non-typeable H. influenza, and M. catarrhalis are in preclinical trials. It is anticipated that these vaccines will lower the disease burden and provide better protection against otitis media. To study disease pathology the rat, mouse, and chinchilla are commonly used to induce experimental acute otitis media to test new therapeutics, including antibiotics and vaccines. Each of these models has its advantages and disadvantages, yet there is still a need to develop an improved animal model providing a better correlated mechanistic understanding of human middle ear infections, thereby underpinning the development of more effective otitis media therapeutics. This review provides an updated summary of current vaccines against otitis media, various animal models of otitis media, their limitations, and some future insights in this field providing a springboard in the development of new animal models and novel vaccines for otitis media.

Resolution of otitis media in a humanized mouse model.

Otitis media (OM) is one of the largest public health problems of children and has devastating impacts in developing countries. The substantial medical and human costs involved have led to research to understand the disease and improve treatment. Animal models of OM have yielded critical information about the immune, inflammatory and genetic mechanisms of OM. However, it is important to link animal studies to human immune and inflammatory responses. In recent years, "humanized" mice have become a valuable tool to study the human immune system in an animal model. Here we describe the first use of humanized mice to study OM. We demonstrate that humanized mice with a sufficient degree of engraftment recapitulate a normal middle ear (ME) inflammatory response to bacterial infection, including the recruitment of human immune cells, and exhibit normal recovery. Moreover, these animals exhibit regulated expression of human-specific immune and inflammatory genes in the ME. In contrast, mice with insufficient engraftment fail to resolve OM. This model has many potential uses in OM research, including using hematopoietic stem cells from patients with differing degrees of OM susceptibility, to understand the role of human immune responses in proneness to this common childhood disease.

15. A Novel Approach to Bacterial Vaccines: Haemophilus influenzae as a Paradigm

BackgroundThe H. influenzae type b vaccines target the type b capsule and therefore have no impact on the nontypable (unencapsulated) H. influenzae (NTHi). NTHi has become the most common cause of otitis media and is the most common isolate from patients with exacerbations of Chronic Obstructive Pulmonary Disease (COPD). Therefore, NTHi is an appropriate target for vaccine development.MethodsTo characterize potential vaccine targets, the core outer proteins of NTHi present in the available sequenced genomes were identified through genomic bioinformatics. The structures of the outer proteins were analyzed through comparison with the available structures of homologues characterized by X-ray crystallography. Sequenced conserved outer regions of these proteins were analyzed for their protective capacity in the infant rat model of H. influenzae infection.ResultsNine peptides that were protective in the infant rat model were used in a novel vaccine to immunize chinchillas, the most established animal model of otitis media. Chinchillas (40 vaccinated and 41 controls) were infected with NTHi 86-028NP. The vaccinated group cleared infection more quickly than the control group as indicated by significantly decreased positive findings on video-otoscopy (p< 0.0001) and tympanometry (p=0.0002) on day 7, and presence of middle ear fluid obtained by aspiration (p=0.0001) on day 10 post infection. Similarly, in the mouse model of NTHi pulmonary clearance, the vaccinated group (n=5) reduced infection more rapidly than the control group (n= 5), p=0.008.ConclusionThese data demonstrate the effectiveness of the Bacterial Vaccine Polypeptide methodology in development of a vaccine against NTHi with protection in relevant preclinical models of both otitis media and pulmonary clearance. The methods are applicable to other bacteria, and this approach to a Bacterial Vaccine Polypeptide against NTHi serves as a paradigm for development of similar vaccines to protect against other bacterial infections.Disclosures All Authors: No reported disclosures

The anti-inflammatory effects of erythromycin, clarithromycin, azithromycin and roxithromycin on histamine-induced otitis media with effusion in guinea pigs.

Otitis media with effusion is a clinical manifestation characterised by inflammation of middle-ear mucosa. This study investigated the therapeutic effect of erythromycin, clarithromycin, azithromycin and roxithromycin on a histamine-induced animal model of otitis media with effusion. The animals were divided into five groups, receiving erythromycin, clarithromycin, azithromycin, roxithromycin or saline solution. The guinea pigs in the study groups received erythromycin (40 mg/kg/day), clarithromycin (15 mg/kg/day), azithromycin (10 mg/kg/day) or roxithromycin (10 mg/kg/day) for 3 days by gastric tube. Four hours after the end of the administration, histamine solution was injected into the right middle ear. The lowest neutrophil density value obtained using stereological techniques was in the azithromycin group (0.86 ± 0.25 × 10-5/μm3), while the highest value was observed in the control group (6.68 ± 3.12 × 10-5/μm3). The anti-inflammatory properties of clarithromycin, azithromycin and roxithromycin were similar to one another, but better than that of erythromycin. The use of macrolide antibiotics is recommended, as they show antibacterial and anti-inflammatory efficacy in otitis media with effusion.

Middle ear irrigation using a hydrodebrider decreases biofilm surface area in an animal model of otitis media.

ObjectiveTo compare the safety and efficacy of manual and powered irrigation of the middle ear using saline or 1% baby shampoo to treat biofilm‐forming bacterial middle ear infections.BackgroundBiofilms play a major role in recalcitrant otitis media and are challenging to treat. Many therapeutic strategies have been attempted and the role of topical therapies is still being investigated. Topical irrigation using saline or 1% baby shampoo and the use of a hydrodebrider have been investigated in biofilms involved in chronic rhinosinusitis and their role within the middle ear is yet to be determined.MethodsTwenty‐two adult chinchillas underwent bilateral trans‐bullar inoculation of non‐typable biofilm forming Haemophilus influenza followed by unilateral middle ear irrigation 5 days later using saline administered via a powered hydrodebrider or manual irrigation of saline or 1% baby shampoo. Contralateral inoculated ears served as control and were not irrigated. Two days following irrigation, the bullae were harvested and processed for scanning electron microscopy to assess biofilm surface area. Auditory brainstem responses were performed before bacterial inoculation and prior to euthanasia.ResultsManual and powered irrigation were effective in reducing the surface area of biofilm when compared to the control group. The hydrodebrider demonstrated to be more effective at eradicating biofilm than manual irrigation, especially in areas of difficult access, such as the ventral portion of the chinchillas' bullae. There was no difference in manual irrigation of saline when compared to 1% baby shampoo. Irrigations either manually or using the hydrodebrider did not affect hearing, the vestibular system or facial function.ConclusionMiddle ear biofilms can be treated safely and effectively with rinses using either normal saline or 1% baby shampoo administered manually or with a powered hydrodebrider.Level of EvidenceNA.

Expression of calcium-binding proteins S100A8, S100A9 and S100A12 in otitis media

ObjectivesCalgranulins (calcium-binding proteins S100A8, S100A9 and S100A12) are predominant cytoplasmic proteins of neutrophils and produced by various cells, playing multiple functions in innate immunity and the inflammatory process. Although up-regulated expression of S100A8 and S100A9 genes were observed in an animal model of otitis media (OM), their expressions have not been studied in human middle ear epithelial cells in response to the OM pathogen or in patients with recurrent or chronic OM (recurrent OM/RecOM or chronic OM with effusion/COME). Study designGene expressions were compared between Streptococcus pneumoniae (SP)-infected and non-infected human middle ear epithelial cells (HMEECs) as well as between chronic OM patients and control patients (CI). MethodsGene expressions were profiled by quantitative real time PCR (qPCR). S100 proteins in OM patient and CI middle ear biopsies were detected by immunostaining. ResultsS100A8, S100A9 and S100A12 gene expressions were elevated in SP-infected HMEECs in time-dependent manner. S100A8 and S100A9 but not S100A12 gene expression was significantly elevated in the middle ear mucosa of OM patients. S100A8 and S100A9 protein were observed in middle ear mucosa of OM, but not CI patients. Minimal co-localization was observed between S100A8 and S100A9 with neutrophil elastase and cytokeratin in ME sections of OM patients. ConclusionElevated S100A8 and S100A9 gene expression in SP-infected HMEECs and in the middle ear mucosa of OM, minor co-localized with neutrophil markers suggests that middle ear epithelial cell secretion of S100A8 and S100A9 may play a role in the pathogenesis of recurrent and chronic OM.

Panel 4: Report of the Microbiology Panel.

Objective To perform a comprehensive review of the literature from July 2011 until June 2015 on the virology and bacteriology of otitis media in children. Data Sources PubMed database of the National Library of Medicine. Review Methods Two subpanels comprising experts in the virology and bacteriology of otitis media were created. Each panel reviewed the relevant literature in the fields of virology and bacteriology and generated draft reviews. These initial reviews were distributed to all panel members prior to meeting together at the Post-symposium Research Conference of the 18th International Symposium on Recent Advances in Otitis Media, National Harbor, Maryland, in June 2015. A final draft was created, circulated, and approved by all panel members. Conclusions Excellent progress has been made in the past 4 years in advancing our understanding of the microbiology of otitis media. Numerous advances were made in basic laboratory studies, in animal models of otitis media, in better understanding the epidemiology of disease, and in clinical practice. Implications for Practice (1) Many viruses cause acute otitis media without bacterial coinfection, and such cases do not require antibiotic treatment. (2) When respiratory syncytial virus, metapneumovirus, and influenza virus peak in the community, practitioners can expect to see an increase in clinical otitis media cases. (3) Biomarkers that predict which children with upper respiratory tract infections will develop otitis media may be available in the future. (4) Compounds that target newly identified bacterial virulence determinants may be available as future treatment options for children with otitis media.

The role of vascular endothelial growth factor and vascular stability in diseases of the ear

Vascular endothelial growth factor (VEGF) is a critical mediator of vascular permeability and angiogenesis and likely plays an important role in cochlear function and hearing. This review highlights the role of VEGF in hearing loss associated with vestibular schwannomas, otitis media with effusion, and sensorineural hearing loss. PubMed literature review. A review of the literature was conducted to determine the role of VEGF in diseases affecting hearing. Therapeutic efficacy has been demonstrated for the anti-VEGF agent bevacizumab in vestibular schwannomas, with tumor size reduction and hearing improvement in patients with neurofibromatosis type 2. The loss of functional Merlin, the protein product of the nf2 gene, results in a decrease in expression of the anti-angiogenic protein SEMA3F through a Rac-1-dependent mechanism, allowing VEGF to promote angiogenesis. Bevacizumab may therefore restore the angiogenic balance through inhibiting the relative increase in VEGF. Many of the clinical findings of otitis media with effusion can be reproduced by delivery of recombinant VEGF through transtympanic injection or submucosal osmotic pump. VEGF receptor inhibitors have been demonstrated to improve hearing in an animal model of otitis media with effusion. VEGF affects both the inner ear damage and repair processes in sensorineural hearing loss. VEGF has an important role in vestibular schwannomas, otitis media with effusion, and sensorineural hearing loss.

NTHi induction of Cxcl2 and middle ear mucosal metaplasia in mice

Chronic otitis media (COM) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Nontypeable Haemophilus influenzae (NTHi), the most common acute otitis media (OM) pathogen, is known to activate inflammation and mucin expression in vitro and in animal models of OM. The goals of this study were to examine histopathological and expression profiling epithelial effects of NTHi challenge in murine middle ears. In vitro and in vivo murine model of OM. Weekly transtympanic inoculation of Balb/c mice with 300 μg/ml of NTHi lysates versus saline was performed. Histopathologic analysis was carried out at 4 weeks. Expression microarray analysis was performed at 1 and 7 days. Microarray findings were validated in independent animal samples and in a cultured murine middle ear epithelial cell (mMEEC) line. Histopathologic analyses revealed middle ear mucosal thickening after NTHi exposure. Microarray analyses of inflammatory response genes which changed significantly demonstrated that the chemokine Cxcl2 had the largest fold-change, with significantly increased expression at 1 and 7 days after NTHi injection compared to either saline or no-injection (P <0.01). Validation by real-time qPCR revealed similar significantly increased relative mRNA levels for Cxcl2. NTHi lysates were also found to significantly upregulate the transcription of Cxcl2 in mMEEC in a time- and dose-dependent manner (P <0.05). Middle ear NTHi challenge in mice leads to chronic epithelial mucosal metaplasia and overexpression of inflammatory mediators, most notably Cxcl2. This finding is parallel to NTHi-mediated pulmonary mucosal metaplasia where Cxcl2 has been identified as an important inflammatory mediator.

Development of Animal Models of Otitis Media

Otitis media is defined as inflammation of the middle ear, including the auditory ossicles and the Eustachian tube. Otitis media is a major health problem in many societies. The causes of otitis media includes infection and anatomic/physiologic, host, and environmental factors. In general, otitis media is a childhood disease, and anatomic and physiologic changes have great effects on its development. Thus, in vitro or human experimental studies of otitis media are difficult. Several experimental animal models have been introduced to investigate the pathogenesis and treatment of otitis media. However, none are ideal. The aim of this review is to provide a brief overview of the current status of animal models of otitis media with effusion, acute otitis media, and cholesteatoma. This review will assist determination of the most appropriate animal models of otitis media.

Haemophilus influenzae OxyR: Characterization of Its Regulation, Regulon and Role in Fitness

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness.

The role of vascular endothelial growth factor in pediatric otitis media with effusion

Objectives Vascular endothelial growth factor (VEGF) is a potent and critical inducer of angiogenesis and vascular permeability, and has been shown to play an important role in inflammatory events, together with hypoxia and inflammatory cytokines. VEGF messenger ribonucleic acid (mRNA) is expressed in the middle ear in an experimental animal model of otitis media with effusion (OME) and in patients with OME. However, the protein levels of VEGF in middle ear effusions (MEEs) are unknown and the role of VEGF in the pathogenesis of OME is unclear. The goals of this study were to determine the VEGF levels in MEEs and to investigate the role of VEGF in production of MEEs by comparing these levels with those of interleukin-8 (IL-8), endotoxin, and albumin. Methods Forty-six MEEs obtained from 33 children (24 boys, 9 girls) were used in the study. The mean age of the subjects was 6.3 years old (range, 1–12 years old). The patients underwent myringotomy and/or insertion of a ventilation tube for treatment of OME. After myringotomy, MEEs were collected with a Juhn Tym-Tap. The samples were divided into serous and mucoid types based on observation by the naked eye. After measuring the weight of the MEE, the sample was diluted with phosphate-buffered saline and frozen until use. The concentrations of VEGF and IL-8 in the MEEs were determined by enzyme-linked immunosorbent assays, endotoxin concentrations were measured by the Limulus Amebocyte Lysate test, and albumin levels were determined using an immunoturbidimetric assay. Results VEGF, endotoxin, IL-8, and albumin were detected in 100%, 89%, 98%, and 100% of the 46 MEEs, respectively. The concentrations of VEGF, endotoxin, and IL-8 were significantly higher in mucoid MEEs than in serous MEEs ( p < 0.01), whereas there was no significant difference in the albumin concentration between mucoid and serous MEEs. The VEGF levels were positively correlated with those of endotoxin ( R 2 = 0.17, p < 0.05) and albumin ( R 2 = 0.65, p < 0.01) in mucoid MEEs, but not in serous MEEs. Conclusions Our results suggest that VEGF is produced in response to bacterial components such as endotoxin in the middle ear cavity and is associated with production of mucoid MEEs by increasing serum exudation and mucosal secretion.

Change in Cochlear Response in an Animal Model of Otitis Media with Effusion

Our previous studies confirm that middle ear mobility is reduced in the presence of otitis media with effusion (OME). Variations in middle ear function may result in changes in cochlear response in OME ears. With the long-term goal of evaluating cochlear function in OME ears, the aim of this study was to measure the displacement of the basilar membrane (BM) in guinea pig ears with OME. Vibrations of the BM at the apex and basal turn were measured in an in vitro preparation extracted 3 and 14 days after injection of lipopolysaccharide in the middle ear of guinea pigs. The results show that the displacement sensitivity of the BM at the apex and the basal turn to sound pressure in the ear canal was reduced up to 25 dB at their characteristic frequencies, respectively. Cochlear gain with respect to umbo movement was also changed in ears with OME in both groups. This study provides data for analysis of the change of BM vibration in a guinea pig OME model.

Immune modulatory oligonucleotides in the prevention and treatment of allergen-induced eustachian tube dysfunction in the animal model

This article reviews the current literature investigating the applications and success of immune modulatory oligonucleotides as immunotherapy to treat and prevent allergen-induced eustachian tube dysfunction in animal models. Synthetic DNA-based immunotherapy agents composed of unmethylated cytosine-guanine dinucleotides (CpG ODNs) that bind to Toll-like receptors have been found to have tremendous potential as therapeutic agents and adjuvants. CpG ODNs can induce a shift in the cytokine profile and immune response that favors the T-helper type 1 pathway and suppresses the T-helper type 2 pathway. This makes CpG ODNs promising candidates for treating allergic diseases. Current CpG ODN studies have demonstrated prevention and treatment of acute allergen inflammation of the eustachian tube in an animal model of otitis media. Immune modulatory oligonucleotides in immunotherapy, administered systemically or topically, have been shown to be safe and effective in the animal model.

Evolution of Middle Ear Changes After Permanent Eustachian Tube Blockage

To develop a valid animal model for otitis media with effusion (OME). Forty specific pathogen-free Wistar rats underwent a procedure based on the permanent obstruction of pharyngeal eustachian tube by means of electrocoagulation without any manipulation. Ear Research Group, Department of Otorhinolaryngology, Puerta de Hierro Hospital, Universidad Autonoma de Madrid, Madrid, Spain. The assessment of OME by otoscopy and tympanometry. The rats were humanely killed at 15 and 90 days, and temporal bones were obtained and processed for histopathologic study. The histopathologic study of the temporal bones demonstrated the occurrence of chronic effusion and mucosal changes owing to mucoperiosteal enlargement. Comparison with other experimental models was made. Our animal model was consistent and reproducible and resembled human OME.

Efficacy of a novel oral carbapenem, tebipenem pivoxil (TBM-PI), against experimental otitis media caused by penicillin resistant Streptococcus pneumoniae in chinchilla

An animal model of otitis media using chinchillas was developed to evaluate the efficacy of tebipenem pivoxil (TBM-PI) against experimental otitis media. Chinchillas inoculated via the transbullar approach with Streptococcus pneumoniae serogroup 6 were included in the efficacy study with TBM-PI, amoxicillin (AMX) or untreated as controls. TBM-PI resulted in survival rate of 83%, compared with 25% survival for AMX and 0% survival for controls ( p < 0.01). Quantitative cultures in the middle ear effusions at day 5 of the TBM-PI group yielded 3.5 ± 2.4 log 10 CFUs/ml. TBM-PI is a promising antibiotic for the treatment of acute otitis media.

Induction of Mucous Cell Metaplasia in the Middle Ear of Rats Using a Three-step Method: An Improved Model for Otitis Media with Mucoid Effusion

Otitis media with mucoid effusion, characterized by mucous cell metaplasia in the middle ear cleft and thick fluid accumulation in the middle ear cavity, is a common otological disease that frequently affects young children. Multiple factors are involved in the development of this disease, especially middle ear infection and Eustachian tube dysfunction. In this study, in order to induce otitis media with effusion in rats, we introduced a three-step method, namely inoculation of Streptococcus pneumoniae at 10 7 colony-forming units (CFU) ear or Haemophilus influenzae at 5 ½ 10 7 CFU ear into the middle ear cavity twice at 2-week intervals, followed by Eustachian tube obstruction (ETO) for 4 and 8 weeks. Animals inoculated with phosphate-buffered saline (PBS) twice in the same manner followed by ETO served as controls. Middle ear effusion and mucosa were harvested for evaluation of carbohydrate concentrations and mucous cell density, respectively. We found that rats inoculated with S. pneumoniae twice, followed by ETO at 8 weeks, yielded the highest carbohydrate concentration in middle ear effusion and the highest goblet cell density in the middle ear cavity compared to the H. influenzae and PBS groups. It is tentatively concluded that inoculation of S. pneumoniae at 10 7 CFU ear into the middle ear cavity of rats twice at 2-week intervals, followed by ETO for 8 weeks, is a promising animal model for otitis media with mucoid effusion which may be valuable for studying the human counterpart.

Middle ear catalase distribution in an animal model of otitis media

Increasing evidence implicates free radicals in the pathogenesis of inflammatory disease, including otitis media. The anti-oxidant enzymes catalase, glutathione peroxidase and superoxide dismutase protect tissues from the destructive effects of free radicals. Our previous work has shown depressed levels of superoxide dismutase in the infected middle ears of a guinea pig model of otitis media in comparison with normal control ears. We studied the distribution and relative abundance of catalase in the middle ear of this animal model in an effort to elucidate the role free radicals play in the pathogenesis of otitis media. Catalase distribution was mapped immunohistochemically in the middle ears of guinea pigs with induced streptococcus otitis media, and compared with normal control ears. In the control ears, catalase was localized to the epithelium of the middle ear mucosa, with scant distribution in the submucosa. The infected ears demonstrated inflammatory cell invasion with hyperemia and submucosal edema. Catalase was localized to the epithelium and had scant distribution in the submucosa. This distribution was similar to that found previously with superoxide dismutase. Enzyme-linked immunosorbent assay of catalase demonstrated a mean value of 1.00 +/- 0.06 microgram/mg protein in the control ears, and 1.06 +/- 0.12 microgram/mg in the infected ears, but these two values were not statistically different.

Identification of surface-exposed B-cell epitopes on high molecular-weight adhesion proteins of nontypeable Haemophilus influenzae.

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of nontypeable Haemophilus influenzae (NTHI) mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to most nontypeable Haemophilus strains. We also reported that immunization with an HMW1-HMW2 mixture modified the course of disease in an animal model of otitis media, suggesting the potential usefulness of these proteins as NTHI vaccine components. Identification of surface-accessible B-cell epitopes could be important to efforts to develop recombinant or synthetic peptide vaccines based upon these high-molecular-weight proteins. Thus, the purpose of the present study was to identify surface-accessible epitopes on the HMW1 and HMW2 proteins by using monoclonal antibodies (MAbs) and to determine the prevalence of these epitopes among the high-molecular-weight proteins expressed by heterologous nontypeable Haemophilus strains. MAbs were generated by immunizing mice with high-molecular-weight proteins purified from prototype strains and were screened by immunoelectron microscopy (IEM) for the ability to recognize surface epitopes. Two MAbs, designated AD6 and 10C5, that recognized surface epitopes by IEM were recovered. In order to map the epitopes recognized by these two MAbs, we constructed a set of HMW1 and HMW2 recombinant fusion proteins using the pGEMEX vectors and examined the reactivity of the MAbs with these fusion proteins. MAb AD6 recognized an epitope in both HMW1 and HMW2 which mapped to the last 75 amino acids at the carboxy termini of the two proteins. When examined for reactivity with heterologous strains, MAb AD6 recognized high-molecular-weight proteins in 75% of 125 unrelated nontypeable Haemophilus strains and, in addition, reacted with three of three such strains when examined by IEM. MAb 10C5 recognized an epitope that mapped to a 155-amino-acid segment near the carboxy terminus of HMW1. This epitope was adjacent to but distinct from the AD6 epitope and was absent from HMW2. The 10C5 epitope was expressed by 40% of the AD6 reactive strains. Identification of shared surface-exposed epitopes on the high-molecular-weight adhesion proteins suggests the possibility of developing recombinant or synthetic peptide-based vaccines protective against disease caused by the majority of NTHI strains.

Superoxide dismutase in an animal model of otitis media.

Cu, Zn superoxide dismutase (SOD) is a metalloprotein that catalyzes the dismutation of the superoxide anion into O2- and H2O2, and therefore functions to maintain a low intracellular concentration of an otherwise toxic metabolite of oxygen. SOD protects living tissue from the destructive effects of free radicals. Increasing evidence implicates free radicals, including the superoxide radical (O2-), in the pathogenesis of disease, including otitis media. In an effort to elucidate the role free radicals play in the pathogenesis of otitis media, SOD was localized immunocytochemically to determine its cellular distribution in specimens of guinea pig middle ear. In normal ears, SOD was found concentrated in the epithelium of the middle ear mucosa. Low quantities were characteristic of connective tissue, bone, and cartilage. In streptococcus-infected ears, SOD localized similarly, concentrating in the epithelium. The infected ears had extensive submucosal edema which stained poorly and appeared to have less SOD than did normal ears. This was confirmed by an assay using laser densitometry of Western blots to quantify the amount of SOD in the mucosa of normal versus infected middle ears. This demonstrated a value of SOD in normal mucosa of 1.77 +/- 0.48 micrograms/mg of protein compared with 1.02 +/- 0.28 micrograms/mg in the infected mucosa. The two groups were significantly different at P < 0.05. These findings are discussed, and suggestions for future experimentation addressed.