Takashi Hirano

Significance of spiral ligament fibrocytes with cochlear inflammation

It has been recently suggested that the spiral ligament fibrocytes, which interconnect with the basal cells of the stria vascularis via gap junctions, may be critical in maintaining cochlear homeostasis. In animal models of pathological conditions such as labyrinthitis and otitis media, reduced immunostaining for gap junction protein connexin 26 is observed in the spiral ligament. This suggests that disruption of the spiral ligament fibrocytes could be among the causes of cochlear dysfunction due to cochlear inflammation. Cultured spiral ligament fibrocytes have been shown to secrete chemokines and other mediators after stimulation of proinflammatory cytokine TNF-alpha or IL-1beta. Each of these mediators might induce inflammatory cell movement, which would prolong the inflammatory response. It is reasonable that such enhanced biological defense ability could be the cause of spiral ligament fibrocyte damage.

Intranasal Immunization Enhances Clearance of Nontypeable Haemophilus influenzae and Reduces Stimulation of Tumor Necrosis Factor Alpha Production in the Murine Model of Otitis Media

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media (OM). One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is considered a candidate for mucosal vaccine. We have previously reported that intranasal immunization with P6 and cholera toxin (CT) could induce P6-specific immunoglobulin A (IgA) antibodies in the middle ear. In the present study, we assessed the effect of intranasal immunization for the protection against NTHi-induced OM. Mice were immunized intranasally with P6 and CT as an adjuvant on days 0, 7, and 14. Control mice were given phosphate-buffered saline (PBS) without antigen. One week after the final immunization, a suspension of live NTHi (107 CFU) was injected into the tympanic cavity to induce experimental OM. On days 3 and 7 after bacterial challenge, mice were killed and middle ear effusions (MEEs) were collected. All immunized mice showed elevated titers of P6-specific antibodies in MEEs. The rank order of specific antibody included, from highest to lowest levels, IgG, IgA, and IgM. In addition, immunized mice showed enhanced clearance of NTHi from the middle ear and the number of NTHi in MEEs of immunized mice was reduced by 97% on day 3 and by 92% on day 7 after bacterial challenge relative the number in the MEEs of control mice. The protective effect of intranasal immunization on the incidence of NTHi-induced experimental OM was evident on day 7 after challenge. By day 7, the number of MEEs in immunized mice was 64% less than that in control mice and the incidence of NTHi culture-positive MEEs in immunized mice was 56% less than that in control mice. Less stimulation of tumor necrosis factor alpha (TNF-α) production in the middle ear was evident on day 3 after challenge. Immunized mice showed lower concentrations of TNF-α in MEEs. These results indicate that intranasal immunization affords protection against experimental OM as evidenced by enhanced clearance of NTHi and less stimulation of TNF-α production in the middle ear. These findings suggest that a nasal vaccine might be useful for preventing OM.

Induction of specific immunoglobulin A and Th2 immune responses to P6 outer membrane protein of nontypeable Haemophilus influenzae in middle ear mucosa by intranasal immunization

ABSTRACT Nontypeable Haemophilus influenzae (NTHI) is a major pathogen of otitis media. One of the outer membrane proteins of NTHI, P6, is an antigen common to all strains and is considered as a candidate for mucosal vaccine. To elucidate the possibility of developing a nasal vaccine against nontypeable Haemophilus influenzae (NTHI) and to investigate mucosal immune responses in the middle ear, mice were immunized intranasally with the P6 outer membrane protein of NTHI, and P6-specific immune responses in the middle ear mucosa were examined. Mice were given with P6 and cholera toxin intranasally as an adjuvant on days 0, 7, and 14 and were killed on day 21. The P6-specific immunoglobulin A (IgA) antibody titer in ear wash was significantly elevated. Mononuclear cells were isolated from middle ear mucosa, and an increase in P6-specific IgA-producing cells was shown with an enzyme-linked immunospot assay. In addition, an increase in memory T cells in middle ear mucosa was detected with flow cytometric analysis after intranasal immunization. Moreover, in vitro stimulation with P6 resulted in proliferation of purified CD4+ T cells from immunized mice, and these T cells expressed Th2 cytokine mRNA. These results indicate that P6-specific IgA–B-cell immune responses and selected Th2 cytokine expressing Th cells were induced in middle ear mucosa by intranasal immunization. These findings suggest that a nasal vaccine is useful for preventing otitis media with effusion.

The influence of pneumococcal otitis media on the cochlear lateral wall.

The cochlear influence of otitis media was investigated in order to identify damaged regions causing cochlear malfunction. BALB/c mice were challenged with viable Streptococcus pneumoniae into the middle ear cavity and were killed 1 day to 1 month later for immunohistochemical analysis. Otitis media was induced in all of the animals, and some showed inflammatory cells in the cochlea. Although other changes were not obvious by hematoxylin and eosin staining, immunohistochemistry showed the presence of fibrinogen in the cochlea, mainly in the lower portion of the spiral ligament and in the spiral limbus. Immunostaining for connexin 26 was decreased in the spiral ligament, accompanied by marked fibrinogen staining. Immunostaining for sodium-potassium-adenosine triphosphatase in the stria vascularis and in the type II fibrocytes of the spiral ligament was not affected obviously. The presence of fibrinogen in the cochlea suggests disruption of the blood-labyrinth barrier caused by the middle ear inflammation. Changes in connexin 26 staining suggest the possibility that the spiral ligament could be among the regions responsible for the cochlear malfunction.

Effects of influenza A virus on lectin-binding patterns in murine nasopharyngeal mucosa and on bacterial colonization

To clarify the role of viral infection in otitis media, we intranasally inoculated mice with influenza A virus and examined histologic changes in the nasopharyngeal mucosa using a battery of lectins. Additionally, live Haemophilus influenzae o r Streptococcus pneumoniae was injected into the nasopharynx after virus inoculation, and the clearance of bacteria from the nasopharynx was examined. Staining of the mucous blanket and epithelial cell surfaces in the nasopharynx with peanut agglutinin, succinyl wheat-germ agglutinin, and Bandeiraea simplicifolia agglutinin was significantly enhanced with intranasal virus inoculation when compared with that in control animals. The nasopharynx was moderately stained with Maachia amurensis agglutinin and wheat-germ agglutinin in control animals, and the staining was enhanced after virus inoculation. These findings were most remarkable 5 and 9 days after virus inoculation. The numbers of bacteria cultured from the nasopharynx were significantly increased when bacteria were injected 5 days after virus inoculation. These results suggest that an alteration in the glycoconjugate structure lining the nasopharyngeal mucosa caused by the influenza virus might be associated with the reduction in bacterial clearance. (Otolaryngol Head Neck Surgery 1999;121:616–21.)

Changes in Immunostaining of Inner Ears After Antigen Challenge Into the Scala Tympani

To study the mechanisms of immune responses and immune injuries in inner ears, labyrinthitis was induced by inoculation of keyhole limpet hemocyanin (KLH) into the scala tympani of systemically sensitized guinea pigs. Inner ears were then immunostained for KLH, immunoglobulin G (IgG), albumin, connexin26 (Cx26), and sodium‐potassium adenosine triphosphate (Na,K‐ATPase). Inflammatory cells containing KLH were observed in the scala tympani and in the collecting venule of the spiral modiolar vein (SMV). Spiral ligament, spiral limbus, and blood vessels including the SMV were diffusely positive for IgG and albumin. Immunoreactivity for Cx26 and Na,K‐ATPase was decreased compared with the normal ears in the fibrocytes of the spiral ligament. These results suggest that inflammatory cells and blood constituents could extravasate into the cochlea from blood vessels and that fibrocyte damage in the spiral ligament could cause cochlear dysfunction.

A new intra-NALT route elicits mucosal and systemic immunity against Moraxella catarrhalis in a mouse challenge model

Mucosally administered antigens are often poorly immunogenic due to the difficulty of transporting antigens through the mucosal epithelium. We investigated a new route of intranasal-associated lymphoid tissue (intra-NALT) administration of antigens to circumvent the antigen transportation barrier. A comparative study was carried out on mice administered with killed whole cells of Moraxella catarrhalis strain 25238 plus cholera toxin (CT) by intra-NALT injection and nasal inoculation. Both routes induced significant elevations of several isotype antibodies against strain 25238 in saliva, lung lavage, and serum as measured by an enzyme-linked immunosorbent assay (ELISA). Most of these antibodies were paralleled by the numbers of their corresponding antibody forming cells in mucosal or systemic lymphoid tissues. However, intra-NALT injection elicited higher levels of immunoglobulin (Ig) A and IgG in saliva, IgA and IgG in lung lavage, and IgG and IgM in sera than nasal inoculation (P<or=0.05). In addition, both routes generated significant reductions of bacteria in lungs following an aerosol challenge with strain 25238 in a mouse model of pulmonary clearance. Once again, intra-NALT route showed better bacterial clearance in mouse lungs than nasal inoculation (P<0.01). These results demonstrate that intra-NALT administration of antigens is a convenient and effective route for mucosal immunization that elicits improved mucosal and systemic immunity. This new route can be used as a model to study mucosal antigens or vaccine candidates for antigen activation and interaction with the NALT that is one of major inductive sites for common mucosal immune system.

Nasal vaccination with P6 outer membrane protein and α-galactosylceramide induces nontypeable Haemophilus influenzae-specific protective immunity associated with NKT cell activation and dendritic cell expansion in nasopharynx

The efficacy of alpha-galactosylceramide (alpha-GalCer) as a mucosal adjuvant was examined. Mice were immunized intranasally with nontypeable Haemophilus influenzae (NTHi) P6 protein and alpha-GalCer. P6-specific antibody responses in the form of P6-specific IgA in nasal washes and serum IgG titers were significantly elevated. Splenic CD4(+) T cells expressed P6-specific Th1 and Th2 cytokine mRNA. In addition, NTHi was quantified in nasal washes following NTHi challenges, and the clearance of NTHi from the nasopharynx was also enhanced. These results indicate that alpha-GalCer might be an effective mucosal adjuvant.

Nasal Vaccination With CpG Oligodeoxynucleotide Induces Protective Immunity Against Nontypeable Haemophilus influenzae in the Nasopharynx

Objectives: Nasal vaccination is an effective therapeutic regimen for preventing otitis media. Since cholera toxin (CT) is toxic, an alternative adjuvant is required for the development of a nasal vaccine. The efficacy of CpG oligodeoxynucleotide (ODN) as a mucosal adjuvant was examined.

Role of interleukin-1β in a murine model of otitis media with effusion

To clarify the role of interleukin-lβ (IL-lβ) in the pathogenesis of otitis media with effusion (OME), we developed and investigated a murine model of this disease. Specific pathogen—free male BALB/c mice received intratympanic injections of 20 μg of endotoxin derived from nontypeable Haemophilus influenzae. Three days after injection, middle ear effusions were observed through the eardrum. Similar pathological changes were observed after inoculation with 100 ng of recombinant IL-lβ. Anti—IL-1 receptor antibodies inhibited the pathological changes induced by the endotoxin. In situ hybridization showed expression of IL-lβ messenger RNA in the epithelium of the middle ear mucosa. These results suggest that IL-lβ might be associated with endotoxin-induced inflammation in the middle ear and might play an important role in the induction of otitis media with effusion.