ratNGF、NaHS对哮喘大鼠气道炎症的调节作用

目的 观察神经生长因子(NGF)及外源性硫化氢钠(NaHS,即H2S供体)处理后哮喘大鼠肺中黏蛋白基因的表达情况,探讨其在哮喘发病中的作用.方法 36只SD大鼠随机分为正常对照组(n=8)和实验组(其中哮喘组9例、NGF干预组10例及NaHS干预组9例),实验组于第1、8天腹腔注射卵白蛋白(OVA)致敏,第15～22天采用1%的OVA溶液雾化激发,每次雾化前30 min哮喘组腹腔注射生理盐水,NGF干预组腹腔注射NGF(80 ng/kg),NaHS干预组腹腔注射NaHS(14 μmol/kg).正常对照组分别于各相对时点以同量生理盐水注射及雾化.各组激发后24 h(第23天)解剖大鼠,用免疫组化的方法测定大鼠肺中黏蛋白基因Muc5ac的表达情况.结果 哮喘组肺Muc5ac蛋白表达高于正常对照组(P＜0.01).与哮喘组相比,NGF干预组肺Muc5ac蛋白含量表达明显增多,而NaHS干预组肺Muc5ac蛋白含量表达减少(P＜0.05).结论 大鼠黏蛋白基因Muc5ac表达和哮喘的发生发展密切相关,NGF可加重气道炎症,而NaHS可以减轻哮喘气道炎症.     

Enhanced desensitization efficacy by liposomal conjugation of a specific antigen

Since liposomes are known as strong adjuvants, we attempted to use liposomes in immunotherapy as adjuvants, and to achieve desensitization in pre-sensitized mice. At first, we sensitized mice with intraperitoneal injection of model antigen, 100 microg ovalbumin (OVA), with Alum and treated them with liposome composed of distearoylphosphatidylcholine (DSPC) and cholesterol (2:1 as a molar ratio), which was coupled with a small amount of OVA (10 microg OVA in 400 nmol DSPC and 200 nmol cholesterol-liposome was injected into 20 g mouse). It is well known that antigen-specific immunotherapy increases IgG blocking antibodies and decreases in IgE antibodies. The treatment with i.v. injection of OVA-liposome at days 8, 10, and 12 after sensitization strongly suppressed OVA-specific IgE production without affecting IgG level after the boost (100 microg OVA with Alum). Moreover, the treatment with high-density OVA-liposome (10 microg OVA in 80 nmol DSPC and 40 nmol cholesterol-liposome/20 g mouse) not only strongly suppressed IgE levels but also reduced IgG production after the boost of OVA-sensitized mice suggesting the importance of liposomal characteristic in desensitization immunotherapy. Next we reduced the dose of OVA-liposome and the desensitization effect was also observed at the dose of as low as 1 microg OVA on OVA-liposome/mouse. On the contrary, free OVA did not affect the production of both IgG and IgE levels. Biodistribution study indicated that OVA-liposome was highly accumulated in spleen of OVA-sensitized mice compared to control liposome at 3 h after i.v. injection. These results suggest that the liposomal OVA effectively interacts with and desensitizes immune cells, therefore, liposomes coupling with a certain antigen may be effective in allergy immunotherapy.     

脂质体包裹可溶性蛋白质抗原诱导特异性CTL的研究

目的：搪塞用可溶性蛋白质抗原诱导特异性ＣＴＬ的途径。方法：用冻融－超声法制备包裹可溶性鸡卵蛋白（ＯＶＡ）和ＣＡ－Ｈｂ３蛋白质抗原的阳离子脂质体，并借助脂南体将抗的导入Ｃ５７ＢＬ／６小鼠脾细胞胞浆内，以此细胞经尾静脉免疫Ｃ５７ＢＬ／６小鼠，取体内初致敏脾细胞，在体外以渗透方式导入ＯＶＡ或ＣＡ－Ｈｂ３抗原的脾细胞再刺激，诱导特异性ＣＴＬ，并进行Ｉ类途径阻断实验和ＭＨＣＩ类分子中和实验，结果：脂质体成功     

变应性鼻炎大鼠模型建造

目的建立稳定的实验性变应性鼻炎（AR）SD大鼠模型。方法将SD大鼠雌雄各60只,随机分为实验组（n=60）和对照组（n=60）。实验组经腹腔隔日1次注射卵清蛋白（OVA）变应原7次后,鼻腔滴入OVA鼻内激发;对照组以生理盐水替代OVA。结果实验组SD大鼠均出现鼻痒、喷嚏、流清涕等变应性鼻炎的临床特征（评分〉5分）,鼻黏膜见大量的嗜酸性粒细胞、淋巴细胞浸润,粘膜水肿增厚、腺体增生、分泌旺盛,鼻黏膜表面纤毛破坏等组织形态学变化。对照组SD大鼠仅轻度抓鼻,且喷嚏少（评分〈5分）,鼻黏膜组织形态学均无实验组上述变化。结论本实验成功建立OVA变应原致敏的变应性鼻炎鼠类模型,具有操作简单、重复性好的优点,为今后变应性鼻炎的病变研究提供了有效的方法及组织形态学依据。     

Murine autoimmune hearing loss mediated by CD4+ T cells specific for β-tubulin

Autoimmune inner ear disease is described as progressive, bilateral although asymmetric, sensorineural hearing loss and can be improved by immunosuppressive therapy. We showed that the inner ear autoantigen β-tubulin is capable of inducing experimental autoimmune hearing loss (EAHL) in mice. Immunization of BALB/c mice with β-tubulin resulted in hair cell loss and hearing loss, effects that were not seen in animals immunized with control peptide. Moreover, the EAHL model showed that β-tubulin responsiveness involved CD4(+) T cells producing IFN-γ, and T cell mediation of EAHL was determined by significantly increased auditory brainstem response after adoptive transfer of β-tubulin-activated CD4(+) T cells into naive BALB/c recipients. The potential mechanisms responsible for the observed pathology of EAHL can be attributed to decreased frequency and impaired suppressive function of regulatory T cells. Our study suggests that EAHL may be a T cell-mediated organ-specific autoimmune disorder of the inner ear.     

IL-10 controls Th2-type cytokine production and eosinophil infiltration in a mouse model of allergic airway inflammation

Interleukin-10 was originally described as a factor that inhibits cytokine production by murine Th1 clones. Recent studies have since shown that IL-10 can also downregulate Th2 clones and their production of IL-4 and IL-5. Because of its immuno-suppressive properties, IL-10 has been suggested as a potential therapy for allergic inflammation and asthma. However, the pathophysiological role of IL-10 in vivo has not been clearly elucidated. We investigated the effects of IL-10 administration on the production of IgE, cytokine and allergen-induced Th2 cytokine production as well as its effects on eosinophilic inflammation. We established GATA-3/TCR double transgenic (GATA-3/TCR-Tg) mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)-specific TCR transgenic mice; these mice were then sensitized using an intraperitoneal injection of OVA adsorbed to alum and challenged with the intratracheal instillation of an allergen. When GATA-3/TCR-Tg mice sensitized with OVA and alum were injected with C57-IL-10 cells before OVA inhalation, the levels of IL-5, IL-13, and IL-4 decreased by 40-85% and number of eosinophils decreased by 70% (P < 0.03) in the murine bronchoalveolar lavage fluid (BALF). These results suggest that IL-10 plays an important role downstream of the inflammatory cascade in the Th2 response to antigens and in the development of BALF eosinophilia and cytokine production in a murine model of asthma. These immunosuppressive properties in animal models indicate that IL-10 could be a potential clinical therapy for the treatment of allergic inflammation.     

Classical MHC class I peptide presentation of a bacterial fusion protein by bone marrow-derived dendritic cells

Dendritic cells (DC) expanded in the presence of GM-CSF from the bone marrow of C57BL/6 mice process Gram-negative bacteria expressing the model antigen Crl-OVA for peptide presentation on MHC class I molecules. Here we show that presentation of OVA(257 – 264) processed by DC co-incubated with E. coli expressing Crl-OVA, which contains the K^b-binding OVA(257 – 264) epitope, occurs by a cytosolic MHC-I presentation pathway. First, we demonstrate the requirement for the transporter associated with antigen processing (TAP) by showing that DC from TAP1^−/− mice co-incubated with E. coli expressing Crl-OVA did not result in K^b presentation of OVA(257 – 264). Second, the proteasome inhibitor MG132 abrogated presentation of OVA(257 – 264) on K^b when C57BL/6 DC phagocytosed and processed E. coli expressing Crl-OVA. Third, inhibiting protein synthesis using cycloheximide or blocking exocytosis of newly synthesized proteins from the endoplasmic reticulum using brefeldin A abrogated presentation of OVA(257 – 264) processed from bacteria expressing Crl-OVA by C57BL/6 DC. Finally, peptide regurgitation and loading of OVA(257 – 264) on neighboring bystander K^b-expressing antigen-presenting cells after BALB/c (H-2^d) DC phagocytosed E. coli expressing Crl-OVA could not be detected. Together, these data support a cytosolic MHC-I presentation pathway for OVA(257 – 264) processed from E. coli expressing Crl-OVA by bone marrow-derived DC.     

Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. I. The primary T cell response is unaffected

Aging is associated with changes in the immune system that lead to decreased immunity in the elderly. Prior studies from humans and mice have shown that aged T cells exhibit numerous defects, including decreased proliferation following in vitro stimulation, suggesting that intrinsic defects exist within aged T cells, leading to defective T cell activation and clonal expansion. In vivo, however, cellular and soluble factors in the lymphoid microenvironment influence T cell function. To investigate the effects of the aged lymphoid microenvironment on T cell function, we monitored the immune response of CD4 T cells from DO11.10 TCR transgenic mice following adoptive transfer into young and aged hosts. After immunization with specific antigen similar rates of donor DO11.10 T cell division were observed in the two host types. However, at the peak of the response, greater numbers of DO11.10 T cells were found in the aged hosts. Regardless of the age of the host, the donor DO11.10 T cell population differentiated into functional effector cells. Despite the increased CD4 T cell growth in aged hosts, similar numbers of memory DO11.10 T cells were found in young and in aged hosts. As CD4 T cell clonal expansion and differentiation is not impaired in the aged microenvironment, our data suggest that diminished T cell immunity during aging is largely due to intrinsic T cell defects, rather than to extrinsic influences associated with the aged lymphoid microenvironment.     

Tolerogenic microenvironment in neonatal period induced by maternal immunization with ovalbumin

Maternal immunization with allergens, such as ovalbumin (OVA), can inhibit the development of an allergic response in offspring. The regulatory mechanisms seem to be mediated by maternal antibodies (MatAbs) and factors generated by the maternal–fetal interface. The aim of this study was to verify the pathways of inhibitory Ab transference after maternal immunization with OVA and the effect of the offspring's dendritic cells (DCs) on the generation of regulatory T (Treg) cells. We verified that preconceptional OVA immunization induces high levels of proinflammatory and regulatory cytokines in the amniotic fluid, allowing the transference of high levels of anti-OVA IgG1 Abs to the offspring. Using an adoptive nursing protocol, we verified that maternal immunization leads to MatAb transference by the placental route and by breastfeeding contribute to the inhibition of anaphylactic IgE and IgG1 Ab responses in immunized offspring. We observed that maternal immunization decreased eosinophil numbers in recovered bronchoalveolar lavage fluid and in the lung tissue, whereas with a lack of control of airway responsiveness to methacholine. Maternal immunization induced in young offspring a decreased percentage of CD11c+ DCs expressing MHC class II and CD40 molecules. Moreover, DCs from both groups of offspring when pulsed with OVA, were able to induce Treg cells in vitro. Similarly, OVA immunization at the neonatal stage increased the frequency of Treg cells, regardless of the mother's immunization status. These findings emphasize that maternal immunization leads to a complex interaction of regulatory factors, with MatAbs, DCs and Treg cells affecting the tolerance of offspring during an allergic response.