Thomas F. DeMaria

Experimental Otitis Media with Effusion following Middle Ear Inoculation of Nonviable H Influenzae

In order to test the hypothesis that nonviable bacteria can induce middle ear inflammation leading to persistent middle ear effusion (MEE), we conducted an animal experiment using formalin-killed Hemophilus influenzae, the bacterium reported to be the most common pathogen isolated from chronic MEEs. Over 70% of the chinchillas injected with formalin-killed H influenzae type b or a nontypeable isolate developed sterile, straw-colored serous MEEs, and exhibited histological evidence of extensive inflammatory changes of the middle ear mucosal connective tissue and epithelium. Control animals injected with pyrogen-free sterile saline did not exhibit any inflammatory changes or effusions in the middle ears. Our data suggest that endotoxin on the surface of H influenzae, a gram-negative bacterium, may be responsible for the induction of the otitis media with effusion. It is suggested that endotoxin (even when the organisms are no longer viable) may be responsible for the production of serous MEE and inflammatory changes in the middle ear.

Effect of Neuraminidase on Receptor-mediated Adherence of Streptococcus pneumoniae to Chinchilla Tracheal Epithelium

The trachea whole organ perfusion technique was used to study the effect of the disruption of the Streptococcus pneumoniae neuraminidase nanA gene on bacterial adherence and alteration of the carbohydrate surface structures of respiratory epithelial cells. Six different lectin probes were used to examine alterations of the cell surface carbohydrates in chinchilla tracheal epithelium incubated in vitro with S. pneumoniae j NA1, a neuraminidase-deficient mutant, or its D39 parent strain. The labeling pattern revealed that the binding of wheat germ agglutinin (WGA), Erythrina cristagalli lectin (ECL), peanut agglutinin (PNA), Bandeiraea simplicifolia lectin II (BSL II) and succinylated WGA was significantly increased in the luminal surface of the trachea in the D39-incubated cohort compared with the uninfected control, which indicated that GlcNAc and D-galactose residues were exposed. Concurrently, decreased labeling with Sambucus nigra agglutinin (SNA) indicated that there were few sialic acid residues remaining in the tracheal epithelium subsequent to incubation with D39. The j NA1 neuraminidase-deficient mutant, however, did not induce any significant changes in the lectin labeling patterns, which were comparable to those of the control cohort. Moreover, adherence data expressed as colony-forming units (CFU) of S. pneumoniae per millimeter of trachea indicated a significant decline in the ability of j NA1 to adhere in vitro . We propose that products of the nanA gene have a significant impact on changes in the carbohydrate moieties in the tracheal epithelium, and may be responsible for the previously reported increased ability of the D39 parent to colonize the nasopharynx and invade the middle ear.

Endotoxin Permeability through the Round Window

The permeability of the round window membrane to Salmonella typhimurium derived endotoxin was examined using a total of 17 chinchillas. One mg of endotoxin was instilled into the tympanic cavity via the superior bulla. Endotoxin activity in middle ear effusions (MEEs), perilymph (both inoculated and non-inoculated side), and sera was determined by Limulus lysate assay after 12, 24, 48, 72, and 120 h following endotoxin instillation. Endotoxin was detected in perilymph on the inoculated side by 12 h after endotoxin instillation and persisted for 5 days during the present measurement period. Endotoxin level peaked at 24-48 h post-instillation, and steadily declined afterwards. This result suggests that the maximum penetration occurred during the active inflammatory stage. Histologic investigation revealed marked pathological changes in the inner ear, including bleeding and inflammatory cell recruitment, mostly in the perilymphatic spaces (e.g. scalae tympani and vestibuli, spiral ligament), strial swelling, and sensory cell degeneration. These results suggest that endotoxin, when introduced into the middle ear, can permeate through the round window membrane and can cause inner ear tissue damage in this animal model.

Effect of Adenovirus Type 1 and Influenza a Virus on Streptococcus Pneumoniae Nasopharyngeal Colonization and Otitis Media in the Chinchilla

Considerable evidence has implicated respiratory tract virus potentiation of bacterial adherence, colonization, and superinfection as a significant factor contributing to the pathogenesis of otitis media (OM). Influenza A and B viruses, adenovirus, and respiratory syncytial virus are the primary respiratory tract viruses associated with this disease. Investigations have established a dramatic increase in the development of experimental OM in chinchillas co-inoculated with influenza A virus and Streptococcus pneumoniae (Spn). The mechanism underlying this phenomenon was suggested to involve, in part, viral compromise of eustachian tube mucosal integrity and function. This study was designed to assess and compare the effect of adenovirus and influenza A virus infection on adherence, the kinetics of colonization, and invasion of the middle ear by Spn in the chinchilla model of OM. Cohorts were inoculated intranasally with adenovirus type 1 or influenza A virus, and then inoculated intranasally 7 days later with Spn 6A. All cohorts were observed over a 14-day period after challenge with Spn, and the incidence and severity of OM were assessed by several methods, including culture of the nasopharynx and middle ear effusions. The data indicated that influenza A virus promotes a significant increase in nasopharyngeal colonization by Spn, an increased incidence and severity of OM, and a sustained presence of Spn in the effusions. Adenovirus infection, however, did not enhance colonization by Spn or result in an increased incidence or severity of OM.

Cytological and Histological Changes in the Middle Ear after Inoculation of Influenza a Virus

Experimental otitis media induced in the chinchilla by inoculation of influenza A virus into the middle ear resulted in capillary engorgement, subepithelial hemorrhage, tissue edema and acute inflammatory cell infiltration. Quantitative morphometric measurements were made for 28 days. Ciliated cells appeared to be the primary target of this strain of influenza virus and demonstrated the greatest degree of damage. Three weeks were required to restore the ciliated epithelium in the tubotympanum to normal levels.

The effect of antecedent influenza A virus infection on the adherence of Hemophilus influenzae to chinchilla tracheal epithelium

The adherence of Hemophilus influenzae (type b and nontypable) to ciliated chinchilla respiratory epithelium was investigated using a whole organ perfusion technique. Nontypable H influenzae (NTHi) were shown to be more adherent than type b to these organized and differentiated tracheal organ cultures. Bacteria were found adhering to ciliated cells. Antecedent influenza A virus infection had no effect on adherence of NTHi for at least 48 hours. However, 72 hours after exposure to the virus, infected tissues demonstrated significantly fewer adherent bacteria than did controls. To summarize, influenza A virus infection was not found to augment the initiation of NTHi adherence to ciliated respiratory epithelium in this model.

Changes in the structure of the cell surface carbohydrates of the chinchilla tubotympanum following Streptococcus pneumoniae-induced otitis media☆

Streptococcus pneumoniae (Spn) are among the most frequently isolated pathogens in acute otitis media (AOM) and in otitis media with effusion (OME). Recently, the specific receptor for Spn has been identified as the trisaccharide unit Gal beta 1-4 GlcNAc beta 1-3 Gal beta with GlcNAc beta 1-3 Gal beta as the principal binding site. During the colonization of mucosal surfaces, pneumococci produce a variety of enzymes. This study was conducted to identify any resulting changes in the cell surface carbohydrate structure due to the action of these enzymes during pneumococcal otitis media (OM) in chinchillas. Using a lectin histochemical method with seven different lectins (SNA, LFA, WGA, Succ WGA, BSL II, PNA, ECL), the labeling pattern revealed not only the removal of the terminal sialic acid, but also the exposure of N-acetyl-glucosamine. These results suggested that Spn-produced enzymes uncover part of their own receptor structure and thus may facilitate adherence and subsequent infection.

Relationship of endotoxin to tumor necrosis factor-α and interleukin-1β in children with otitis media with effusion

Sixty-five middle ear effusions and paired sera from 41 children with chronic otitis media with effusion were assayed for endotoxin and for tumor necrosis factor–α (TNF-α) and interleukin-1β (IL-1β) in order to establish whether a correlation exists between the concentrations of endotoxin and of these cytokines. Endotoxin concentration was determined by means of a chromogenic limulus amebocyte lysate assay, and the cytokine concentration by means of a quantitative enzyme-linked immunosorbent assay. Forty percent of the effusions had detectable levels of endotoxin, with a mean concentration of 2.9 ± 7.8 endotoxin units per milligram of total protein. The mean concentration of TNF-α was 1.24 ± 3.1 pg/mg total protein, and that of IL-1β was 18.79 pg/mg total protein. A strong, statistically significant correlation exists between the concentrations of endotoxin and TNF-α (r =.89) and IL-1β (r =.72). The data indicate that endotoxin may contribute to the pathogenesis of chronic otitis media with effusion by stimulating the sustained production of TNF-α and IL-1β in the middle ear.

Experimental otitis media with effusion induced by nonviable hemophilus influenzae: cytologic and histologic study

In an earlier study the authors demonstrated that formalin-killed Hemophilus influenzae induces serous-type middle ear effusion in chinchillas and provides an excellent model for the study of human otitis media with effusion. The present study was initiated to evaluate the morphologic and histologic changes that occur in the middle ear after injection of this organism. All of the experimental animals injected with formalin-killed H. influenzae in the present study had straw-colored serous-type effusions within four days after injection. The submucosal thickness, mononuclear cell density, and capillary permeability all increased dramatically in the experimental animals. Marked bleeding, tissue edema, and cellular infiltration in the submucosa were prominent findings after injection of the inactivated bacteria. Half of the experimental animals had histologic evidence of marked proliferation of epithelial cells resembling adhesive otitis media. These findings suggest that nonviable H. influenzae are capable of inducing severe inflammatory changes in the middle ear and may play an important role in the pathogenesis of otitis media with effusion and its sequelae.

Quantitative cytologic and histologic changes in the middle ear after the injection of nontypable Hemophilus influenzae endotoxin.

This study was undertaken to investigate and quantify the morphologic changes in the middle ear mucosa and connective tissue after the inoculation of graded doses (0.001 to 100 micrograms) of endotoxin prepared from Hemophilus influenzae. Histopathologic changes were observed in the middle ear mucosa in all animals. Marked bleeding and new bone formation in the submucosa were prominent at days 4 through 14. These findings indicate that the endotoxin from nontypable H influenzae is capable of inducing inflammation or pathologic changes in the middle ear mucosa and may play an important role in the pathogenesis of otitis media with effusion and its sequelae.