Katsuhiro Toyama

Expression of Mucins in Mucoid Otitis Media

A hallmark of mucoid otitis media (MOM, i.e., chronic otitis media with mucoid effusion) is mucus accumulation in the middle ear cavity, a condition that impairs transduction of sounds in the ear and causes hearing loss. The mucin identities of mucus and the underlying mechanism for the production of mucins in MOM are poorly understood. In this study, we demonstrated that the MUC5B and MUC4 were major mucins in MOM that formed distinct treelike polymers (mucus strands). The MUC5B and MUC4 mRNAs in the middle ear mucosa with MOM were up regulated 5-fold and 6-fold, compared with the controls. This upregulation was accompanied by the extensive proliferation of the MUC5B- and MUC4-producing cells in the middle ear epithelium. Further study indicated that the mucin hyperproduction was significantly linked to CD4+ and CD8+ T cells and/or CD68+ monocyte macrophages. It suggests that MUC5B and MUC4 expression may be regulated by the products of these cells.

Pneumococcal Peptidoglycan-Polysaccharides Regulate Toll-Like Receptor 2 in the Mouse Middle Ear Epithelial Cells

Toll-like receptor 2 (TLR2) plays a key role in the host defense against Gram staining positive (Gram+) bacteria and their cell wall envelope components. However, little is known about the expression of TLR2 in the middle ear under otitis media (OM) conditions, and its role in the persistent otitis media with effusion (OME). In this study, we demonstrated that the pneumococcal cell wall component, peptidoglycan-polysaccharides (PGPS), activated the expression of TLR2 in the middle ear epithelial cells through the nuclear factor kappa B (NF-κB)-cytokine signaling pathway while I kappa B alpha mutant (IκBαM), a dominant negative inhibitor of NF-κB, abrogated the expression of TLR2 induced by PGPS. This study suggests that the existence of residual PGPS may maintain a low profile of cytokine production in the middle ear mucosa and thus contribute to the pathogenesis of OME.

Increase in glutamate-aspartate transporter (GLAST) mRNA during kanamycin-induced cochlear insult in rats

Kanamycin (KM)-induced changes in expression of the gene for glutamate-aspartate transporter (GLAST) in the rat cochlea were analyzed by Northern blotting. With the administration of KM (600 mg/kg/day) once daily for 20 days, the expression of GLAST mRNA gradually increased and reached a peak on day 20. Although the expression of GLAST mRNA remained at a high level until 12 days after the completion of the KM treatment, it then fell to the normal level within 2 months. Such KM treatment resulted in loss of both inner and outer hair cells and a concomitant profound permanent threshold shift. The present findings suggest that during KM administration, high concentrations of extracellular glutamate released by collapsing hair cells induced GLAST mRNA expression. Increased GLAST mRNA might play an important role in the prevention of the secondary death of spiral ganglion neurons from glutamate neurotoxicity.

Expression of the integrin genes in the developing cochlea of rats.

Integrins play an important role in the development of the cochlea. However, little is known about the expression pattern of integrins in the developing cochlear tissue. In this study, we investigated the dynamic expression profile of the integrin genes in the developing cochlear tissue of rats by Affymetrix microarrays and explored the role of the integrin genes in vitro by using antisense oligonucleotides. It was demonstrated that the alpha1, alpha7, alphav, beta3, and beta4 genes were expressed in the developing cochlear tissue of rats. Inhibition of the integrin expression with antisense oligonucleotides against alphav, alpha7, beta3, and beta4, respectively, in cochlear sensorineural epithelial cells significantly decreased the [3H]thymidine incorporation, suggesting that these integrins are involved in cell growth and proliferation. Inhibition of the alphav and beta4 integrins significantly decreased the transcription of nuclear factor-kappa B (NF-kappaB, a signal molecule involved in cell growth and proliferation) induced by epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), respectively. It suggests that EGF-induced cell growth is dependent upon the alphav integrin whereas bFGF-induced cell growth is dependent upon the beta4 integrin in the cochlear tissue during the development of the inner ear.

Pneumococcal Peptidoglycan-Polysaccharides Induce the Expression of Interleukin-8 in Airway Epithelial Cells by way of Nuclear Factor-κB, Nuclear Factor Interleukin-6, or Activation Protein-1 Dependent Mechanisms

Cell envelope compounds of bacteria trigger immune and inflammatory reactions by way of chemokines/cytokines. In this study, we demonstrated that pneumococcal peptidoglycan‐polysaccharides (PGPS) induced the production of interleukin (IL)‐8 by way of nuclear factor (NF)‐κB, nuclear factor interleukin (NF‐IL)6, and activation protein (AP)‐1 dependent mechanisms in the human bronchial epithelial cells (NL‐20) in a dose‐ and time‐dependent manner in vitro, and the mutation of either the NF‐κB, NF‐IL6, or AP‐1 binding sites in the promoter of IL‐8 abrogated the IL‐8 transcriptional activity. In a similar way, lipopolysaccharides induced the promoter activation of IL‐8 in NL‐20. However, the PGPS‐induced IL‐8 promoter activation in rodent middle ear epithelial cells required NF‐κB and NF‐IL6 but not AP‐1.

Temperature-Sensitive Sv40-Immortalized Rat Middle Ear Epithelial Cells

The proliferation and differentiation of middle ear epithelial cells are essential in both normal and diseased middle ears. The normal situation involves physiologic growth and renewal of the epithelium, and the diseased situation involves pathological changes of the epithelium such as mucous cell metaplasia and ciliated cell proliferation in otitis media. In this study, we used a temperature-sensitive large T antigen (the SV40 mutant) to transduce and immortalize the primary culture of middle ear epithelial cells. SV40-immortalized middle ear epithelial cells have been cultured for more than 50 passages and are stable morphologically. Their nonimmortalized parent cells died at the second passage. Immortalized middle ear epithelial cells carrying the S V40 mutant show a monolayer, cobblestonelike morphology. The cell line expresses characteristic middle ear mucosal molecules such as mucins, keratins, and collagens. It also responds to temperature changes; namely, cells proliferate at 33°C, when the SV40 antigen is active, and differentiate at 39°C, when the SV40 antigen is inactive. Therefore, we conclude that a temperature-sensitive middle ear epithelial cell line has successfully been established.