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

1)儿童腺样体在分泌性中耳炎（OME）中的作用：为了研究腺样体是否是OME的活性因子，比较了有和无OME的儿童腺样体的大体大小、细菌学检查、上皮的网状结构和纤毛上皮的百分比。腺样体大小无明显差异。不可分型的H.流感病毒（NTHi）在OME患儿的腺样体标本中培养更频繁。OME患儿的复层鳞状上皮增加，纤毛上皮减少的趋势明显。网状上皮的扩展是更大的儿童OME比没有。这些结果表明，腺样体炎症与OME的发病机制有关，腺样体通过作为NTHi的储存库在OME的病因中起重要作用。2）流感病毒对鼻咽细菌定植的影响：为了阐明病毒感染在OME发病中的作用， ...更多信息 在中耳炎中，将流感病毒接种小鼠，并检查鼻咽部的组织学变化。病毒接种后将活菌注入鼻咽部，并检查鼻咽部细菌的清除情况。与对照动物相比，鼻内病毒接种后，花生凝集素、琥珀酰麦胚凝集素和Bandeiraea simplicifolia aggulutinin对鼻咽粘液毯和上皮细胞表面的染色显着增强。对照组动物鼻咽部有中等程度的山慈菇凝集素和麦胚凝集素染色，病毒接种后染色增强。这些结果在接种病毒后5天和9天最为显著。病毒接种后5天注射细菌时，鼻咽部培养的细菌数量显著增加。这些结果表明，由流感病毒引起的鼻咽粘膜的糖缀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，开发并检查了小鼠OME模型。小鼠接受内毒素鼓室内注射。注射后三天，观察到中耳积液。重组IL-1β接种后，也观察到类似的病理变化。抗IL-1受体抗体可抑制内毒素引起的病理改变。原位杂交显示IL-1β mRNA在中耳粘膜上皮中表达。提示IL-1β可能与内毒素诱导的中耳炎有关，并在中耳炎的发生中起重要作用。4）中耳粘膜免疫为了阐明中耳粘膜的免疫功能、淋巴细胞亚群、细胞因子mRNA、并在无特定病原体的小鼠中研究了中耳粘膜中抗原特异性IgA产生细胞的诱导。流式细胞仪分析显示，在CD 3 + T细胞中有一定数量的γδT细胞。P6特异性IgA产生细胞通过鼻内免疫与P6和霍乱毒素一起诱导。RT-PCR检测黏膜T细胞Th 2型T细胞mRNA表达。这些发现支持中耳是粘膜免疫的潜在效应部位的事实。5）中耳粘膜中对NTHi的特异性粘膜免疫应答的诱导为了阐明开发针对NTHi的鼻疫苗的可能性，用NTHi的P6鼻内免疫小鼠，并检查中耳中的粘膜免疫应答。耳洗液中P6特异性伊加滴度显著升高。中耳粘膜中P6特异性IgA产生细胞增加。P6体外刺激免疫小鼠纯化的CD 4 + T细胞增殖，这些T细胞表达Th 2细胞因子mRNA。这些结果表明，鼻内免疫在中耳粘膜中诱导了P6特异性IgA-B细胞免疫应答和选择的表达Th 2细胞因子的Th细胞。6）通过鼻内免疫从中耳清除NTHi为了评估用P6鼻内免疫对NTHi诱导的中耳炎的保护作用，用P6鼻内免疫小鼠，并在免疫后一周将活NTHi的悬浮液注射到鼓室中以诱导实验性OM。免疫小鼠显示出从中耳的NTHi清除增强。激发后对中耳TNF-α产生的刺激作用减弱。这些结果表明，鼻内免疫提供针对实验性中耳炎的保护。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