大鼠白念珠菌支气管肺感染时肺组织Toll样受体2的表达及意义

目的 探讨Toll样受体2(TLR2)在白念珠菌支气管肺感染大鼠肺组织模型中的表达及意义.方法 建立白念珠菌支气管肺感染大鼠模型,观察感染后肺组织病理形态学改变,采用免疫组织化学法观察感染后不同时间(第3天、第7天)白念珠菌支气管肺感染大鼠肺组织TLR2的表达,并与未感染组进行比较.结果 大鼠白念珠菌支气管肺感染后肺组织TLR2表达水平明显升高.结论 白念珠菌支气管肺感染肺组织TLR2表达增高可能参与感染的炎症反应.     

Phagocytosis of yeast by human oocytes: Fine structural observations

Fine-structure observations have been made on the interaction between invasive yeast cells and human oocytes. The yeast appear to make their way through the zona pellucida and once in the perivitelline space are incorporated into phagocytic vacuoles by surface activity of the oocyte. The yeast attach to the vitelline membrane via fuzzy surface material on the cell wall, and incorporation appears to be aided by oocyte microvillar activity. Coated pits in the oocyte plasma membrane are incorporated into the phagosomes, but no lysosomal activity is seen, and neither oocytes nor yeast cels appear to undergo degeneration in the time frame investigated.     

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms

Aim: The purpose of this work was to evaluate the size‐dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms. Methods and Results: The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4–3·3 μg ml^?1). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 μg ml^?1. In general, all SN suspensions promoted significant log₁₀ reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 μg ml^?1. Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms. Conclusions: This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. Significance and Impact of the Study: SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.     

抗白念珠菌感染的疫苗及抗体研究进展

白念珠菌是与人类共生的条件致病真菌,能引起免疫力低下患者皮肤黏膜和全身系统性持续感染.系统性念珠菌病是引起免疫力低下患者死亡的主要原因之一.由于临床缺乏念珠菌病的早期诊疗手段、可用的抗真菌药物种类有限且毒副作用大、耐药菌株越来越普遍、新药研发难度大等因素,抗真菌治疗依然面临着严峻挑战.目前有较多研究者致力于阐明白念珠菌感染的宿主免疫应答机制,并试图研发抗白念珠菌感染的免疫治疗方法,使免疫治疗有望成为预防和治疗真菌感染的有效手段.该文将几种抗白念珠菌感染的疫苗和抗体研究进展作简要概述,旨在为新型抗白念珠菌感染疫苗及抗体的研究提供参考.     

抗系统性白念珠菌感染的先天性免疫

白念珠菌是引起浅部、深部真菌感染常见的病原菌.先天免疫反应在宿主抗系统性白念珠菌感染中起主导作用.介导宿主抗念珠菌感染的先天性免疫包括一系列真菌识别受体及免疫效应细胞.宿主对系统性白念珠菌感染的免疫反应是决定患者预后的关键.本文就宿主抗系统性白念珠菌感染的先天性免疫机制进行综述.     

腹部大手术术后深部真菌感染的临床与真菌学分析

目的探讨腹部大手术术后深部真菌感染的危险因素及诊治措施。方法回顾性分析2006年1月～2009年6月期间我科腹部大手术术后合并深部真菌感染48例患者的临床和真菌学资料。结果 48例真菌感染患者共分离出56株菌株,其中白念珠菌占41.1%,是最主要的致病菌株。患者基础疾病和术后长期、多种广谱抗生素联合使用是深部真菌感染的重要因素。结论深部真菌感染是腹部大手术术后的重要并发症,白念珠菌仍然是主要病原菌。对术后患者深部真菌感染应采取积极预防、及时发现和有效治疗。     

大鼠支气管肺白念珠菌感染后血清IL-17水平及意义

目的探讨大鼠支气管肺白念珠菌感染后血清白细胞介素17(IL-17)的变化及意义。方法将24只Sprague Dawley(以下简称SD)健康雄性鼠随机分为3组,每组8只,免疫低下+白念珠菌感染组(阳性对照组);免疫低下+生理盐水组(阴性对照组);免疫正常+白念珠菌感染组(实验组)。因为实验过程中存在大鼠死亡,最终每组各有6只大鼠完成实验。系统应用糖皮质激素建立免疫低下的大鼠模型;实验组及阳性对照组使用标准白念珠菌菌株混悬液经气管插管注入而建立感染模型;阴性对照组使用生理盐水作对照。感染后第1、3、5、7天使用尾静脉取血分离血清;酶联免疫吸附法(ELISA)检测血清IL-17水平;第7天取血后处死大鼠,取右肺下叶组织进行HE染色观察。结果第3、5天实验组血清IL-17水平高于对照组,而第1、7天3组间无差异;两对照组IL-17水平无差异。肺组织病理:实验组呈明显炎症反应;阳性对照组呈轻微炎症反应;阴性对照组未见明显炎性细胞浸润。结论大鼠白念珠菌支气管肺感染后,免疫正常的大鼠在感染中期血清IL-17水平升高,IL-17可能通过促进中性粒细胞在气道的聚集而参与炎症反应;IL-17的水平与机体炎症反应呈正相关;IL-17在气道白念珠菌感染后炎症介导及机体防御机制的调动中起一定作用。     

Immunomodulatory and protective effect of probiotic Lactobacillus casei against Candida albicans infection in malnourished mice

The effect of Lactobacillus casei CRL 431 (Lc), when administered as a supplement to a repletion diet, on the resistance of malnourished mice to Candida albicans infection was studied. Weaned mice were malnourished by being given a protein-free diet (PFD) for 21 days. The malnourished mice were then fed a balanced conventional diet (BCD) for 7 days or BCD for 7 days with supplemental Lc on days 6 and 7 (BCD+Lc). Malnourished (MNC) and well-nourished (WNC) mice were used as controls. At the end of the treatments the mice were infected intraperitoneally with C. albicans. Animals that had received probiotics had improved survival and resistance against this infection compared to those in the BCD and MNC groups. The number and fungicidal activity of phagocytes, and the concentrations of tumor necrosis factor-α, interferon-γ and interleukin-6 (IL-6), increased in blood and infected tissues in all experimental groups, but MNC mice showed lower concentrations than those in the WNC group. BCD and BCD+Lc mice showed higher concentrations of these variables than those in the MNC group, but only the BCD+Lc group presented values similar to the WNC mice. Malnutrition also impaired the production of IL-17 and IL-10 in response to infection. Both repletion treatments normalized IL-17 concentrations, but IL-10 in the BCD+Lc group was significantly higher than in WNC mice. The addition of L. casei to the repletion diet normalized the immune response against C. albicans, allowing efficient recruitment and activation of phagocytes, as well as effective release of pro-inflammatory cytokines. In addition, probiotic treatment induced an increase in IL-10 concentrations, which would have helped to prevent damage caused by the inflammatory response.     

住院患者光滑念珠菌检出的回顾性分析

目的调查住院患者光滑念珠菌检出的特征。方法回顾性调查分析白求恩国际和平医院2008年1月～2009年4月住院患者中光滑念珠菌检出阳性者的临床资料,以同期白念珠菌检出患者为对照。结果其间共有52例详细病史资料记录的光滑念珠菌检出患者,以60岁以上老年人为主,占65.4%;主要分离自痰标本,占76.9%。患者患有多种基础疾病,以肺部感染(28例,53.8%)、恶性肿瘤(20例,38.5%)、脑梗死(15例,28.8%)常见。使用抗生素(52例,100%)、留置导尿管(15例,28.8%)是光滑念珠菌检出者的主要实施医疗措施。氟康唑是临床最常用的治疗光滑念珠菌感染药物(23例,44.2%)。光滑念珠菌检出患者死亡率高(14例,26.9%),高于同期白念珠菌检出对照组(6.2%,P=0.004)。结论光滑念珠菌检出患者与白念珠菌检出具有相似的临床流行病学特征。     

Cleavage of human big endothelin-1 by Candida albicans aspartic proteinase

Abstract A Candida albicans aspartic proteinase (CAP), one of the secretory proteinases of Candida albicans , is thought to be a possible virulence factor in Candida albicans infection. Whereas endothelin-1 is found as an endothelium-derived strong vasoconstrictive peptide, it is known to have a role in the maintenance of vascular homeostasis and tissue survival. Endothelin-1 is generated from a precursor form of endothelin-1, the so-called big endothelin-1. It has recently been reported that cathepsin D, E and pepsin, which are aspartic proteinases, convert big endothelin-1 to endothelin-1. In this study, the relationship between CAP and big endothelin-1 was studied. High performance liquid chromatography analysis revealed that big endothelin-1 was cleaved into several amino acid sites by CAP, but endothelin-1 was not converted from big endothelin-1. CAP cleaved big endothelin-1 at different sites when compared with that of other known aspartic proteinases, and it suppressed endothelin-1 production through the degradation of big endothelin-1. CAP may break homeostatic mechanism of endothelin-1 in Candida albicans infectious lesion.     

白念珠菌ALS3、SSA1基因表达在念珠菌性阴道炎免疫机制中的作用

目的探讨白念珠菌ALS3、SSA1基因缺失对阴道上皮细胞激发免疫反应的作用。方法培养白念珠菌野生株及ALS3、SSA1基因敲除株(SC5314、Δals3、Δssa1),对其进行形态测定。按不同MOI感染人阴道上皮细胞系VK2/E6E7细胞,通过台盼蓝染色观察和乳酸脱氢酶(LDH)活性检测,评价不同MOI白念珠菌对上皮细胞的损伤作用;使用酶联免疫吸附试验(ELISA)评估感染过程中炎性细胞因子及趋化因子在共培养上清中的差异。结果 ALS3基因的缺失对白念珠菌芽管长度影响差异无统计学意义,而SSA1基因的缺失与其他两个菌株相比芽管长度减少约30%～40%(P<0.001)。台盼蓝染色观察及LDH测定发现,3株菌在感染上皮细胞时,其细胞损伤能力均与菌载量成正比;与野生型相比,Δssa1突变体在相同比率感染上皮细胞时,细胞损伤能力明显降低,且差异有统计学意义(P<0.05),Δals3突变株影响较小,甚至略微升高。检测炎性细胞因子及趋化因子发现,突变株在诱导上皮细胞产生促炎因子及趋化因子(GM-CSF、G-CSF、IL-1α、IL-8)的能力上明显减弱,差异均有统计学意义(P<0.05)。结论 ALS3和SSA1基因表达在阴道上皮细胞抗白念珠菌感染的局部免疫应答过程中可能起到重要作用,且SSA1基因表达意义更大。     

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.     

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years^18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies^33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors³³.An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida^{3,4,11,16,17,19,21,25,37}. This model has also been employed to test potential antifungal therapies in vivo^13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection^6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties^3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.     

深圳市人民医院连续13年真菌血症病原谱分析

目的 调查深圳市人民医院真菌血症病原谱。方法 收集2004‒2016年深圳市人民医院真菌血症患者血液分离的真菌,使用VITEK-2、VITEK-MS系统并结合真菌内转录间隔区（ITS）测序技术进行菌种鉴定。结果 2004‒2016年共发生真菌血症295例,分离296株真菌,假丝酵母菌占89.9%（266/296）。最常见前5位真菌依次为白假丝酵母菌（36.1%,107/296）、近平滑假丝酵母菌种复合群（19.3%,57/296）、热带假丝酵母菌（13.9%,41/296）、光滑假丝酵母菌种复合群（11.8%,35/296）和菌膜假丝酵母菌（4.7%,14/296）;非白假丝酵母菌占53.7%（159/296）。69.8%（206/295）真菌血症来自重症医学科（ICU）（89例）、新生儿科（33例）、血液科（30例）、胃肠外科（29例）和呼吸科（25例）。结论 假丝酵母菌是真菌血症最重要的病原菌,其中非白假丝酵母菌占比过半。     

SCID小鼠肠道菌群失调对深部白色念珠菌感染的影响研究

目的建立重度联合免疫缺陷（SCID）小鼠白色念珠菌感染模型,探讨肠道菌群失调与深部白色念珠菌感染的联系。方法SCID小鼠随机口服万古霉素水溶液7d,饥饿24h后给予白色念珠菌灌胃,建立小鼠白色念珠菌感染模型,观察小鼠死亡情况。荧光定量PCR检测肠道细菌总量、基质辅助激光解析电离飞行时间质谱仪鉴定肠道菌群种类,并应用扫描电镜观察肠壁黏膜组织超微结构的改变。结果应用万古霉素可致肠道菌群失调,肠道黏膜完整性受损。在万古霉素致肠道菌群失调的基础上,外源性白色念珠菌攻击可加重肠道菌群失调和肠壁黏膜损伤程度,促进深部白色念珠菌感染的发生。结论肠道菌群失衡可以导致深部白色念珠菌感染的发生,肠壁黏膜的完整性可能参与了肠道白色念珠菌播散过程。     

濮阳市部分浅部真菌病中癣菌和念珠菌混合感染的分析

目的了解濮阳地区部分浅部真菌病中两种及两种以上真菌混合感染的情况。方法对临床确诊为浅部真菌病的患者456例,取标本行10%KOH直接镜检、真菌培养及菌种鉴定。登记确诊患者相关临床资料,分析其中混合感染的发病特点。结果确诊皮肤癣菌和念珠菌混合感染病例36例,分离率为7.89%,多见于股部(58.33%)和足部(27.78%);分离菌株72株,皮肤癣菌中以红色毛癣菌为主(75.00%),念珠菌中以非白念珠菌为主(72.22%)。结论濮阳地区部分浅部真菌病中混合感染主要为皮肤癣菌和念珠菌的混合感染,多见于股部和足部;致病菌以红色毛癣菌和非白念珠菌为主。     

Purification and germination of Candida albicans and Candida dubliniensis chlamydospores cultured in liquid media

Candida albicans and Candida dubliniensis are the only Candida sp. that have been observed to produce chlamydospores. The function of these large, thick-walled cells is currently unknown. In this report, we describe the production and purification of chlamydospores from these species in defined liquid media. Staining with the fluorescent dye FUN-1 indicated that chlamydospores are metabolically active cells, but that metabolic activity is undetectable in chlamydospores that are >30 days old. However, 5–15-day-old chlamydospores could be induced to produce daughter chlamydospores, blastospores, pseudohyphae and true hyphae depending on the incubation conditions used. Chlamydospores that were preinduced to germinate were also observed to escape from murine macrophages following phagocytosis, suggesting that these structures may be viable in vivo . Mycelium-attached and purified chlamydospores rapidly lost their viability in water and when subjected to dry stress, suggesting that they are unlikely to act as long-term storage structures. Instead, our data suggest that chlamydospores represent an alternative specialized form of growth by C. albicans and C. dubliniensis .