白念珠菌的烯醇化酶

烯醇化酶是生物体内糖酵解途径中的关键酶。文中就白念珠菌烯醇化酶的生物学活性、酶蛋白的一级和二级结构特征、基因组成、与白念珠菌过敏患者IgE结合的特点和位点以及其作为白念珠菌感染宿主的特征性标志物在诊断深部念珠菌病中的价值等方面加以综述。     

白念珠菌的稀醇化酶

烯醇化酶是生物体内糖醇解途径中的关键酶。文中就白念珠菌烯醇化酶的生物学活性，酶蛋白的一级和二级结构特征，基因组成，与白念珠菌过敏患者ＩｇＥ结合的特点和位点以及其作为白念珠菌感染宿主的特征标志物在诊断深部念珠菌病中的价值等方面加以综述。     

白念珠菌烯醇化酶的分离与纯化

卫生部重点项目并得到南京大学医药生物技术国家重点实验室开放基金资助烯醇化酶 (enolase)，又称 2－磷酸甘油水解酶，是糖酵解所必需的胞内酶，能催化磷酸甘油酸盐向磷酸烯醇式丙酮酸转变，同时也在糖异生过程中催化逆向反应。白念珠菌烯醇化酶具有高度的保守性，在白念珠菌系统感染时能激发机体产生特异的免疫反应，白念珠菌烯醇化酶的分离与纯化是研究它的前提和基础。 1：标准分子量 2：白念珠菌全菌蛋白 3：经硫酸铵沉淀后的菌体蛋白 4：经 DEAE－ Sephadex A－ 50凝胶柱 (pH7.8)层析后的菌体蛋白 5：经 DEAE－ Sephadex A－ 50…     

白念珠菌芽管特异性抗原快速检测方法的建立和初步评价北大核心CSCD

目的建立白念珠菌芽管特异性抗原快速检测系统,探讨其特异性和敏感性。方法以单克隆抗体McAb03．2C1-C2作为一抗,辣根过氧化物酶标记McAb03．2C1-C2为二抗,建立双抗体夹心ELISA实验方法并检测6只动物模型和5例系统性白念珠菌感染患者的血清标本。结果筛选出McAb03．2C1-C2最佳稀释度和检测包被抗原浓度分别为l：4000和1．25-40μg,／ml;双抗体夹心ELISA检测动物模型的特异性和敏感性分别为95％、92％,其对阳性临床标本的检测结果与常规鉴定方法一致。结论初步建立白念珠菌芽管特异性抗原的双抗体ELISA快速检测系统,动物模型实验证实该系统有较好的特异性和敏感性。     

抗白念珠菌卵黄免疫球蛋白对白念珠菌生长及黏附的影响

白念珠菌黏附于宿主上皮是其在宿主中形成集落及入侵体内的第一步,也是其具有强致病性的标志,因此,抑制其黏附和生长是防治念珠菌病的有效途径。抗白念珠菌卵黄免疫球蛋白（immunoglobulin of egg yolk,IgY）是白念珠菌免疫产蛋母鸡后从鸡蛋中提取的一种卵黄抗体,能与白念珠菌特异性结合。为此,笔者通过黏附试验观察了IgY对白念珠菌黏附于人口腔颊黏膜细胞的影响,并观察IgY对体外培养白念珠菌生长的影响．现报告如下。     

白念珠菌系统性感染中白念珠菌相关抗原检测的意义

甘露聚糖和甘露糖蛋白、葡聚糖、烯醇化酶、磷脂酶和蛋白酶等是白念珠菌胞壁或细胞内的成分。文中就其生物学特性及其作为在感染宿主过程中的特征性标志物在诊断系统性白念珠菌感染中的方法、价值和意义等方面加以综述。     

重要条件致病真菌感染组织免疫组化研究

目的 探索在组织中鉴定重要条件致病真真的组织病理学方法。方法 以实验小鼠白念珠菌感染组织和可疑念珠菌、隐球菌和曲霉感染的临床组织病理标本进行ＰＡＳ染色和免疫组化染色，并比较了两种方法的敏感性。结果 兔抗白念株菌多克隆抗体、兔抗新生隐球菌多克隆抗体和鼠抗曲霉单克隆抗体可以分别鉴定组织中的白念珠菌、新生隐球菌和曲霉。免疫组经和ＰＡＳ染色的敏感性差异无显著性。结论 免疫组化为组织中确定真菌和鉴定真菌种属     

白念珠菌系统性感染中白念珠菌相关抗原检测的意义

甘露聚糖和甘露糖蛋白、葡聚糖、烯醇化酶、磷脂酶和蛋白酶等是白念珠菌胞壁或细胞内的成分。文中就其生物学特性及其作为在感染宿主过程中的特征性标志物在诊断系统性白念珠菌感染中的方法、价值和意义等方面加以综述。     

白念珠菌芽管特异性抗原的研究

运用ⅡＦ、ＳＤＳ－ＰＡＧＥ和免疫印迹技术，证实８株临床分离的白念珠菌芽管表面存在芽管特异性抗原。白念珠菌菌丝相胞壁提取物中含有比孢子相更多的、不同的蛋白成份；发现白念珠菌菌丝相胞壁提取物中有一分子量大约为３９ ０００蛋白抗原成份很可能是白念珠菌芽管特异性抗原；认为白念珠菌孢子相和其它念珠菌孢子相胞壁中有以隐蔽状态存在的芽管抗原成份。     

抗白念珠菌人鼠嵌合抗体真核表达载体的构建与表达

目的构建抗白念珠菌人鼠嵌合抗体的真核表达载体,并实现真核表达。方法从含有单克隆天然抗白念珠菌抗体3B4可变区基因的载体pMDT-V2H和pUC-VL中PCR克隆单抗3B4的可变区VH和VL基因,依次插入含有人免疫球蛋白κ轻链恒定区基因和γ1重链恒定区基因的真核表达载体pMH-CA。经DNA序列测定正确后,电穿孔转染J558L细胞进行嵌合抗体表达,RT-PCR,ELISA方法对抗体表达进行鉴定。结果成功构建了抗白念珠菌人鼠嵌合抗体,转染真核细胞后RT-PCR显示人鼠嵌合抗体重链和轻链mRNA的转录,ELISA证实了抗白念珠菌人鼠嵌合抗体的表达以及对白念珠菌的识别。结论成功构建抗白念珠菌人鼠嵌合抗体的真核表达载体,并表达出具有结合活性的基因工程抗体。     

An assessment of the role of Candida albicans antigen in atopic dermatitis.

An immediate hypersensitivity reaction to Candida albicans (C. albicans) antigen has been observed in patients with atopic dermatitis. Recent data from a comparative study of the immune response to C. albicans antigen in patients with atopic dermatitis and non-atopics suggest a shift form type 1 helper T cells to type 2 helper T cells in the immune response to C. albicans antigen in atopic dermatitis. To delineate the role of C. albicans in the pathogenesis in atopic dermatitis, we evaluated skin reaction of C. albicans antigen, as well as the serum IgE antibody level against C. albicans in patients with atopic dermatitis, patients with nasal allergy, and non-atopics. In addition, the clinical effect of antifungal drugs was evaluated in the patients with atopic dermatitis. As a result, we found that immediate hypersensitivity to C. albicans antigen is strongly correlated with the patients with atopic dermatitis. On the other hand, the delayed-type hypersensitivity to this antigen, which is highly prevalent in atopics without dermatitis as well as non-atopics, was reduced in most of these patients. Antifungal drugs markedly improved the skin manifestations in patients with atopic dermatitis that have IgE antibodies against C. albicans, and the serum IgE levels also decreased. These results suggest that C. albicans antigen is a potent intrinsic factor in inducing skin lesions in atopic dermatitis because of IgE-mediated hypersensitivity of C. albicans antigen.     

Activation of complement by Pityrosporum orbiculare

The ability to activate complement in human serum was evaluated for the two yeast-like organisms Pityrosporum orbiculare, the presumed etiologic agent of tinea versicolor, and Candida albicans. Complement activation was measured by: (a) using inhibition of rabbit red blood cell lysis by human serum after incubation with the organisms, and (b) quantitation of the amount of C3 deposited on the surface of the yeast by an enzyme-linked immunoabsorbent assay. It was found that both organisms had approximately equal ability to activate complement in normal serum or serum having only the alternative pathway intact, even though extracts of C. albicans contained significantly greater amounts of both carbohydrate and antigenic material capable of combining with the antibody present in normal human serum. The marked difference in inflammation in the cutaneous lesions produced by these two organisms does not appear to be related to their complement-activating ability and is more likely due to some other factor such as differences in invasiveness or in ability to elicit other immunologic reactions.     

Antigen distribution and antigen-presenting cells in skin biopsies of human chromoblastomycosis

BACKGROUND: Chromoblastomycosis is a chronic, suppurative, granulomatous mycosis usually confined to skin and subcutaneous tissues. The host defense mechanisms in chromoblastomycosis have not been extensively investigated. The purpose of the present study was to determine the distribution and pathways of the fungal antigen(s) and the possible role of the different immunocompetent cells in antigen processing in skin lesions. METHODS: The distribution of Fonsecaea pedrosoi antigen(s) in human skin was studied in 18 biopsies from 14 patients with chromoblastomycosis. A purified polyclonal immune serum raised in rabbits against metabolic antigen(s) of F. pedrosoi was used to detect yeast antigen(s) by immunohistochemical procedures. Double immunolabeling was performed with yeast antigen(s) and Langerhans' cells [labeled with anti-S100 protein monoclonal antibody (MoAb)], yeast antigen(s) and factor XIIIa+ dermal dendrocytes (immunolabeled with anti-factor XIIIa polyclonal antibody), and yeast antigen(s) and macrophages (labeled with CD 68 monoclonal antibody). RESULTS: The F. pedrosoi antigen(s) accumulated in the skin macrophages and, in a few instances, in factor XIIIa+ dendrocytes and Langerhans' cells. CONCLUSIONS: The data obtained suggest that chiefly macrophages, also Langerhans' cells and factor XIIIa+ dermal dendrocytes, function as antigen-presenting cells in chromoblastomycosis.     

Contact sensitivity to Candida albicans—comparative studies in man and animal (guinea-pig)

We studied the elicitation of contact sensitivity to Candida albicans antigen in guinea-pigs with experimental cutaneous candidiasis and in humans, using commercially available potent 1:100 C. albicans antigen (Torii) by patch testing on abraded skin. In guinea-pigs, non-immune animals became patch test-reactive 4-5 days after topical application of viable C. albicans, either under occlusion or without occlusive dressings, concurrently with the demonstrability of delayed responses to intradermally injected 1:10 000 Candida antigen. In humans, all healthy adults who showed delayed hypersensitivity reactions to intradermally injected 1:10 000 C. albicans antigen demonstrated positive patch-test reactions to 1:100 C. albicans antigen. There was a significant correlation between the magnitude of responses to these tests. In contrast, no positive patch test reactions were elicited to the 1:100 C. albicans antigen on neonatal skin, emphasizing the lack of irritability of this test agent. These results also indicate that in humans contact sensitivity to Candida antigen occurs during later life because C. albicans is a ubiquitous organism. The practical value of this Candida patch test for evaluation of patients' immune function was assessed by a prospective study in patients with various skin disorders. The results obtained suggested some potential value of the test for evaluation of cell-mediated immune function of patients with regard to ubiquitous recall antigens.     

应用胶体金免疫层析技术快速检测杜克雷嗜血杆菌方法的建立

目的建立快速检测杜克雷嗜血杆菌的胶体金免疫层析试验(GICA)系统。方法采用胶体金颗粒标记抗rHgbA-IgG抗体,制备出免疫胶体金;采用双抗体夹心模式建立适用于杜克雷嗜血杆菌检测的胶体金免疫层析技术;以ELISA试验作为平行对照,对其检测的灵敏度及特异性进行评价。结果以该方法检测杜克雷嗜血杆菌、流感嗜血杆菌以及其他7种生殖器溃疡相关细菌,杜克雷嗜血杆菌显示强阳性,其余8种均为阴性,无交叉反应发生,GICA与ELISA实验结果相符合;GICA和ELISA检测灵敏度分别为:纯化抗原实验GICA为6.25ng/mL,ELISA为1.56ng/mL;细菌学实验GICA为2×106cfu/mL,ELISA为2×105cfu/mL;模拟样本检测实验GICA为1×107cfu/mL,ELISA为1×106cfu/mL。GICA能在15min内完成检测,稳定性良好。结论建立的检测杜克雷嗜血杆菌的GICA系统能够特异、快速检测杜克雷嗜血杆菌。该检测系统的灵敏度还不能满足所有临床标本的需要,有待于进一步研究。     

A human monoclonal antibody reacting with Merkel cells: immunofluorescence, immunoperoxidase, and immunoelectron microscopy

A human monoclonal, mu, kappa, cold agglutinin antibody of the rare specificity Pr h (serum and proper eluates) was used in immunofluorescence and immunoperoxidase techniques and in immunoelectron microscopy on rabbit lip specimens. Pr h antibody strongly reacted with scattered cells in epidermis, which were demonstrated to be Merkel cells by electron microscopy; no nerve fibers were stained. In immunoelectron microscopy (IEM), a strong reaction was seen within the cytoplasm and around the granules. This is the first IEM staining of Merkel cells (MC) so far reported; it demonstrates the expression of a carbohydrate differentiation antigen in MC. The availability of a potent monoclonal antibody reacting with MC but not with neighboring epidermal cells in rabbit lip offers a new tool for the study of several aspects of MC biology, including antigenic properties and kinetics.     

白念珠菌保护性单抗的研究

目的 探讨抗白念珠菌单克隆抗体在系统性念珠菌感染动物中的保护作用。方法 制备抗白念珠菌单抗,观察单抗对系统性念珠菌感染小鼠存活时间,组织病理改变以及组织中菌落形成单位的影响。结果 制备出3株抗白念珠菌胞壁外膜抗原单克隆抗体-1B5、3E8、4C7;1B5、3E8两株单抗能显著延长致死量白念珠菌感染小鼠存活时间,减少感染小鼠主要脏器组织中白念珠菌菌落形成单位,减轻组织病理改变;1B5单抗能识别白念珠菌胞壁外膜上相对分子质量约为32000抗原;并在体外能抑制白念珠菌孢子对人颊粘膜上皮细胞,胎儿脐静脉内皮细胞的粘附。结论 1B5、3E8是具有保护作用的抗白念珠孢子对人颊粘膜上皮细胞,胎儿脐静脉内皮细胞的粘附。结论 1B5、3E8是具有保护作用的抗白念珠菌单抗;其中1B5是抗白念珠菌胞壁外膜上相对分子质量为32000的抗原的单抗;1B5单抗可通过抑制白念珠菌对上皮细胞,内皮细胞的粘附,降低该菌的侵袭力。     

抗白念珠菌芽管单抗MAb03.2C1-C2特异性及临床初步应用研究

目的分析抗白念珠菌芽管胞壁外膜抗原单克隆抗体MAb03.2C1-C2的特异性及其应用于实验室检测的可行性。方法用临床分离白念珠菌、致病非白念的念珠菌的孢子、菌丝及常见的酵母菌、细菌做抗原包被,用间接免疫荧光(IIF)方法,对单抗特异性进行分析。取临床口腔念珠菌病患者的真菌涂片菌丝阳性的标本,用IIF方法检测。结果MAb03.2C1-C2仅与白念珠菌芽管或菌丝特异性地结合,与白念珠菌的孢子不发生结合。13种非白念的念珠菌孢子和菌丝、新型隐球菌、大肠杆菌、金黄色葡萄球菌、绿脓杆菌均不能与该单抗相结合。59例口腔念珠菌病真菌涂片IIF试验阳性的标本最终鉴定为白念珠菌(100%)。结论MAb03.2C1-C2对白念珠菌芽管有高度的特异性,并可用于口腔念珠菌病真菌涂片中白念珠菌的早期诊断。