P. aeruginosa infection increases morbidity in experimental cholesteatomas.

OBJECTIVES: Clinicians have long noted that infected cholesteatomas are more aggressive than uninfected ones without data to support these observations. The purpose of this study is to determine the etiological role of biofilm forming P. aeruginosa (PA) and the virulence factor, type IV pili (TFP), in the pathogenesis of experimental cholesteatomas. DESIGN: We evaluated three different PA strains and one Escherichia coli strain in cholesteatoma progression: PA14, a well-characterized wound isolate, OPPA8, an otopathogenic strain from a human cholesteatoma, OPPA8-NP, an isogenic TFP deletion mutant, and DH5α, an E. coli strain. METHODS: Cholesteatomas were induced in gerbils. We inoculated the right ear with bacteria and the left with vehicle. After 6 weeks their cholesteatomas were evaluated by micro-CT scanning. Cholesteatoma size and bone resorption were analyzed digitally. RESULTS: Results demonstrate that PA infection increases cholesteatoma size when compared to uninfected controls: OPPA8 showed an 8.9-fold increase, PA14 a 2.6-fold increase, OPPA8-NP a 1.9-fold increase, while DH5α was not increased over controls. Additionally, infected bullae showed 10 to 50% more cholesteatoma-induced bone resorption. CONCLUSIONS: In this model, PA infected cholesteatomas enlarge more rapidly and are more destructive than uninfected controls. OPPA8, the strain from a human cholesteatoma, showed the greatest enlargement and bone destruction. Additionally, we demonstrate that TFP is a virulence factor in this model because the nonpiliated isogenic mutant, OPPA8-NP, was significantly less aggressive than the wild-type OPPA8 indicating that type IV pili may be a virulence factor in this disease.

Radiographic and micro-computed tomographic imaging of lipopolysaccharide-mediated bone resorption.

OBJECTIVES: Chronic otitis media and cholesteatomas cause hearing loss as a result of bony erosion. This bone resorption is known to be more aggressive when cholesteatomas become infected. The most common organism isolated from both diseases is the gram-negative bacterium Pseudomonas aeruginosa. Lipopolysaccharide (LPS), a major virulence factor found in the gram-negative bacterial cell wall, is well known to incite inflammatory bone resorption. The mechanisms underlying this process, however, are poorly understood. In this study, we developed a mouse model of calvarial osteolysis in which resorption was reliably imaged by plain radiography and micro-computed tomography (micro-CT). METHODS: A murine calvarial model was developed to study bone resorption induced by P aeruginosa LPS. Calvariae from wild-type and knockout mice used in this model were imaged by plain radiography and micro-CT. RESULTS: A high degree of correlation between plain radiography and micro-CT was identified (R2 = 0.8554). Furthermore, maximal LPS-induced bone resorption required functioning toll-like receptor (TLR) 2, TLR4, and myeloid differentiation factor 88 (MyD88). CONCLUSIONS: We have developed a successful model of inflammatory osteolysis in which plain radiography can reliably delineate induced bone resorption. In vivo, we have shown that P aeruginosa LPS signals via TLR2, as well as TLR4 through MyD88.

Lipopolysaccharide-induced osteoclastogenesis from mononuclear precursors: a mechanism for osteolysis in chronic otitis.

Osteoclasts are the only cells capable of carrying out bone resorption and therefore are responsible for the osteolysis seen in infectious diseases such as chronic otitis media and infected cholesteatoma. Pseudomonas aeruginosa is the most common organism isolated from these infectious middle ear diseases. In this study, we examined the mechanisms by which P. aeruginosa lipopolysaccharide (LPS) stimulates osteoclastogenesis directly from mononuclear osteoclast precursor cells. Osteoclast precursors demonstrated robust, bone-resorbing osteoclast formation when stimulated by P. aeruginosa LPS only if previously primed with permissive, sub-osteoclastogenic doses of receptor activator of NF-kappaB ligand (RANKL), suggesting that LPS is osteoclastogenic only during a specific developmental window. Numerous LPS-elicited cytokines were found to be released by osteoclast precursors undergoing P. aeruginosa LPS-mediated osteoclast formation. Two lines of evidence suggest that several cytokines promote Oc formation in an autocrine/paracrine manner. First, inhibition of several cytokine pathways including TNF-alpha, IL-1, and IL-6 block the osteoclastogenesis induced by LPS. Secondly, increased expression of the receptors for TNF-alpha and IL-1 was demonstrated by real-time quantitative polymerase chain reaction. Such a mechanism has not previously been established and demonstrates the ability of osteoclast precursors to autonomously facilitate bone destruction.

Bacterial biofilms may explain chronicity in osteoradionecrosis of the temporal bone.

OBJECTIVE: To identify biofilm formation within a case of temporal bone osteoradionecrosis. PATIENT: Single-case patient presenting with temporal bone osteoradionecrosis. INTERVENTION(S): Antibiotic therapy and then surgical debridement of the temporal bone. MAIN OUTCOME MEASURE(S): Histologic identification of biofilm formation within the affected temporal bone specimen. RESULTS: Positive identification of biofilm formation in multiple sections of the temporal bone specimen removed from a patient affected by osteoradionecrosis. CONCLUSION: This is the first evidence that temporal bone osteoradionecrosis involves biofilm formation. Such a pathogenic mechanism may explain the recalcitrance of this disease process and offer new strategies in formulating therapeutic interventions.

Otopathogenic Pseudomonas aeruginosa strains as competent biofilm formers.

OBJECTIVE: To determine whether Pseudomonas aeruginosa, a common cholesteatoma pathogen, known to form biofilms in other chronic infections, is capable of contributing to biofilm formation in cholesteatoma. DESIGN: We tested 12 OPPA isolates for several aspects of biofilm formation, including adherence to human keratinocytes, expression of quorum-sensing genes, twitching motility, and production of extracellular matrix as determined by both crystal violet staining and carbazole reaction. RESULTS: Ten OPPA strains demonstrated increased adherence (1.5- to 12-fold) to human keratinocytes relative to PAO1, a laboratory strain. Expression of las and rhl quorum-sensing products were detected in 11 OPPA strains. By crystal violet staining, we found biofilm formation in all OPPA strains equal to or greater than that found in PAO1 (2- to 18-fold). In addition, OPPA strains demonstrated mucoid characteristics, including down-regulation of twitching motility and increased alginate production. CONCLUSIONS: Strains of OPPA isolated from cholesteatoma are strongly adherent to keratinocytes and capable of forming biofilm. In addition, OPPA strains have mucoid characteristics in vitro. When these bacteria assume a biofilm phenotype, they are highly resistant to antibiotics and host defenses. These data suggest that OPPA can contribute to biofilm formation in cholesteatoma, leading to the persistence of this infection.

Smoke/burn injury-induced respiratory failure elicits apoptosis in ovine lungs and cultured lung cells, ameliorated with arteriovenous CO2 removal.

STUDY OBJECTIVE: s: The purpose of this study was to examine the effects of two supportive therapies, conventional mechanical ventilation (CMV) and arteriovenous CO(2) removal (AVCO(2)R), during treatment of severe smoke/burn injury-induced ARDS. DESIGN: Sheep were exposed to a smoke/burn injury (lethal dose causing death in 40% of animals); lung tissue and blood was collected prior to injury (control), when an ARDS criterion was met (PaO(2)/fraction of inspired oxygen ratio < 200), then after 72 h of either CMV (group 1) or AVCO(2)R (group 2). Lung tissue was studied by standard histopathologic techniques; cultured lung cells were studied in media supplemented with serum from all four groups. MEASUREMENTS AND RESULTS: In vivo assays demonstrate less apoptotic cell death, and in vitro assays show significantly greater (p < 0.05) cell survival in group 2 (AVCO(2)R) than in group 1 (CMV) or baseline. Differential gene expression demonstrates significantly higher messenger RNA levels of proapoptotic and tumor necrosis factor (TNF)-alpha in cells incubated in baseline media. After exposure of cultured lung cells to conditioned media, protein expression assay of the culture medium revealed no TNF-alpha, TNF receptor (TNFR)-1, or TNFR-2, however, cultured cell lysate reveals elevated levels of TNF-alpha, TNFR-1 and caspase-3 in all groups; most occurred in cells incubated in baseline media (p < 0.05). HOECHST stain, DNA fragmentation, and caspase-3 cleavage show that AVCO(2)R ameliorates apoptosis in this model. CONCLUSIONS: This in vitro work specifically examines cell death in lung cells as a result of smoke/burn injury and effects of therapeutic interventions. Our in vivo studies temporally correlate the clinical pathology to that studied in these lung cells and show that both in vivo and in vitro cell death is predominantly apoptotic and is significantly reduced by AVCO(2)R.

Molecular characterization of mutations in patients with benign and aggressive recurrent respiratory papillomatosis: a preliminary study.

This study was performed to determine whether genetic polymorphisms within the human papillomavirus (HPV) can predict the disease course in patients with recurrent respiratory papillomatosis. The HPV type and genomic variations were determined by comparing the sample sequence to a prototypical HPV in the database of the National Center for Biotechnology Information. The results were correlated with the clinical course. Seven children and 6 adults were studied. Six of the 7 children had aggressive disease associated with HPV type 11. The remaining child had HPV type 6. Five of the 6 adult patients had HPV type 6; 1 had a history of juvenile recurrent respiratory papillomatosis. The remaining adult had an aggressive disease course associated with HPV type 11. The HPV type and specific polymorphisms were conserved over time in serial isolates. The age of onset and medical therapy did not appear to affect the polymorphisms present. Future studies may find that the presence of certain polymorphisms is associated with different geographic locations and possibly with the disease course.