The Role and Mechanism of Mucosal Immune Responses against Outer Membrane Proteins of Haemophilus influenzae.

1)人扁桃体对流感嗜血杆菌的黏膜免疫反应：为了阐明腭扁桃体在上气消化道中的免疫作用，研究了扁桃体淋巴细胞对非分型流感嗜血杆菌（NTHi） P6的免疫反应。采集扁桃体切除术患者的鼻分泌物和血清，采用ELISA法检测P6抗体滴度。采用不连续梯度离心法分离腭扁桃体单个核细胞，采用ELISPOT法检测抗原特异性抗体分泌细胞的数量。用抗CD4微珠分离CD4^+T细胞，用P6孵育，ELISA检测细胞因子的产生。结果表明，不含NTHi的样品中p6特异性iga分泌细胞的数量大于含细菌的样品。扁桃体中p6特异性IgA分泌细胞的数量与鼻分泌物中IgA抗体滴度显著相关。CD4^+T细胞在P6刺激下产生th2和th1型细胞因子。上述结果提示，腭扁桃体作为粘膜免疫反应的效应位点和诱导位点，对上消化道细菌感染具有重要的预防作用。2)用流感嗜血杆菌和霍乱毒素（CT）的外膜蛋白（OMP）经鼻、口服、气管内或腹腔内免疫小鼠，比较Th1/Th2和B细胞免疫应答，探讨鼻腔黏膜免疫应答的机制，以及鼻内免疫诱导OMP特异性IgA应答的有效性。在第0、7和14天接种OMP和CT联合疫苗。采用ELISA法测定唾液、鼻洗液、支气管肺泡灌洗液和血清中抗原特异性抗体滴度。第21天，用omp特异性ELISPOT分析从鼻腔、肺、肠固有层和脾脏分离的单个核细胞。用OMP培养脾脏CD4^+T细胞4 d， ELISA检测细胞因子浓度。免疫后，将用于制备OMP的同一菌株的活菌悬液接种鼻腔，观察细菌从鼻腔中清除的情况。鼻内免疫小鼠的唾液、鼻洗液和支气管肺泡灌洗液中的抗omp IgA抗体滴度以及鼻道中omp特异性IgA产生细胞的数量最高。细胞因子分析显示，IFN-g、IL-2、IL-5和IL-6在鼻内免疫后主要产生。在清除试验中，鼻内免疫小鼠的鼻子中存在的细菌明显少于对照小鼠。这些发现表明，鼻子是诱导IgA免疫反应的一个强大的诱导和效应部位，鼻内免疫增强了对鼻子致病菌的清除。少

1)Mucosal immune responses against Haemophilus influenzaein human tonsils :In order to clarify the immunological role of palatine tonsils in upper aerodigestive tract, the immune responses of tonsillar lymphocytes against P6 of nontypeable Haemophilus influenzae (NTHi) were investigated. Nasal secretions and sera were obtained frome the patients who underwent tonsillectomy and the antibody titers against P6 were determined by ELISA.Mononuclear cells were isolated from palatine tonsils by discontinuous gradient centrifugation and the numbers of antigen-specific antibody-secreting cells were determined by ELISPOT.CD4^+T cells isolated by use of anti-CD4 microbeads were incubated with P6 and the production of cytokines was determined by ELISA.The results showed that the number of P6-specific IgA-secreting cells was greater in the samples having no NTHi than that in the samples having the bacteria. The numbers of P6-specific IgA-secreting cells in tonsils were significantly correlated with … More IgA antibody titers in nasal secretions. CD4^+T cells produced Th2-and Th1-type cytokines in response to the stimulation with P6. Those findings suggest that palatine tonsils play an important role as an effector site and an inductive site for mucosal immune responses, and prevent bacterial infection in upper aerodigestive tract.2)Mucosal immune responses against Haemophilus influenzaein miceMice were immunized intranasally, orally, intratracheally, or intraperitoneally with outer membrane proteins (OMP) of Haemophilus influenzae and cholera toxin (CT), and Th1/Th2 and B cell immune responses were compared to investigate the mechanisms of mucosal immune responses in the nose and the usefulness of intranasal immunization for inducing OMP-specific IgA responses. A combined vaccine of OMP and CT was administered on days 0,7, and 14. Antigen-specific antibody titers in saliva, nasal wash, bronchoalveolar lavage, and serum were determined by ELISA.On day 21, mononuclear cells isolated from nasal passages, lung, intestinal lamina propria, and spleen were analyzed by OMP-specific ELISPOT.CD4^+T cells isolated from spleen were cultured with OMP for 4 days, and the concentrations of cytokines in the culture supernatants were examined by ELISA.Following the immunization, a live bacterial suspension of the same strain as used for the preparation of OMP was inoculated into the nose, and the clearance of the bacteria from the nasal cavity was observed. Anti-OMP IgA antibody titers in saliva, nasal wash, and bronchoalveolar lavage and the numbers of OMP-specific IgA-producing cells in nasal passages were highest in mice immunized intranasally compared to the other groups of mice. Cytokine assay showed that IFN-g, IL-2, IL-5, and IL-6 were predominantly produced following intranasal immunization. In the clearance assay, significantly fewer bacteria were present in the nose of the intranasally immunized mice than in the control mice. These findings suggest that the nose is a powerful inductive and effector site for inducing IgA immune responses and that intranasal immunization enhances the clearance of pathogenic bacteria from the nose. Less