烟草表达的粉尘螨Ⅰ类变应原重组蛋白的致敏效果研究

目的探讨烟草表达的粉尘螨Ⅰ类变应原重组蛋白对小鼠的致敏性。方法 40只BALB/c小鼠随机分为4组,分别为PBS阴性对照组、卵白蛋白阳性对照组、原核表达Der f1(pDer f1)组、烟草表达Der f1(tDerf1)组,各组小鼠分别于第0、7、14天腹腔注射,PBS组给予PBS,其余3组分别用卵白蛋白、pDer f1、tDerf1致敏,第21天起连续7 d雾化吸入激发,24 h后处死。通过肺组织病理切片观察小鼠肺部炎症,收集支气管肺泡灌洗液(BALF)进行总细胞及分类计数,以ELISA法检测BALF、脾细胞培养上清(SCCS)的特异性细胞因子IL-2、IL-4、IL-5、IL-17和IFN-γ和血清中变应原特异性IgE、IgG1及IgG2a抗体浓度。结果 PBS组小鼠肺部未见明显病理改变,卵白蛋白、pDer f1、tDer f1组小鼠肺部出现明显的炎症。tDer f1组小鼠BALF中的细胞总数、嗜酸性粒细胞、中性粒细胞、淋巴细胞、抗原特异性细胞因子IL-4、IL-5、IL-17和SCCS中IL-4、IL-5、IL-17均明显高于阴性对照组(P<0.01),而与阳性对照组相比无统计学意义;tDer f1组小鼠BALF和SCCS中IL-2、IFN-γ均低于阴性对照组,差异有统计学意义(P<0.01);卵白蛋白、pDer f1、tDerf1组特异性IgE和IgG1抗体水平均高于PBS组(P<0.01)。结论从烟草中表达的重组Derf1蛋白对小鼠具有致敏作用,其可作为变应原进一步研究。     

屋尘螨和粉尘螨Ⅰ类变应原研究进展

屋尘螨和粉尘螨是最常见的室内过敏原之一，尘螨最主要的致敏成分是尘螨Ⅰ类变应原。该文从屋尘螨和粉尘螨Ⅰ类变应原的研究概况、在螨体内的定位、结构和生化特性研究与同源性和多态性分析、应用于尘螨变应原研究的新方法等4个方面进行综述。     

变应性哮喘患儿的变应原分析

目的分析变应性哮喘患儿的变应原分布特点,为防治儿童哮喘提供理论依据。方法对2007年1月至2009年6月就诊的483例变应性哮喘患儿采用CAP方法进行吸人性变应原和食物性变应原检测。结果483例患儿系统变应原过筛总阳性率为84．5％（408／483）,其中吸人性变应原阳性率为81．0％（391／483）,食物性变应原阳性率为40．6％（196／483）;〈3岁、3—7岁、〉7岁患儿吸人性变应原阳性率、食物性变应原阳性率差异均有统计学意义（P〈0．05）。结论吸人性变应原是儿童哮喘的主要变应原,明确自身的变应原,减少接触变应原的机会是防止儿童哮喘的有效手段之一。     

变应性哮喘患儿的变应原分析

目的 分析变应性哮喘息儿的变应原分布特点,为防治儿童哮喘提供理论依据.方法 对2007年1月至2009年6月就诊的483例变应性哮喘患儿采用CAP方法进行吸人性变应原和食物性变应原检测.结果 483例患儿系统变应原过筛总阳性率为84.5%(408/483),其中吸入性变应原阳性率为81.0%(391/483),食物性变应原阳性率为40.6%(196/483);＜3岁、3～7岁、＞7岁患儿吸人性变应原阳性率、食物性变应原阳性率差异均有统计学意义(P＜0.05).结论 吸入性变应原是儿童哮喘的主要变应原,明确自身的变应原,减少接触变应原的机会是防止儿童哮喘的有效手段之一.     

舌下含服粉尘螨滴剂治疗单一和多重过敏的儿童变应性鼻炎的疗效观察

目的评估标准化粉尘螨滴剂舌下特异性免疫治疗(SLIT)对单一和多重过敏的变应性鼻炎(AR)儿童的临床疗效。方法 68例尘螨过敏的AR儿童,根据变应原皮肤点刺试验结果,将其分为两组:单一过敏组35例和多重过敏组33例,采用标准化粉尘螨滴剂进行舌下特异性免疫治疗(SLIT)1年。对治疗前与接受SLIT治疗3、6、9及12个月后的两组临床资料进行对比研究与分析。结果 SLIT治疗3、6、9及12个月后,两组内症状评分和药物评分均低于治疗前,差异有统计学意义(P<0.01);两组间症状评分和药物评分差异无统计学意义(P>0.05)。结论舌下含服粉尘螨滴剂治疗单一和多重过敏的儿童变应性鼻炎的临床治疗效果相当,因此,舌下含服粉尘螨滴剂可以应用于临床治疗尘螨合并多重过敏的儿童变应性鼻炎。     

舌下免疫治疗对粉尘螨过敏哮喘患儿气道炎症及免疫失衡的影响

目的探讨粉尘螨过敏哮喘患儿采用舌下免疫治疗后气道炎症及免疫失衡的改变情况。方法选取2014年6月—2016年6月收治的94例粉尘螨过敏哮喘患儿为研究对象,随机分为观察组和对照组,各47例,两组均采取糖皮质激素吸入治疗方案,观察组同时给予舌下含服粉尘螨滴剂治疗。观察两组治疗后气道炎症及免疫功能指标改善情况。计量资料比较采用t检验,P0.05为差异有统计学意义。结果治疗后,观察组呼出气一氧化氮(Fe NO)、IL-4水平下降,且低于对照组(均P0.01),尘螨SIg G4、干扰素-γ水平升高,且高于对照组(均P0.01);观察组日间和夜间哮喘症状评分低于治疗前,且低于对照组(均P0.01)。结论舌下免疫治疗粉尘螨过敏性哮喘可明显改善患儿气道炎症反应,促进机体免疫趋向平衡。     

淄博市变应性鼻炎和哮喘患者变应原类型的比较及分析

目的：比较淄博地区单纯性变应性鼻炎、支气管哮喘及同时患有变应性鼻炎和哮喘的患者其变应原阳性率,分析机体对变应原反应程度与疾病发生、发展的关系,并探讨变应性鼻炎与哮喘发生的相互关系,为临床治疗提供依据。方法选取2010年12月至2013年9月在我科变态反应室行变应原皮肤点刺试验的患者共247例,行皮肤点刺试验。结果单纯性变应性鼻炎组、支气管哮喘组和变应性鼻炎合并哮喘组,最常见变应原均为屋尘螨、粉尘螨和热带螨。结论淄博市单纯性变应性鼻炎、支气管哮喘和变应性鼻炎合并哮喘三类患者中,常见11种变应原的阳性检出率具有相似的变化规律。     

致敏花粉与变应性鼻炎发病关系及花粉变应原免疫治疗效果的研究

应用重力测量法,对淄博市空气中致敏花粉进行一年的曝片调查.全年曝片365张,共检出花粉10898粒,36科属;全年均可见到花粉飘散,并出现两次花粉飘散高峰期,分别在3～6月和7～9月.其主要致敏花粉为篙属、大麻律草属、悬铃木属和扬属等.应用本地优势致敏花粉抗原为376例变应性鼻炎患者做皮内试验,阳性率为745%,对照组为97%(P<0.01).采用花粉抗原为864例变应性鼻炎患者进行特异性免疫治疗,临床控制率为76.7%,对照组为24.0%(P<0.01).结果初步证实致敏花粉是变应性鼻炎的重要病因,花粉的飘散高峰期与花粉性变应性鼻炎病人的发病季节相一致.应用花粉抗原进行免疫治疗取得了显着的疗效     

经口致敏食物过敏大鼠模型的建立

目的探讨经口致敏食物过敏大鼠模型的建立方法,以及适宜的评价指标。方法以卵清蛋白(OVA)作为致敏原,将16只3周龄Brown-Norway(BN)大鼠随机分为3组:阴性对照组(生理盐水灌胃)、阳性对照组(OVA腹腔注射组)和实验组(OVA灌胃组),共灌胃9周。在第4、5、6、7、8、9周分别采用双抗体夹心ELISA法及皮肤过敏实验法(PCA)测定实验动物血清OVA特异性IgE(OVA-IgE)抗体滴度,以判断动物是否致敏成功,在第13周各组动物给予100mg/mlOVA1ml灌胃激发后测血清OVA-IgE滴度。结果ELISA结果显示在灌胃第6周时实验组动物OVA-IgE滴度水平达到最高,且显著高于阴性对照组(P<0.05),致敏率为60%(3/5);第7、第8周时OVA-IgE滴度水平略有下降,但是仍然显著高于阴性对照组(P<0.05),致敏率达到80%(4/5),OVA-IgE水平和致敏率与阳性对照组之间差异无显著性;实验组PCA结果在第6、7、8周均显示为阴性,而阳性对照组PCA显示为阳性。结论经口致敏可作为一种可行的建立食物过敏动物模型的方法,符合食物过敏发生的自然生理过程,建议适宜致敏时间为6周;采用ELISA法检测血清OVA-IgE抗体水平较PCA法能更加灵敏地反映经口致敏模型是否成功。     

小鼠背部变应性接触性皮炎模型的建立

目的建立小鼠背部变应性接触性皮炎(allergic contact dermatitis,ACD)模型.方法用二硝基氟苯(dinitrofluorobenzene,DNFB)分别建立小鼠背部及耳廓ACD模型,比较两种模型在不同时间点皮肤肿胀度及HE染色炎细胞数量变化情况.结果①背部ACD肿胀度大于耳廓ACD肿胀度,配对t检验,t=6.39,P＜0.01,并且两种模型的肿胀度变化呈正相关. ②背部ACD较耳廓ACD单一核细胞数量少,多形核细胞数量多,前者t=8.59,P＜0 .01,后者t=6.05,P＜0.01;两模型的细胞数量变化呈正相关.结论背部ACD模型可以代替耳廓ACD模型用于评价抗炎药物.背部模型肿胀度高,适合肉眼判断皮炎程度.     

珠海及周边地区哮喘患儿过敏原状况及粉尘螨滴剂舌下含服疗效研究

[目的]探讨珠海及周边地区哮喘患儿过敏原状况以及粉尘螨滴剂舌下含服在哮喘治疗中的意义.[方法]应用屋尘螨、粉尘螨等25种常见过敏原对367例哮喘的患儿进行皮肤点刺过敏原检测.检测血清IgE,同时对粉尘螨阳性的哮喘患儿采用粉尘螨滴剂舌下含服治疗. [结果]过敏原检测阳性率为71.3%.阳性率最高依次为屋尘螨、粉尘螨和海产品类.粉尘螨脱敏治疗有效率为86.53%. [结论]珠海及周边地区哮喘患儿的过敏原最常见为屋尘螨,粉尘螨及海产品;粉尘螨滴剂舌下含服是治疗哮喘的重要措施之一.     

潍坊地区过敏性鼻炎和支气管哮喘患儿过敏原检测结果分析

目的 了解潍坊地区过敏性鼻炎、支气管哮喘患儿的主要过敏原,为过敏性疾病的预防及诊治提供参考。方法 对234例患儿(71例过敏性鼻炎、84例支气管哮喘、46例过敏性鼻炎合并支气管哮喘)进行皮肤点刺试验(SPT),并对试验结果进行统计学分析。结果 234例被检测患儿中阳性201例(85.90%)。最常见的两种过敏原为屋尘螨和粉尘螨,分别占总阳性例数的79.10%、77.11%;其余依次为带鱼(17.91%)、狗上皮(17.41%)、海虾(16.92%)、牛奶(13.43%)、青霉菌(12.44%)等;吸入性过敏原组SPT阳性率显著高于食物类过敏原组(P＜0.001);过敏性鼻炎、支气管哮喘、过敏性鼻炎合并支气管哮喘三组,SPT阳性率分别为88.75%、86.60%、80.70%,且三组间SPT阳性率差异均无统计学意义(P＞0.05)。结论 屋尘螨、粉尘螨是潍坊地区最常见的过敏原;随年龄的增长吸入性过敏原皮肤点刺的阳性率有增高趋势,食物类过敏原阳性率随年龄的增长而减少;吸入性过敏原是儿童过敏性鼻炎和支气管哮喘最常见的诱发因素。     

小鼠过敏性肠炎模型的免疫学研究

目的 评价卵清蛋白(OVA)饲喂致敏OVA特异性T细胞受体转基因小鼠(OVA23-3)的方法建立过敏性肠炎模型的可行性,并观察其免疫学指标变化.方法 采用高OVA饲料饲喂OVA23-3小鼠的方法,建立小鼠过敏性肠炎模型.通过ELISA法检测血清OVA特异性IgE抗体和细胞培养上清细胞因子的含量,流式细胞术检测细胞内细胞因子.结果 高OVA饲料饲喂引起OVA23-3小鼠体重下降,并发生以小肠为主的出血性炎症.与对照组比较,实验组血清OVA特异性IgE含量明显增高,细胞培养上清IL-4水平增高,而IFN-β水平降低,同时CD4+T细胞中IL-4产生细胞百分率显著增加,IFN-γ产生细胞百分率显著减少.结论 高OVA饲料饲喂OVA23-3小鼠诱发过敏性肠炎的方法是一种理想快速简便的方法,Th2型细胞反应占优势是其重要特征.     

Neonatal colonization of germ-free mice with Bifidobacterium longum prevents allergic sensitization to major birch pollen allergen Bet v 1

The main goal in reversing the allergy epidemic is the development of effective prophylactic strategies. We investigated the prophylactic effect of neonatal mother-to-offspring mono-colonization with Bifidobacterium longum ssp. longum CCM 7952 on subsequent allergic sensitization. Adult male and female germ-free (GF) mice were mono-colonized with B. longum, mated and their offspring, as well as age-matched GF controls, were sensitized with the major birch pollen allergen Bet v 1. Furthermore, signaling pathways involved in the recognition of B. longum were investigated in vitro. Neonatal mono-colonization of GF mice with B. longum suppressed Bet v 1-specific IgE-dependent β-hexosaminidase release as well as levels of total IgE and allergen-specific IgG2a in serum compared to sensitized GF controls. Accordingly, Bet v 1-induced production of both Th1- and Th2-associated cytokines in spleen cell cultures was significantly reduced in these mice. The general suppression of Bet v 1-specific immune responses in B. longum-colonized mice was associated with increased levels of regulatory cytokines IL-10 and TGF-β in serum. In vitro, B. longum induced low maturation status of bone marrow-derived dendritic cells and production of IL-10 in TLR2-, MyD88-, and MAPK-dependent manner. Our data demonstrate that neonatal mono-colonization with B. longum reduces allergic sensitization, likely by activation of regulatory responses via TLR2, MyD88, and MAPK signaling pathways. Thus, B. longum might be a promising candidate for perinatal intervention strategies against the onset of allergic diseases in humans.     

雌激素诱导白假丝酵母菌阴道炎小鼠模型中环氧化酶-2的表达

目的:探讨雌激素对白假丝酵母菌性阴道炎模型小鼠阴道组织中环氧化酶-2(COX-2)表达的影响及其在发病机制中的意义。方法:80只小鼠随机分为雌激素化未感染白假丝酵母菌组(E组)、未雌激素化感染白假丝酵母菌组(I组)、雌激素化感染白假丝酵母菌组(EI组)和空白对照组(C组)。制作白念珠菌性阴道炎小鼠模型,每组分别于接种后第2天、第4天、第7天和第14天取小鼠阴道组织,用免疫组化方法检测组织中COX-2的表达。结果:除C组外,各组小鼠阴道组织内第2天出现COX-2的阳性表达,持续升高至第7天达到高峰,第14天时开始下降;E组在第4、第7天COX-2免疫组化检测OD值分别为0.157±0.017和0.161±0.014,与相应C组(0.101±0.014和0.106±0.014)比较均具有显著性统计学差异(P均<0.05)。EI和I组第7天的OD值分别达到0.275±0.059和0.234±0.063,与对照组相比明显上调(P均<0.01),且EI组显著高于I组(P<0.05)。结论:在白假丝酵母菌性阴道炎小鼠模型中,雌激素能通过诱导阴道组织中COX-2的表达在其发病机制中起重要作用。     

Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice

Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans.     

Voluntary exercise under a food restriction condition decreases blood branched-chain amino acid levels,in addition to improvement of glucose and lipid metabolism,in db mice,animal model of type 2 diabetes

Objectives

Exercise is effective for preventing the onset and development of type 2 diabetes mellitus (T2DM) in human cases; however, the effect of exercise on the pathophysiology using animal models of T2DM has not been fully evaluated.

Methods

We applied voluntary exercise under pair-fed (P) conditions in db mice, an animal model of T2DM. Exercising (Ex) and sedentary (Se) mice were placed in a cage, equipped with a free or locked running wheel, for 4 weeks, respectively. The amount of food consumed by ad libitum-fed wild-type mice under the Se condition (ad-WT) was supplied to all mice, except ad libitum db mice (ad-db). Blood parameters and expression of the genes involved in nutrient metabolism were analyzed.

Results

PEx-db (pair-fed and exercising) mice showed significantly lower HbA1c, body weight and liver weight than PSe-db and ad-db mice. Decreased hepatic triglycerides in PEx-db mice corresponded to a lower expression of lipogenic enzyme genes in the liver. Moreover, PEx-db mice showed significantly lower plasma branched-chain amino acids (BCAA), arginine, proline, and tyrosine, in addition to increased skeletal muscle (SM) weight, than PSe-db and ad-db mice, in spite of little influence on the expression of the BCAA transaminase gene, in SM and WAT.

Conclusion

We found that exercise under a food restriction condition decreases several amino acids, including BCAA, and may improve insulin sensitivity more than mere food restriction. We propose that the decreased concentration of blood amino acids may be a valuable marker evaluating the effects of exercise on diabetic conditions.

Electronic supplementary material

The online version of this article (doi:10.1007/s12199-014-0400-z) contains supplementary material, which is available to authorized users.     

免疫低下模型的建立及流式细胞术的检测研究

目的：探讨环磷酰胺致免疫低下模型的建立,并试图研究应用流式细胞术检测小鼠自然杀伤细胞（NK）和T淋巴细胞的应用。方法：以环磷酰胺强化注射、少量多次注射、一次大剂量注射分别建立免疫低下模型,30 d后处死,取外周血和脾,分别检测小鼠外周血自然杀伤细胞（NK）亚群和外周血T淋巴细胞亚群,同时进行脾NK细胞活性测定、ConA诱导的小鼠脾淋巴细胞转化试验,观察环磷酰胺对外周血T淋巴细胞、NK细胞免疫功能的影响。结果：环磷酰胺强化注射、少量多次注射均可使小鼠外周血和脾的活化的NK细胞和T淋巴细胞比例及T淋巴细胞转化增殖能力、脾NK细胞活性降低。结论：环磷酰胺强化注射可以长时降低小鼠细胞免疫功能的作用,适用于建立保健食品免疫低下模型;流式细胞术检测T淋巴细胞和NK细胞分化抗原在保健食品免疫调节功能评价中有较好应用价值。     

Evaluation of novel recombinant porcine circovirus type 2d (PCV2d) vaccine in pigs naturally infected with PCV2d

IntroductionThe pathogenic porcine circovirus type 2 (PCV2) causes significant economic losses in pig production. Emergence of the PCV2d genotype has been linked with PCV2-associated disease (PCVAD) outbreaks. However, no study has been conducted efficacy of an experimental PCV2d-based subunit vaccine in pigs. Therefore, PCV2b- and PCV2d-based capsid (CP) proteins were generated using a baculovirus (Bac) expression system, and we evaluated the protective immune responses in a commercial pig farm where predominant PCV2d is circulating.MethodsEighteen 3-week-old pigs with maternal antibodies were randomly divided into four groups, and were immunized with purified Bac-2dCP, mixed 1:1 ratio with purified Bac-2bCP and Bac-2dCP (Bac-mCP), a commercial PCV2a-based subunit vaccine (VAC) or phosphate-buffered saline (PBS) as controls.ResultsThe Bac-2dCP and Bac-mCP groups had significantly higher PCV2b- or PCV2d- specific IgG and neutralizing antibody without interference by maternal antibody compared to control group in pigs naturally infected with PCV2d. Interestingly, not only serum IL-4 level was significantly increased in the Bac-2dCP group, but also PCV2d viremia level was significantly reduced than the control group.ConclusionsThe recombinant Bac-2dCP subunit vaccine is a good candidate for the effective reduction against PCV2d infection.