Elucidation of molecular-biological dynamics in the onset of otitis media

1)儿童腺样体在中耳炎伴积液（OME）中的作用：为了探讨腺样体是否是OME的活性因子，我们比较了患有和未患有OME的儿童腺样体的宏观大小、细菌学检查、上皮网状形成、纤毛上皮百分比。腺样体大小差异无统计学意义。不可分型流感嗜血杆菌（NTHi）在OME患儿的腺样体标本中培养更为频繁。在OME患儿中，分层鳞状上皮明显增加，纤毛上皮明显减少。患有OME的儿童的网状上皮扩展比未患OME的儿童大。这些发现表明，腺样体炎症与OME的发病机制有关，并且腺样体作为NTHi的储存库在OME的发病中起重要作用。2)甲型流感病毒对鼻咽部细菌定植的影响：为了明确病毒感染在中耳炎中的作用，我们接种了流感病毒，观察了鼻咽部的组织学变化。接种病毒后，将活菌注入鼻咽，观察鼻咽细菌的清除情况。鼻内接种花生凝集素、琥珀酰小麦胚芽凝集素和单纯斑蝇凝集素对鼻咽部粘膜层和上皮细胞表面的染色效果较对照组显著增强。对照动物鼻咽部用马achia amurenis凝集素和小麦胚凝集素进行中度染色，接种病毒后染色增强。这些结果在病毒接种后第5天和第9天最为显著。在病毒接种后第5天注射细菌，培养出的细菌数量显著增加。这些结果表明，流感病毒引起的鼻咽粘膜糖结合物结构的改变可能与细菌清除率的降低有关。3) IL-1β在OME模型中的作用为了阐明IL-β在OME发病机制中的作用，我们建立并检测了小鼠OME模型。小鼠鼓室内注射内毒素。注射后3 d，观察中耳积液。接种重组IL-1β后观察到类似的病理变化。抗IL-1受体抗体抑制内毒素引起的病理改变。原位杂交显示IL-1β信使RNA在中耳黏膜上皮中表达。这些结果提示IL-1β可能与内毒素诱导的中耳炎症有关，并可能在内毒素诱导的中耳粘膜免疫中发挥重要作用。4)中耳粘膜免疫为了阐明中耳粘膜在免疫潜能方面的特点，我们对特定无病原体小鼠中耳粘膜淋巴细胞亚群、细胞因子mRNA和抗原特异性iga产生细胞的诱导进行了研究。流式细胞术分析显示CD3_+ T细胞中存在一定量的γδT细胞。P6联合霍乱毒素经鼻免疫诱导产生P6特异性iga细胞。粘膜T细胞RT-PCR检测Th2型细胞因子mRNA。这些发现支持了中耳是粘膜免疫的潜在影响部位这一事实。5)在中耳粘膜诱导对NTHi的特异性黏膜免疫反应为探讨研制抗NTHi鼻用疫苗的可能性，采用NTHi P6经鼻免疫小鼠，观察中耳粘膜免疫反应。耳液中P6特异性IgA滴度显著升高。中耳黏膜p6特异性iga生成细胞增多。P6体外刺激可使免疫小鼠的纯化CD4_+ T细胞增殖，这些T细胞表达Th2细胞因子mRNA。这些结果表明，鼻内免疫可诱导中耳黏膜产生p6特异性iga - b细胞免疫应答和表达Th2细胞因子的选择性Th2细胞。6)鼻内免疫清除中耳NTHi为评价P6鼻内免疫对NTHi诱导中耳炎的保护作用，采用P6鼻内免疫小鼠，免疫1周后将活的NTHi活悬液注入鼓室诱导实验性OM。免疫小鼠显示中耳对NTHi的清除增强。刺激后中耳TNF-α产生的刺激减少。这些结果表明鼻内免疫对实验性中耳炎具有保护作用。少

1) Role of child adenoids in otitis media with effusion (OME) :In order to investigate whether the adenoid is an active agent of OME, the adenoids of children with and without OME were compared regarding the macroscopic size, the bacteriological examination, reticular formation of the epithelium, and the percent of ciliated epithelium. There was no significant difference in the size of adenoids. Nontypeable H. influenzas (NTHi) was cultured more frequently in adenoid specimens from children with OME. A tendency toward increased stratified squamous epithelium and decreased ciliated epithelium was apparent in children with OME. Reticular epithelium extension was greater in children with than without OME. These findings suggested that adenoid inflammation is implicated in the pathogenesis of OME and the adenoids have an important role in the cause of OME by being a reservoir for NTHi.2) Effects of influenza A virus on nasopharyngeal bacterial colonization :To clarify the role of viral inf … More ection in otitis media, influenza virus was inoculated mice and examined histologic changes in the nasopharynx. Live bacteriae were 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 aggulutinin was significantly enhanced with intranasal virus inoculation when compared with that in control animals. The nasopharynx was moderately stained with Maachia amurensis aggulutinin 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 hi bacterial clearance.3) Role of IL-1β in a OME modelTo clarify the role of IL-β in the pathogenesis of OME, a murine OME model was developed and examined. Mice received intratympanic injections of endotoxin. Three days after injection, middle ear effusions were observed. Similar pathological changes were observed after inoculation with recombinat IL-1β. Anti- IL-1 receptor antibodies inhibited the pathological changes induced by the endotoxin. In situ hybridization showed expression of IL-1β messenger RNA in the epithelium of the middle ear mucosa. These results suggest that IL-1β might be associated with endotoxin-induced iflammation in the middle ear and might play an important role in the induction of OME.4) Mucosal immunity of the middle earIn order to clarify the characteristics of the middle ear mucosa with respect to immune potential, lymphocyte subsets, mRNA of cytokines, and induction of antigen-specific IgA-producing cells in the middle ear mucosa were investigated in specific pathogen-free mice. Flow cytometric analysis showed a certain amount of γδT cells among CD3_+ T cells. P6-specific IgA-producing cells were induced by intranasal immunization with P6 together with choleratoxin. RT-PCR assay of mucosal T cells detected mRNA of Th2 type cytokined. These findings support the fact that the middle ear is a potentially an effector site of the mucosal immunity.5) Induction of specific mucosal immune responses to NTHi in middle ear mucosaTo elucidate the possibility of developing a nasal vaccine against NTHi, mice were immunized intranasally with the P6 of NTHi and mucosal immune responses in the middle ear were examined. The P6 specific IgA titier in ear wash was significantly elevated. An increase in P6-specific IgA-producing cells in middle ear mucosa was detected. 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 indicated that P6-specific IgA-B-cell immune responses and selected Th2 cytokine expressing Th cells were induced in middle ear mucosa by intranasal immunization.6) Clearance of NTHi from the middle ear by intranasal immunizationTo assess the effect of intranasal immunization with P6 for the protection against NTHi-induced otitis media, mice were immunized intranasally with the P6 and one week after the immunization a suspension of live of live NTHi was injected into the tympanic cavity to induce experimental OM. Immunized mice showed enhanced clearance of NTHi from the middle ear. Less stimulation of TNF-α production in the middle ear was shown after challenge. These results indicated that intranasal immunization affords protection against experimental otitis media. Less