用免疫蛋白质组学技术筛选烟曲霉分泌蛋白中的免疫优势抗原

摘要：目的：用免疫蛋白质组学技术筛选烟曲霉分泌蛋白中的免疫优势抗原,用于侵袭性曲霉病（IA）的诊断标志物及疗效监测。 方法：YEPG培养基培养14 d的真菌分泌蛋白质进行二维电泳（2-DE）分离,用抗曲霉抗体阳性的IA确诊患者混合血清进行免疫印迹,再用质谱分析及生物信息学方法鉴定阳性反应点。 结果：2-DE、免疫印迹结果显示,确诊IA患者血清与分泌蛋白质中多种抗原反应强烈。40个蛋白质点被抗体阳性IA患者血清识别。对40个蛋白质点质谱分析,成功鉴定了39个免疫反应阳性的蛋白质点,代表17种蛋白质,包括分泌型二肽基肽酶V（DppV）、NAD-依赖型苹果酸脱氢酶、硫氧还蛋白还原酶、醛脱氢酶、果糖二磷酸醛缩酶、3-羟丁酰辅酶A脱氢酶等。在鉴定出的17种蛋白质中,有2个为已知抗原,15个首次报道为免疫原性蛋白质。有7种免疫原性蛋白质显示有多个蛋白质点。且在新发现的免疫原性蛋白质中,2号蛋白质（共13个蛋白质点）免疫反应最强,鉴定结果为硫氧还蛋白还原酶。 结论：免疫蛋白质组学技术是筛选病原体免疫优势抗原的一种有效方法,此次鉴定的抗原可能是潜在的疾病标志物,但还需进一步地实验加以评估。     

Colonization With Small Conidia Aspergillus Species Is Associated With Bronchiolitis Obliterans Syndrome: A Two‐Center Validation Study

Aspergillus colonization after lung transplantation may increase the risk for bronchiolitis obliterans syndrome (BOS), a disease of small airways. We hypothesized that colonization with small conidia Aspergillus species would be associated with a greater risk of BOS, based upon an increased likelihood of deposition in small airways. We studied adult primary lung recipients from two large centers; 298 recipients at University of California, Los Angeles and 482 recipients at Duke University Medical Center. We grouped Aspergillus species by conidia diameter ≤3.5 μm. We assessed the relationship of colonization with outcomes in Cox models. Pre‐BOS colonization with small conidia Aspergillus species, but not large, was a risk factor for BOS (p = 0.002, HR 1.44, 95% CI 1.14–1.82), along with acute rejection, single lung and Pseudomonas. Colonization with small conidia species also associated with risk of death (p = 0.03, HR 1.30, 95% CI 1.03–1.64). Although other virulence traits besides conidia size may be important, we have demonstrated in two large independent cohorts that colonization with small conidia Aspergillus species increases the risk of BOS and death. Prospective evaluation of strategies to prevent Aspergillus colonization of small airways is warranted, with the goal of preserving lung allograft function as long as possible.     

Microsatellite typing of Aspergillus fumigatus isolates recovered from deep organ samples of patients with invasive aspergillosis

Microsatellite typing was used to analyze 41 Aspergillus fumigatus isolates from 9 patients with proven invasive aspergillosis hospitalized in 2 different centers. No strains were shared between patients. For 8 of 9 patients, a single genotype was found for the isolates recovered from all anatomic sites involved.     

曲霉菌对唑类抗真菌药物的耐药机制研究进展

侵袭性曲霉病是由致病曲霉菌引起的危及生命的真菌感染,主要致病菌为烟曲霉,而唑类抗真菌药物是临床治疗的首选。目前已上市的唑类抗真菌药物包括伊曲康唑,伏立康唑,泊沙康唑和伊沙康唑。然而,流行病学研究发现曲霉菌对唑类抗真菌药物的耐药率呈逐年上升趋势,给临床治疗造成了威胁。其中,Cyp51A蛋白突变、外排系统高表达以及环境耐药机制等多种因素都参与唑类抗真菌药物的耐药。因此,本文综述近年来有关曲霉菌对唑类抗真菌药物的耐药机制,以期为耐药监测、耐药菌控制和新药研发提供理论依据。     

Pharmacodynamic studies of voriconazole: informing the clinical management of invasive fungal infections

Introduction: Voriconazole is a broad-spectrum antifungal agent commonly used to treat invasive fungal infections (IFI), including aspergillosis, candidiasis, Scedosporium infection, and Fusarium infection. IFI often occur in immunocompromised patients, leading to increased morbidity and mortality.

Areas covered: The objective of this review is to summarize the pharmacodynamic properties of voriconazole and to provide considerations for potential optimal dosing strategies. Studies have demonstrated superior clinical response when an AUC/MIC >25 or C_min/MIC >1 is attained in adult patients, correlating to a trough concentration range as narrow as 2–4.5 mg/L; however, these targets are poorly established in the pediatric population. Topics in this discussion include voriconazole use in multiple age groups, predisposing patient factors for IFI, and considerations for clinicians managing IFI.

Expert commentary: The relationship between voriconazole dosing and exposure is not well defined due to the large inter- and intra-subject variability. Development of comprehensive decision support tools for individualizing dosing, particularly in children who require higher dosing, will help to increase the probability of achieving therapeutic efficacy and decrease sub-therapeutic dosing and adverse events.     

烟曲霉的微管蛋白基因鉴定及唑类抗真菌药物的耐药性研究

目的建立烟曲霉临床分离菌株的微管蛋白基因鉴定方法,并分析烟曲霉对唑类抗真菌药物的耐药性。方法对表型鉴定为烟曲霉的菌株,采用十六烷基三甲基溴化铵(CTAB)方法提取烟曲霉菌株DNA,并采用对微管蛋白基因进行PCR扩增和测序的方法对烟曲霉菌株进行分子生物学鉴定,采用琼脂稀释法检测烟曲霉菌株对伊曲康唑和伏立康唑的耐药性。结果 91株临床分离株表型鉴定为烟曲霉,这些菌株在35℃和48℃生长。其中88株(96.70%)经微管蛋白基因鉴定为烟曲霉。87株烟曲霉菌株对4μg/mL伊曲康唑和1μg/mL伏立康唑敏感,检出1株对4μg/mL伊曲康唑和1μg/mL伏立康唑耐药菌株。结论微管蛋白基因鉴定方法可以对烟曲霉进行准确鉴定。     

Fluvastatin potentiates the activity of caspofungin against Aspergillus fumigatus in vitro

Statins (anticholesterol drugs) inhibit HMG-CoA reductase, which is a key rate-limiting enzyme in the synthesis of sterols in fungi. We therefore investigated the in vitro inhibitory activity of various statins against Aspergillus fumigatus alone and in combination with antifungal drugs. Fresh conidial suspensions from 10 clinical isolates of A. fumigatus were obtained. The MIC and minimum fungicidal concentration (MFC) were determined by the Clinical and Laboratory Standards Institute M-38A protocol and the fungicidal activity by time–kill study. Fluvastatin (FST) showed good activity (MIC, 2 mg/L; MFC, 4 mg/L) against A. fumigatus; other statins had no activity (MIC ≥256 mg/L). FST enhanced the activity of caspofungin (CFG) against A. fumigatus; subinhibitory concentrations of FST in combination with CFG showed >99.9% killing of A. fumigatus conidia, whereas either drug alone showed poor activity at subinhibitory concentrations. FST potentiated the antifungal activity of CFG but displayed no specific interaction with voriconazole or amphotericin B.     

TLR4基因沉默下调TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用

目的 研究单核巨噬细胞RAW264.7受烟曲霉孢子刺激时,TLR2和TLR4信号通路发挥的作用,以及沉默TLR4基因后,对TLR2信号通路的影响.方法 利用RNAi技术将TLR4-siRNA转染RAW264.7细胞24h后给予烟曲霉孢子刺激12h,将细胞随即分为正常组(N组)、正常+烟曲霉孢子刺激组(N+Af组)、正常+TLR4-siRNA组[TLR4(RNAi)组]、正常+TLR4-siRNA+烟曲霉孢子刺激组[ TLR4(RNAi) +Af组],RT-PCR和Western blot法检测细胞受烟曲霉孢子刺激后TLR2、TLR4、MyD88 mRNA及TNF-α蛋白的表达变化.结果 (1)TLR4基因沉默前:与N组比较,N+Af组TLR2、TLR4、MyD88 mRNA及TNF-α蛋白表达量均显著升高(P＜0.05).(2)TLR4-siRNA( 100nmoL/L)转染RAW264.7细胞,沉默效率达83％.(3)TLR4基因沉默后:与N组比较,TLR4( RNAi)组TLR2、MyD88 mRNA的表达量均显著降低(P＜0.05);与N+Af组比较,TLR4( RNAi)+Af组的TLR2、MyD88 mRNA和TNF-α蛋白的表达量均显著降低(P＜0.05);与TLR4( RNAi)组比较,TLR4(RNAi)+ Af组MyD88 mRNA表达量显著升高(P＜0.05),而TLR2 mRNA及TNF-α蛋白表达量却无显著变化(P＞0.05).结论 RAW264.7细胞受烟曲霉孢子刺激时,TLR2和TLR4信号通路被激活,通过释放促炎细胞因子TNF-α发挥抗烟曲霉孢子刺激作用;当沉默TLR4基因后,TLR2信号通路不能被很好地激活来抵抗烟曲霉孢子对细胞的刺激作用,沉默TLR4基因下调了TLR2信号通路在RAW264.7细胞中的抗烟曲霉孢子刺激作用,可能TLR4较TLR2在抵抗烟曲霉孢子刺激时发挥更重要的作用.     

常见侵袭性真菌疫苗的研究现状

临床上存在抗肿瘤药物及抗生素滥用现象,使得条件致病性真菌感染疾病发病率升高,研究针对条件致病性真菌敏感的制剂-抗真菌药物及疫苗就显得十分重要。而临床应用抗真菌药物治疗的疗效不佳。出现较多耐药菌株,同时药物本身的毒副反应使患者无法耐受。因此,抗真菌治疗依然面临着严峻挑战。这无疑给研发真菌疫苗进行早期预防开辟了一条新的挑战途径。本文对真菌菌体疫苗、亚单位疫苗、DNA疫苗、树突状细胞与真菌疫苗以及含有失活基因的真菌疫苗进行综述。     

曲霉生物膜研究进展

真菌生物膜的形成是与临床死亡率密切相关的一个重要问题。近年来,曲霉生物膜日益受到研究人员的关注,研究发现,在侵袭性曲霉病感染中大多数存在着生物膜的感染源。曲霉形成生物膜以后可以逃逸宿主的免疫作用,在感染部位难以清除,是临床上难治性感染的重要原因。概述曲霉生物膜形成过程及结构、曲霉生物膜形成的影响因素以及生物膜形成导致抗真菌药耐药的机制等几个方面的研究进展。