Viability and virulence of pneumolysin, pneumococcal surface protein A, and pneumolysin/pneumococcal surface protein A mutants in the ear.

IMPORTANCE: Understanding how pneumococcal proteins affect the pathology of the middle ear and inner ear is important for the development of new approaches to prevent otitis media and its complications. OBJECTIVES: To determine the viability and virulence of Streptococcus pneumoniae mutants deficient in pneumolysin (Ply-) and pneumococcal surface protein A (PspA-) in the chinchilla middle ear. DESIGN: Bullae of chinchillas were inoculated bilaterally with wild-type (Wt), Ply-, PspA-, and Ply-/PspA- strains. Bacterial colony-forming units (CFUs) in middle ear effusions were counted at 48 hours. The CFUs of the PspA- group were also counted at 6 to 36 hours after inoculation. Temporal bone histopathological results were compared. SETTING AND PARTICIPANTS: Twenty-seven chinchillas in an academic research laboratory. EXPOSURE: Chinchilla middle ears were inoculated with S pneumoniae to produce sufficient volumes of effusions and noticeable histopathological changes in the ears. MAIN OUTCOMES AND MEASURES: The CFU counts in the middle ear effusions and histopathological changes were compared to determine the effect of pneumococcal protein mutations on chinchilla ears. RESULTS: At 48 hours, CFUs in middle ears were increased for the Wt and Ply-/PspA- strains, but Ply- remained near inoculum level. No bacteria were detected in the PspA- group. The CFUs of PspA- decreased over time to a low level at 30 to 36 hours. In vitro, PspA- in Todd-Hewitt broth showed an increase in bacterial growth of 2 logs at 43 hours, indicating PspA- susceptibility to host defenses in vivo. The PspA- and Ply- groups had fewer pathologic findings than the Wt or Ply-/PspA- groups. Histopathological analysis showed significant differences in the number of bacteria in the scala tympani in the Wt group compared with the Ply-, PspA-, and Ply-/PspA- groups. The PspA- strain was the least virulent. CONCLUSIONS AND RELEVANCE: The PspA- mutant was much less viable and less virulent in the ear than the Wt, Ply-, and Ply-/PspA- strains. There was no significant attenuation in the viability and virulence of the Ply-/PspA- mutant compared with the Wt or single mutants. The viability and virulence of pneumococcal mutants seemed to be protein and organ specific.

Invasive and noninvasive Streptococcus pneumoniae capsule and surface protein diversity following the use of a conjugate vaccine.

The 13-valent pneumococcal conjugate vaccine (PCV13) was introduced in the United States in 2010 for the prevention of invasive pneumococcal disease (IPD) and otitis media. While many studies have reported its potential efficacy for IPD, not much is known about the epidemiology of noninvasive disease following its introduction. We characterized the capsular types and surface protein genes of noninvasive pediatric pneumococcal isolates collected between 2002 and 2010 (n = 1,058) at Children's of Alabama following the introduction of PCV7 and tested a subset of noninvasive and previously characterized IPD isolates for the presence of the pspA, pspC, and rrgC genes, which encode protection-eliciting proteins. PCV7 serotypes had dramatically decreased by 2010 (P < 0.0001), and only 50% of all noninvasive infections were caused by the PCV13 capsular serotypes. Serotype 19A accounted for 32% of the noninvasive isolates, followed by serotypes 35B (9%), 19F (7%), and 6C (6%). After 7 years of PCV7 usage, there were no changes in the frequencies of the pspA or pspC genes; 96% of the strains were positive for family 1 or 2 pspA genes, and 81% were also positive for pspC. Unexpectedly, more noninvasive than invasive strains were positive for rrgC (P < 0.0001), and the proportion of rrgC-positive strains in 2008 to 2010 was greater than that in 2002 to 2008 (IPD, P < 0.02; noninvasive, P < 0.001). Serotypes 19F, 19A, and 35B were more frequently rrgC positive (P < 0.005) than other serotypes. A vaccine containing antigens, such as PspA, PspC, and/or RrgC, can provide coverage against most non-PCV13-type pneumococci. Continued surveillance is critical for optimal future vaccine development.

Panel 4: Recent advances in otitis media in molecular biology, biochemistry, genetics, and animal models.

BACKGROUND: Otitis media (OM) is the most common childhood bacterial infection and also the leading cause of conductive hearing loss in children. Currently, there is an urgent need for developing novel therapeutic agents for treating OM based on full understanding of molecular pathogenesis in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. OBJECTIVE: To provide a state-of-the-art review concerning recent advances in OM in the areas of molecular biology, biochemistry, genetics, and animal model studies and to discuss the future directions of OM studies in these areas. DATA SOURCES AND REVIEW METHODS: A structured search of the current literature (since June 2007). The authors searched PubMed for published literature in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. RESULTS: Over the past 4 years, significant progress has been made in the areas of molecular biology, biochemistry, genetics, and animal model studies in OM. These studies brought new insights into our understanding of the molecular and biochemical mechanisms underlying the molecular pathogenesis of OM and helped identify novel therapeutic targets for OM. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Our understanding of the molecular pathogenesis of OM has been significantly advanced, particularly in the areas of inflammation, innate immunity, mucus overproduction, mucosal hyperplasia, middle ear and inner ear interaction, genetics, genome sequencing, and animal model studies. Although these studies are still in their experimental stages, they help identify new potential therapeutic targets. Future preclinical and clinical studies will help to translate these exciting experimental research findings into clinical applications.

Activation of the transforming growth factor beta pathway in bacterial otitis media.

OBJECTIVES: Granulation tissue is common in otitis media (OM), yet little is known about the signaling pathways in the formation of granulation tissue in response to infections. In this study, we sought to investigate the activation of the transforming growth factor beta (TGF-beta) signaling pathway in the formation of granulation tissue in response to middle ear pathogens. METHODS: Rat OM models were made by inoculating pneumococcus type 6A or nontypeable Haemophilus influenzae into the middle ear cavity or by obstructing the eustachian tube. Various pathway activities in the middle ear mucosa were analyzed with microarrays. RESULTS: The TGF-beta signaling pathway was highly regulated in the middle ear cleft with bacterial OM, but not in the ears with eustachian tube obstruction. In ears with bacterial OM, the TGF-beta signaling pathway products were higher in Haemophilus-infected ears than in pneumococcus-infected ears. CONCLUSIONS: Bacterial OM triggers granulation tissue to thrive in the middle ear cleft of rats. Nontypeable H influenzae is more potent than pneumococcus type 6A in the formation of granulation tissue. Eustachian tube obstruction alone did not contribute to granulation tissue formation in the middle ear.

Effect of apolactoferrin on experimental pneumococcal otitis media.

OBJECTIVE: To find the effect of apolactoferrin administration on the middle and inner ears after experimentally induced pneumococcal otitis media. DESIGN: Histopathologic and morphometric analysis of the middle and inner ears. SETTING: University of Minnesota, Minneapolis. SUBJECTS: Ten chinchillas. INTERVENTIONS: The middle ear cavities of chinchillas were inoculated bilaterally with type 2 wild-type Streptococcus pneumoniae. Twenty-four hours later, the ears of 5 of the animals were injected with phosphate-buffered saline (PBS) and the other 5 with human apolactoferrin. The animals were killed 24 hours after the last injection. Bacterial counts were made of the middle ear effusions, and the cochleae were processed for histologic analysis. The thickness of the round window membranes and bacterial and inflammatory cell infiltration of the round window membranes, and scala tympani and damage of the hair cells and stria vascularis were compared for these 2 groups of animals. MAIN OUTCOME MEASURES: Comparison of inflammatory and bacterial cells in the middle and inner ears, and damage to inner ear structures. RESULTS: Bacterial plate counts of middle ear effusions (P  = .005) and the number of inflammatory cells in the round window membrane (P  = .047) were significantly lower in the apolactoferrin group compared with the group treated with PBS. CONCLUSION: Further investigation of apolactoferrin as a nonantibiotic approach for the treatment of otitis media and its complications is needed to confirm its safety and efficacy.

A combination antioxidant therapy prevents age-related hearing loss in C57BL/6 mice.

OBJECTIVE: Age-related hearing loss (ARHL) is characterized by gradual, progressive sensorineural hearing loss, which impairs communication, lending to clinical depression and social withdrawal. There are currently no effective treatments for ARHL. The purpose of this study is to evaluate the potential of a combination antioxidant therapy in preventing ARHL. STUDY DESIGN: Randomized controlled trial. SETTING: Animal study. SUBJECTS AND METHODS: C57BL/6 mice, a recognized animal model of ARHL, were assigned to one of three groups: early treatment (n = 12), late treatment (n = 9), or control group (n = 9). Treatment groups of mice were fed with a combination agent comprising six antioxidant agents that target four sites within the oxidative pathway: L-cysteine-glutathione mixed disulfide, ribose-cysteine, NW-nitro-L-arginine methyl ester, vitamin B12, folate, and ascorbic acid. Auditory brainstem response (ABR) thresholds were recorded at baseline and every three months following initiation of treatment. RESULTS: Threshold shifts from baseline were decreased in the treatment groups when compared to the control group at all tested frequencies (P < 0.001). The ABR threshold shift at 12 months of age for the control group was 34.7 dB with a 95% confidence interval (CI) of +/-1.6. The mean threshold shifts for the early and late treatment groups were 7.5 dB (+/-0.87, 95% CI) and 9.2 dB (+/-1.6, 95% CI). CONCLUSION: Combination antioxidant therapy effectively decreased threshold shifts on ABR within an animal model of ARHL. Combination antioxidant therapy, with further research and investigation, may provide a safe and cost-effective method of preventing presbycusis in the growing elderly population.

Cochlear pathology in human temporal bones with otitis media.

CONCLUSION: Middle and inner ear interactions in otitis media can lead to cochlear pathology. More severe pathological changes observed in the basal turn of the cochlea are consistent with prevalence of sensorineural hearing loss at higher frequencies in patients with otitis media. METHODS: Of 614 temporal bones with otitis media, 47 with chronic and 35 with purulent otitis media were selected following strict exclusion of subjects with a history of acoustic trauma, head trauma, ototoxic drugs, and other diseases affecting the cochlear labyrinth. Temporal bones with labyrinthine inflammatory changes were further evaluated for loss of hair cells and other histopathologic changes compared to age-matched controls. RESULTS: In all, 19% of temporal bones with chronic and 9% with purulent otitis media showed labyrinthine inflammatory changes. In chronic otitis media, inflammatory changes were: 56% localized purulent, 22% localized serous, 11% generalized seropurulent, and 11% generalized serous. Inflammatory changes in temporal bones with purulent otitis media included 67% localized purulent and 33% were generalized seropurulent. Pathological findings included: serofibrinous precipitates and inflammatory cells in scala tympani of basal turn and cochlear aqueduct, significant loss of outer and inner hair cells, and significant decrease in area of stria vascularis in the basal turn of the cochlea, as compared to controls.

Effect of lipooligosaccharide mutations of Haemophilus influenzae on the middle and inner ears.

OBJECTIVE: The purpose of this study was to determine the virulence of nontypeable Haemophilus influenzae 2019 (NTHi 2019) and its two lipooligosaccharide (LOS) mutant strains, B29 (gene htrB) and DK1 (gene rfaD), and compare their effect on the middle ear, round window membrane, and inner ear. RESULTS: Fifteen chinchillas were divided into three equal groups and their bullas inoculated bilaterally with 0.5 ml of 10(2)CFU/ml of parent NTHi 2019, B29 or DK1 mutant strains. Two days after inoculation all animals had otitis media and inflamed middle ear mucosa. There was a trend of greater thickness and infiltration of the round window membrane in animals inoculated with the wild-type NTHi strain compared to the mutant strains and a significant increase in both inflammatory cell infiltration and bacteria presence in the scala tympani area of the inner ear. Strial edema was only observed in the wild-type-inoculated group. CONCLUSIONS: LOS mutants of NTHi appear to have a reduced ability to pass through the round window membrane resulting in less inner ear inflammation and pathological changes.

Expression of fibroblast growth factor binding protein in head and neck cancer.

OBJECTIVES: To characterize the expression of fibroblast growth factor binding protein (FGF-BP) messenger RNA (mRNA) in head and neck squamous cell carcinoma (HNSCC) and to study the association of FGF-BP with vascularity. DESIGN: The expression of FGF-BP mRNA in HNSCC was studied in 35 primary and 8 metastatic HNSCC specimens and 7 control tissues using in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). Microvessels in tumor specimens were identified with endothelial cell markers (von Willebrand factor [vWF] and CD34-specific antibodies). Correlates between FGF-BP and microvessel counts were evaluated statistically. SETTING: University of Minnesota Hospitals and Clinics. PATIENTS: Forty-two surgically treated patients with HNSCC. INTERVENTIONS: The patients were routinely treated in the study hospitals and clinics. MAIN OUTCOME MEASURES: The expression of FGF-BP and angiogenesis in tumors were evaluated. RESULTS: In situ hybridization and RT-PCR demonstrated that FGF-BP mRNA transcripts were expressed in 34 of 35 primary HNSCC specimens and 5 of 8 metastatic tumor specimens but not in adjacent control tissues. The microvessel counts in HNSCC specimens were closely related to the expression level of FGF-BP (P < .001). CONCLUSION: The expression of FGF-BP is statistically linked to the angiogenesis of HNSCC, suggesting that FGF-BP participates in the angiogenesis of HNSCC.

Ototoxicity after combined platinum and fractionated radiation in a novel guinea pig model.

PURPOSE: Cis-platinum and radiation in combination are current organ preservation treatment strategies for head and neck cancer. Their individual ototoxicity has been investigated, with recent demonstration of ototoxicity in clinical studies. Currently, no ototoxicity studies have been performed in animals receiving similar schedules of radiation or cis-platinum to those patients with head and neck cancer. MATERIALS AND METHODS: In the present study, an animal model was developed to investigate the effects of combined modality therapy on hearing. Albino guinea pigs were given equivalent protocol dosages of cis-platinum (3 parenteral courses), fractionated radiation (25 fractions over 5 weeks), or both. Click and tone burst auditory brainstem response (ABR) measurements were performed before and 6 weeks after the completion of treatment. RESULTS: Animals receiving radiation or cis-platinum and radiation experienced permanent significant ABR shifts at all frequencies, with 33% of the animals experiencing complete unilateral sensorineural hearing loss at 2 or more frequencies in the ear receiving the full radiation dose (7075 cGy over 25 fractions) (P < .05, paired t test analysis). The animals receiving 3 doses of cis-platinum had no significant ABR threshold shifts at 6 weeks. These data suggest that cis-platinum and radiation cause greater ototoxicity than cis-platinum alone. These findings correlate closely with sensorineural hearing loss in combined modality patients at our institution and in recent studies. CONCLUSIONS: We conclude that the current animal results parallel those seen clinically and serve as a model for ototoxicity from combined modality therapies in future protocols.

Role of pneumococcal proteins in sensorineural hearing loss due to otitis media.

HYPOTHESIS: Two Streptococcus pneumoniae proteins, pneumococcal surface protein A (PspA) and pneumolysin (Ply), have functional and histopathologic effects on the inner ear. BACKGROUND: Temporary or permanent sensorineural hearing loss is known to be a sequela of pneumococcal otitis media. Several pneumococcal proteins such as PspA and Ply have been shown to contribute to the pathogenesis of the middle ear; however, effects of these proteins on the inner ear and hearing loss are unknown. METHODS: Middle ears of chinchillas were inoculated with either wild-type S. pneumoniae or its mutants, deficient in PspA or Ply proteins. After 28 days, auditory brainstem response of animals was tested, and their bullae were processed for histopathologic analysis by light microscopy. RESULTS: Twenty-eight days after instillation of 20 colony-forming units of wild-type pneumococci, auditory brainstem response test showed threshold changes of 10 to 15 dB for 4 to 32 kHz and more than 20 dB for 1 to 2 kHz. No significant hearing loss was observed after instillation of the same or even higher doses of isogenic S. pneumoniae mutants of PspA or Ply proteins, or saline injection, after the same period. Histologic analysis showed no fluid, inflammatory cells, or bacteria in the middle ear, indicating that hearing loss was sensorineural. Inner ear morphology showed pathologic changes in the stria vascularis, suggesting it as the target of otitis media-induced damage, which may lead to sensorineural hearing loss. CONCLUSION: The virulence PspA and Ply proteins of S. pneumoniae affect the inner ear and auditory function.

Up-regulation of macrophage migration inhibitory factor induced by endotoxin in experimental otitis media with effusion in mice.

CONCLUSION: Injection of endotoxin into the middle ear causes production of macrophage migration inhibitory factor (MIF) in an experimental mouse model of otitis media with effusion (OME). Down-regulation of MIF may become a new approach for the management of OME. OBJECTIVE: To determine the role of MIF in OME. MATERIALS AND METHODS: Mice were divided into two groups and their middle ears were injected with either endotoxin or phosphate-buffered saline (PBS). Mice were sacrificed at 6 h, 12 h, or 1, 3, 7, or 14 days after injection and concentrations of MIF, interleukin-1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in middle ear effusions were measured by enzyme-linked immunosorbent assay. RESULTS: Concentrations of MIF in the endotoxin group at 1 day and 3 days were significantly higher than in the PBS control group. Concentrations of IL-1beta in the endotoxin group at 6 h, 12 h, 1 day, and 3 days were significantly higher than in controls. Concentrations of TNF-alpha in the endotoxin group at 1 day and 3 days were significantly higher than in controls. Concentration of MIF in the endotoxin group was positively correlated with that of IL-1beta and TNF-alpha.

The role of inflammatory mediators in the pathogenesis of otitis media and sequelae.

This review deals with the characteristics of various inflammatory mediators identified in the middle ear during otitis media and in cholesteatoma. The role of each inflammatory mediator in the pathogenesis of otitis media and cholesteatoma has been discussed. Further, the relation of each inflammatory mediator to the pathophysiology of the middle and inner ear along with its mechanisms of pathological change has been described. The mechanisms of hearing loss including sensorineural hearing loss (SNHL) as a sequela of otitis media are also discussed. The passage of inflammatory mediators through the round window membrane into the scala tympani is indicated. In an experimental animal model, an application of cytokines and lipopolysaccharide (LPS), a bacterial toxin, on the round window membrane induced sensorineural hearing loss as identified through auditory brainstem response threshold shifts. An increase in permeability of the blood-labyrinth barrier (BLB) was observed following application of these inflammatory mediators and LPS. The leakage of the blood components into the lateral wall of the cochlea through an increase in BLB permeability appears to be related to the sensorineural hearing loss by hindering K(+) recycling through the lateral wall disrupting the ion homeostasis of the endolymph. Further studies on the roles of various inflammatory mediators and bacterial toxins in inducing the sensorineumral hearing loss in otitis media should be pursued.

Peripheral facial nerve regeneration using collagen conduit entubulation in a cat model.

OBJECTIVES: Facial nerve (FN) injuries are functionally, psychologically, and financially debilitating. Facial nerve autograft repairs produce significant donor nerve morbidity and functional results that rarely exceed House-Brackmann (HB) grade III over VI. In this study we sought to enhance FN regeneration via collagen conduit entubulation. METHODS: Five control cats underwent right ("cut-side") FN transection and immediate microsurgical anastomosis repair. Five experimental cats underwent identical repairs plus collagen conduit entubulation of each anastomosis. RESULTS: Postoperative behavioral observations revealed gradual FN functional recovery in all cats, who attained adapted HB grades of II to III over VI after 6 weeks. Electromyographic latencies and amplitudes from the bilateral orbicularis oculi and orbicularis oris muscles indicated restoration of FN continuity in all 10 cats. In comparison with FN repairs without conduits, repairs with conduits significantly enhanced recovery of amplitude in cut-side orbicularis oculi muscles (p = .037) and latency in cut-side orbicularis oris muscles (p = .048). In comparison with intact left ("uncut-side") FN latencies and amplitudes, more statistically significant differences in cut-side FN function were observed in repairs without conduits than in repairs with conduits. Conduits therefore facilitated a more complete return of electrophysiological function. Histologic analyses confirmed FN continuity and revealed more organized FN regenerative architecture in conduit-implanted repairs. CONCLUSIONS: The overall results support enhanced FN regeneration with collagen conduit entubulation.

Induction of heat shock protein 70 inhibits NF-kappa-B in squamous cell carcinoma.

OBJECTIVE: To determine the relationship between heat shock proteins (HSPs) and the proinflammatory, anti-apoptosis mediator NF-kappa-B in squamous cell carcinoma. STUDY DESIGN AND SETTING: CA-9-22 cells were exposed to heat stress to induce the production of HSPs. Immunoblot and reporter gene experiments determined the inducibility of HSP production and the activation of cytokine-induced NF-kappa-B. Immunoblot experiments determined the presence of the inhibitor-kappa-B-alpha (IkappaB alpha). RESULTS: CA-9-22 cells can be induced by heat stress to produce HSPs at 100-fold above baseline levels. The induction of HSPs prevents the activation and nuclear translocation of NF-kappa-B despite stimulation with IL-1beta and TNF-alpha. CONCLUSIONS: Constitutive activation of NF-kappa-B is prevented by HSP induction through an increase in IkappaB alpha synthesis. SIGNIFICANCE: The induction of HSP70 alters the inflammatory milieu associated with squamous cell carcinoma progression through the inhibition of NF-kappa-B and may ultimately promote apoptosis in head and neck carcinoma.

Pseudomonas aeruginosa lipopolysaccharide induction of keratinocyte proliferation, NF-kappa B, and cyclin D1 is inhibited by indomethacin.

NF-kappaB is activated during acute inflammatory states as well as in other injury response disease states. Several pathologic states in squamous tissue injury response are characterized by increased squamous proliferation. This study was performed to investigate the hypothesis that Pseudomonas aeruginosa LPS is able to activate a proliferative phenotype in squamous cells via NF-kappaB induction and that this NF-kappaB-mediated response may be abrogated with the classic anti-inflammatory agent indomethacin. EMSA, luciferase reporter gene experiments, Western blots, and cellular proliferation assays were performed in normal and transformed human keratinocytes after stimulation with P. aeruginosa LPS. EMSA and luciferase reporter gene assays showed a 3- to 5-fold induction of active NF-kappaB in human keratinocyte cell lines after stimulation with P. aeruginosa LPS. The stimulation correlated with significantly increased cellular proliferation. As one potential mechanism for this increase in proliferation, an NF-kappaB-specific activation of cyclin D1 was observed. Both the NF-kappaB induction and proliferation response were inhibited with indomethacin and in dominant negative stable transfection clones. P. aeruginosa LPS activates proliferation of human keratinocytes, potentially through the induction of NF-kappaB and cyclin D1. These findings suggest that bacterial components can contribute to proliferative disease states in squamous epithelium through NF-kappaB activation.

Expression of platelet-derived growth factor in the developing cochlea of rats.

OBJECTIVE: Platelet-derived growth factor (PDGF) is involved in the control of cell proliferation, differentiation and survival in various tissues of vertebrates. However, little is known about the expression of PDGF in the developing cochlea of rodents. MATERIAL AND METHODS: We examined the expression of PDGF family genes in the developing cochlear tissue of rats using microarrays and tested their role in the proliferation of progenitor hair cells using cellular and molecular biology techniques. RESULTS: It was found that the genes for PDGF-A, PDGF receptor (R)-alpha and PDGFR-beta were highly expressed in the rapidly growing otocyst on embryonic Days 12-14 and weakly expressed thereafter. Reverse transcriptase polymerase chain reaction demonstrated the expression of PDGF-C and confirmed the expression of PDGF-A and PDGFR-alpha and -beta in the developing cochlear tissue of rats and the cultured progenitor hair cells. Inhibition of the expression of PDGFs in the cultured progenitor hair cells with antisense oligonucleotides reduced the DNA synthesis. CONCLUSION: PDGFs and their receptors may play a role in the proliferation of developing cochlear hair cells.