Tumor necrosis factor during experimental lipopolysaccharide-induced otitis media.

Studies in our laboratory and others have indicated that endotoxin is present in a high percentage of human middle ear effusions, including those that are culture negative. Endotoxin has been identified as one of the most potent inducers of tumor necrosis factor (TNF), but this relationship has not been investigated in regard to the pathogenesis of otitis media. The purpose of this study was to determine whether endotoxin induces the production of TNF in the middle ear. Otitis media was induced in chinchillas by the injection of isolated lipopolysaccharide and middle ear fluids (MEFs) and serum harvested and assayed for TNF content by means of a cytolytic assay using L-929 cells and the Cell Titer 96 assay (Promega, Madison, WI). The MEF pools' TNF concentration ranged from 200 to 1,100 pg/mL MEF. Serum pools did not contain any detectable TNF. The results of the present study suggest that endotoxin induces TNF production locally in the middle ear cleft, which may contribute to the pathogenesis of otitis media via any number of established inflammatory pathways.

Immunization with outer membrane protein P6 from nontypeable Haemophilus influenzae induces bactericidal antibody and affords protection in the chinchilla model of otitis media.

The role of nontypeable Haemophilus influenzae (NTHi) outer membrane protein (OMP) P6 in the pathogenesis of otitis media (OM) has not been defined. OMPs, fimbriae, pili, and lipooligosaccharide are several types of surface antigens of NTHi that are currently being evaluated as potential vaccine candidates. P6 is antigenically conserved among both nontypeable and type b H. influenzae strains and elicits bactericidal as well as protective antibodies; however, initial evaluation of a vaccine mixture of P6 combined with other NTHi OMPs failed to induce bactericidal antibody or protection in the chinchilla model of OM. We undertook an assessment of the ability of immunization with isolated P6 lipoprotein alone to confer protection. Chinchillas were immunized with P6 and challenged 10 days after the final immunization with either 3 x 10(3) CFU of NTHi delivered directly into the middle ear to induce OM or 5 x 10(8) CFU of NTHi delivered intranasally to establish nasopharyngeal colonization. All immunized animals responded with elevated serum titers of anti-P6 antibody, which also demonstrated bactericidal activity against homologous as well as a heterologous NTHi isolate. By 14 days post-transbullar challenge, the number of chinchillas with middle ear fluid and the incidence of NTHi culture-positive middle ear fluids were reduced 48 and 51%, respectively, in the P6-immunized chinchillas relative to the sham-immunized cohort. Nasopharyngeal colonization levels were comparable in the two cohorts. These data demonstrate that active immunization with P6 results in the production of NTHi-specific bactericidal antibody in the chinchilla and also affords a reduction in the incidence of NTHi-induced OM; however, parenteral immunization does not appear to affect the extent or duration of nasopharyngeal colonization by NTHi.

Adenovirus serotype 1 does not act synergistically with Moraxella (Branhamella) catarrhalis to induce otitis media in the chinchilla.

A chinchilla model of otitis media in which adenovirus compromise of the tubotympanum facilitates the subsequent induction of middle ear disease was used to investigate Moraxella (Branhamella) catarrhalis pathogenesis. Intranasally inoculated M. catarrhalis did readily colonize the nasopharynx of this host; however, despite evidence of viral infection and tubotympanal compromise, M. catarrhalis did not induce culture-positive otitis media in this model.

Role of fimbriae expressed by nontypeable Haemophilus influenzae in pathogenesis of and protection against otitis media and relatedness of the fimbrin subunit to outer membrane protein A.

Nontypeable Haemophilus influenzae is a primary pathogen in both acute otitis media (OM) and chronic OM, yet the pathogenesis of this disease is not fully understood. Although fimbriae have been observed on all clinical OM isolates examined to date, their role in pathogenesis remains unclear. Therefore, the gene which codes for the fimbrial subunit protein (fimbrin) in nontypeable H. influenzae 1128 was isolated, cloned, and sequenced. The nucleotide sequence of the fimbrin gene was found to contain an open reading frame of 1,077 bp which would encode a mature fimbrin protein consisting of 338 amino acid with a calculated molecular mass of 36.4 kDa. The translated amino acid sequence was found to be homologous with various OmpA proteins of other gram-negative bacteria, and algorithmic analysis predicted that this protein is organized as a coiled coil. To directly test whether fimbriae are involved in pathogenesis, the fimbrin gene was disrupted, and the biological consequences of disruption were absence of both expression of the fimbrial appendage and the specific immunogold labeling thereof with antisera directed against isolated fimbrial protein, reduced adherence to human oropharyngeal cells in vitro, augmented clearance from the tympanum post-transbullar inoculation, and significantly reduced induction of OM post-intranasal inoculation in a chinchilla model compared with the fimbriated parent strain. We additionally find that either passive immunization or active immunization against isolated fimbrial protein confers partial protection against transbullar challenge. A Western blot (immunoblot) indicated a degree of serological relatedness among fimbrin proteins of 15 nontypeable and type b isolates. These data suggest that fimbrin could be useful as a component of a vaccine to protect against OM.