健康人外周血烟曲霉硫氧还蛋白还原酶抗原特异性T 淋巴细胞检测与结果评价

目的：为评估烟曲霉硫氧还蛋白还原酶（ thioredoxin reductase , TR）抗原是否具有免疫保护作用,文中建立并优化检测健康人外周血单个核细胞（peripheral blood mononuclear cell , PBMC）中TR抗原特异性T淋巴细胞（TR／AST）分泌干扰素γ（interferon γ, IFN-γ）与白细胞介素-4（interleukin-4, IL-4）的酶联免疫斑点法（enzyme linked immunospot assay , ELISPOT）检测技术,以探讨TR抗原特异性T淋巴细胞在侵袭性曲霉病（ invasive aspergillosis , IA）中的作用。方法采用方阵滴定法优化ELISPOT反应条件。以烟曲霉TR为特异性刺激物,阳性细胞刺激剂为对照,用ELISPOT技术检测20名健康人外周血PBMC分泌IFN-γ、IL-4的阳性斑点形成细胞（spot forming cell, SFC）频数。结果方阵滴定法结果显示,10μg TR抗原、健康人PBMC终浓度为3×105／孔时ELISPOT检测结果最佳。20名健康人特异性分泌IFN-γ和IL-4的SFC频数分别为15（3．5,59．5）个和0（0,0）个,IFN-γ的SFC频数显著高于IL-4,差异有统计学意义（P＜0．001）。 TR可刺激所有20名健康人产生IFN-γ应答,其中9例产生强IFN-γ应答（SFC＞20个／3×105 PBMC）,占45％;而19名健康人在TR刺激后不产生IL-4应答,仅1例产生弱IL-4应答（SFC＝1）。结论烟曲霉TR抗原诱导的T细胞免疫以Th1型免疫应答占绝对优势,因此,该抗原具有成为保护性抗原的潜能。     

重症肺部感染后继发曲霉菌感染的临床研究

目的研究重症肺部感染后患曲霉菌感染的危险因素、临床特征、影像学特点,以做到早期诊断和治疗。方法回顾分析2005年1月—2011年12月在呼吸重症监护室（RICU）的重症肺部感染继发曲霉菌感染患者,随机抽取同一时期未并发真菌感染的重症肺炎为对照组。记录患者临床资料,包括一般资料、基础疾病、治疗相关因素进行统计分析,以及血液指标、细菌培养结果和影像学资料。结果监护病房住院天数、广谱抗生素、糖皮质激素、机械通气（MV）、感染性休克、肝功能不全、糖尿病、免疫性疾病以及慢性呼吸道疾病（CRD）在2组比较中差异有统计学意义（P〈0．05）;而年龄、留置静脉导管、肠外营养以及实体肿瘤之间差异无统计学意义（P〉0．05）。临床以发热、呼吸困难及肺部哮鸣音为主;外周血白细胞升高、CRP、IGE升高占较大比例;同时影像学具有不典型性,以肺纹理增重、片状渗出和实变等非特异性表现。抢救成功8例中7例为伏立康唑治疗。结论重症肺部感染后存在上述相关危险因素时需注意易患曲霉菌感染可能;由于其临床表现及影像学具有不典型性,因此临床医师应认识其好发因素、观察临床病情的变化、多次查痰培养,同时气管镜检查观察黏膜、PBS及活检获得病理不失为一个比较安全的方法,抢先治疗成为降低病死率的关键。     

异绿原酸对烟曲霉生物被膜的体外作用

目的：探讨金银花活性成分异绿原酸对早期、成熟期烟曲霉生物被膜的体外作用。方法采用微量液基稀释法测定异绿原酸对受试烟曲霉菌株最低抑菌浓度（MIC）和最低杀菌浓度（MFC）;构建体外早期、成熟期静止生物被膜模型并用不同浓度（64、128、256、512、1024μg/mL）的异绿原酸组分别作用48h,激光共聚焦显微镜（CLSM）检测生物被膜内存活菌;扫描电镜（SEM）观察生物被膜形态学改变;结晶紫染色法定量生物被膜。比较各组MIC和MFC。结果异绿原酸对受试烟曲霉菌株的MIC和MFC均大于1024μg/mL;不论早期或成熟期,空白对照组和异绿原酸各浓度组生物被膜内菌体在CLSM下均以绿色的活菌为主;电镜观察证实异绿原酸可破坏早期、成熟期的生物被膜结构,使胞外基质减少,菌体轮廓清晰;当异绿原酸浓度达到256μg/mL时,可以使载体上不同时期生物被膜的量明显降低,256、512、1024μg/mL异绿原酸组生物被膜定量与空白对照组及组间两两比较,差异均有统计学意义（P〈0.05）。结论异绿原酸在体外不能杀死烟曲霉生物被膜内菌体,但可对烟曲霉早期和成熟期生物被膜产生破坏作用,其作用呈浓度依赖性。     

烟曲霉对小鼠巨噬细胞自噬流的影响初步研究

【摘要】 目的 探讨烟曲霉对小鼠骨髓来源巨噬细胞（BMDM）自噬流的影响。方法 灭活烟曲霉体外诱导小鼠BMDM不同时间（0、0.5、4、12 h）,提取细胞蛋白,Western印迹法检测自噬关键蛋白LC3-Ⅰ型/Ⅱ型的转换及磷酸化雷帕霉素靶蛋白（p-mTOR）Ser2481的蛋白水平。用烟曲霉和溶酶体阻断剂［包括半胱氨酸蛋白酶抑制剂E-64d + 胃蛋白酶抑制剂pepstatin、巴佛洛霉素-A1（BAF-A1）、氯化铵、氯喹］单独或联合体外诱导小鼠BMDM不同时间（4、12 h）后,Western印迹法检测烟曲霉对新生的LC3-Ⅱ、细胞基础自噬流的影响,并通过共聚焦荧光显微镜观察烟曲霉与LC3、Rubicon（RUN domain Beclin-1-interacting and cysteine-rich-domain-containing protein）的共定位关系。不同处理时间数据结果采用非匹配t检验分析,烟曲霉孢子和自噬溶酶体阻断剂两因素处理数据结果采用2 × 2析因分析方法。结果 Western印迹显示,与对照组（0 h组）比较,烟曲霉体外诱导小鼠BMDM 0.5、4、12 h后细胞内LC3-Ⅱ表达逐渐增加,差异均有统计学意义（t值分别为6.58、3.28、3.02,均P < 0.05）,但各组p-mTOR蛋白水平差异无统计学意义（t值分别为0.441、0.477、0.382,P值分别为0.682、0.660、0.722）。与单独氯喹处理BMDM 4 h和12 h相比,烟曲霉联合氯喹处理后 LC3-Ⅱ进一步增高,差异均有统计学意义（t = 2.13、2.78,均P < 0.05）。与单独氯化铵处理BMDM 4 h和12 h相比,烟曲霉联合氯化铵处理后 LC3-Ⅱ进一步增高,差异均有统计学意义（t = 2.92、2.92,均P < 0.05）。与单独BAF-A1处理BMDM 4 h和12 h相比,烟曲霉联合BAF-A1处理后LC3-Ⅱ进一步增高,差异有统计学意义（t = 2.13、2.13,均P < 0.05）。与单独E-64d + pepstatin处理BMDM 4 h和12 h相比,烟曲霉联合E-64d + pepstatin处理后 LC3-Ⅱ进一步增高,差异有统计学意义（t = 2.13、2.92,均P < 0.05）。用钙荧光白荧光标记的烟曲霉孢子刺激BMDM 8 h后,共聚焦荧光显微镜显示LC3、Rubicon主要包绕于烟曲霉周围,与烟曲霉均有共定位关系。结论 烟曲霉体外诱导可增加小鼠BMDM基础自噬流。     

NADPH oxidase 2 plays a protective role in experimental Aspergillus fumigatus keratitis in mice through killing fungi and limiting the degree of inflammation

AIM: To explore whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease. METHODS: NOX2 expression was detected in C57BL/6 mice. After testing the inhibitory effect of diphenyleneiodonium chloride (DPI) on NOX2, its impact on clinical performance, myeloperoxidase levels, the number of colonies forming units, the level of H3, the generation of reactive oxygen species (ROS) and the release of cytokines [NF-κB, interleukin-17A (IL-17A), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), Nrf2, IL-10, and TGF-β] were compared. A one-way ANOVA and an unpaired, two-tailed Student’s t-test was used to determine the statistical significance. RESULTS: NOX2 expression was significantly increased after Aspergillus fumigatus injection in corneas and that this increase could be reduced by treatment with DPI. DPI treatment produced more severe inflammation and resulted in higher clinical scores, more neutrophils infiltration, a weakened ability to clear fungi, the release of fewer ROS and the formation of neutrophil extracellular traps. Treatment with DPI increased the expression of the proinflammatory cytokines NF-κB, IL-17A, IL-6, and TNF-α and decreased the expression of the anti-inflammatory cytokines Nrf2, IL-10 and TGF-β compared to their expression levels without DPI treatment. CONCLUSION: NOX2 plays an important role against Aspergillus fumigatus in the mouse cornea through killing fungi and limiting the degree of inflammation.     

The Role of SREC-Ⅰ in Innate Immunity to Aspergillus fumigatus Keratitis

PurposeTo determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-Ⅰ) in vitro and in a mouse model of Aspergillus fumigatus keratitis.MethodsSREC-Ⅰ mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-Ⅰ expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-Ⅰ small interfering RNA, then the mRNA levels of LOX-1, IL-1β, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-Ⅰ mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1β, and TNF-α mRNA expression levels were tested before and after anti–SREC-Ⅰ treatment.ResultsSREC-Ⅰ expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-Ⅰ mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-Ⅰ small interfering RNA treatment inhibited the expressions of LOX-1, IL-1β, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1β, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-Ⅰ–neutralizing antibody treatment.ConclusionsSREC-Ⅰ is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-Ⅰ blockade could be a potential therapeutic approach for FK.     

Unusual trifecta of infections,aspiration, and metastatic prostatic adenocarcinoma in a bronchoalveolar lavage specimen

Bronchoalveolar lavage (BAL) is a useful procedure to evaluate lung infiltrates in order to identify infection, foreign body aspiration, and neoplasms. However, it is indeed unusual to find all three in the same sample. We report such a case in a 68‐year‐old male with a history of metastatic prostate adenocarcinoma and longstanding chronic obstructive pulmonary disease who presented with features of pneumonia. BAL revealed Aspergillus and parainfluenza infections, food particle aspiration pneumonia, as well as metastatic prostatic adenocarcinoma. The food particles were initially confused for yeast infection, but we finally identified them as nut products. This may be the first documented case of nut product aspiration diagnosed on BAL. The potential pitfalls that may complicate the evaluation are also discussed.     

The domestic pig as human-relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus

Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus-specific CD4⁺ T cells in blood are described to reflect the actual host–pathogen interaction status, little is known about Aspergillus-specific pulmonary T-cell responses. Here, we exploit the domestic pig as human-relevant large animal model and introduce antigen-specific T-cell enrichment in pigs to address Aspergillus-specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus-exposed pigs, the fungus-specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 10⁶ cfu/m³ conidia induces a long-lasting accumulation of Aspergillus-specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus-exposure showed a drastic reduction in the lung-infiltrating antifungal T-cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human-relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal-specific T-cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response.     

黑曲霉孢子悬液贮存期的验证

目的：对贮存期的黑曲霉孢子悬液的质量进行考察和研究。方法：采用计数法及药敏实验对黑曲霉孢子悬液在贮存期的变化进行分析。结果：低温（4℃）保存6个月内黑曲霉孢子悬液菌落计数结果稳定,药物敏感性未出现变化,符合药典的规定。结论：在实验中,验证过的储存期内黑曲霉孢子悬液可直接稀释使用。     

IL-36α Exerts Proinflammatory Effects in Aspergillus fumigatus Keratitis of Mice Through the Pathway of IL-36α/IL-36R/NF-κB

PurposeTo explore the role of IL-36α in corneas infected by Aspergillus fumigatus.MethodsThe experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1β, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry.ResultsIL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1β, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra.ConclusionsIL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1β, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.