T-cell-derived cytokines, including IFN-, can induce secretory component in epithelial cells and major histocompatibility complex class II expression on antigen-presenting cells (1, 15). detected with circulation cytometric analysis after intranasal immunization. Moreover, in vitro activation 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 IgACB-cell immune responses and selected Th2 cytokine expressing Th cells were induced in middle ear mucosa by intranasal immunization. Pseudoginsenoside-F11 These findings suggest that a nasal vaccine is useful for preventing otitis media with effusion. Nontypeable (NTHI) is usually Pseudoginsenoside-F11 a major pathogen of otitis media with effusion (OME) and other upper respiratory tract diseases (10, 30). In patients with OME, this bacterium is frequently isolated from your nasopharynx, as well as from middle ear effusions, and the inhibition of NTHI colonization in the upper respiratory tract is considered effective in preventing OME. Due to Pseudoginsenoside-F11 the increase of antibiotic-resistant strains of NTHI in recent years, the development of a vaccine against this bacterium is considered an important goal for public health. Since NTHI lacks capsular antigens, Pseudoginsenoside-F11 the chief antigenicity is present in the outer membrane proteins (OMPs). One of the OMPs of NTHI, P6, is usually a common antigen to all strains and is considered as a candidate for mucosal vaccine (7, 9, 10, 11, 30, 31). In the mucosal surface, secretory immunoglobulin A (IgA) plays a major role in protective immunity. We previously exhibited that intranasal immunization was an effective regimen for inducing mucosal IgA immune responses in the upper respiratory tract (26) and that the nasal mucosal IgA immune responses induced by intranasal immunization were effective for the clearance of bacteria in the nasopharynx. The mucosal Pseudoginsenoside-F11 immune system is considered as a separate functional entity quite independent of the systemic immune system because the mucosal immune system possesses unique anatomic features and is composed of specialized subsets of lymphoid cells (21, 27, 34). Despite the recent emphasis on a better understanding of molecular and cellular aspects of the mucosal immune system, little information is currently available regarding the middle ear mucosa (MEM). Several histologic studies have indicated that this MEM has a function as a mucosal effector site, as does the nasal mucosa (19, 29, 36, 37, 41); however, immune responses by mucosal lymphocytes in the middle ear have not been studied because of the difficulty in isolating cells from your MEM. Recently, we established a method for isolating lymphocytes from your MEM and analyzed mucosal lymphocytes at the single cell level in the middle ear of normal mice. Results of Fgfr2 that study showed that MEM has characteristics of a mucosal effector site (S. Suenaga, S. Kodama, S. Veyama, M. Suzuki, and G. Mogi, submitted for publication). Several studies concerning the prevention of OME by mucosal immunization have been reported, and these reports have suggested that intranasal immunization with P6 is effective for the prevention of OME (7, 9, 12, 18, 35). However, studies with mice have not investigated immune responses in the middle ear (18), and studies using chinchilla models have not analyzed immunological aspects (3, 12, 35). In the present study, we investigated antigen-specific immune responses in the middle ear by intranasal immunization for the ultimate purpose of developing a mucosal vaccine for preventing OME. P6-specific T- and B-cell immune responses in the MEM were examined at the cellular and transcriptional levels. MATERIALS AND METHODS Animals. BALB/c mice were purchased from Charles River Japan (Atsugi, Japan). The mice were managed under specific-pathogen-free conditions. Small adult mice between.