A型新生隐球菌质粒克隆库的构建及其特征分析

新生隐球菌是一种条件致病菌，当机体免疫力减低时就可能侵犯人体，引起严重的隐球菌病，特别是隐球菌性脑膜炎的死亡率极高。隐球菌属包括１７个种７个变种，然而能够致病的只有新生隐球菌及其变种，且以血清型Ａ型最常见。我们以Ａ型新生隐球菌标准菌为实验菌株，构建了质粒克隆库，这是进一步进行分子生物学研究的基础，如在核酸水平探索致病机理，建立快速诊断方法等。一、材料和方法（一）菌株和质粒：①Ａ型新生隐球菌标准株Ｄ４８由世界卫生组织（ＷＨＯ）提供，本研究室保藏；②质粒ＰＵＣ１８及宿主菌ＪＭ１０３由复旦大学遗传学研…     

新生隐球菌AD血清型是新生变种?

新生隐球菌根据传统的分类方法和以往的观点,血清型AD型新生隐球菌一直被视为新生隐球菌新生变种\,但事实上,AD型新生隐球菌无论在地区分布、致病特征等方面,均与新生隐球菌新生变种中的A型和D型有明显不同^[2,3].本文采用变性梯度胶电泳(denaturing gradient gel electrophoresis,DGGE)技术和核酸序列分析,首次发现新生隐球菌AD型28S rDNA基因型与新生隐球菌新生变种的A、D型不同,而与新生隐球菌格特变种B、C型完全一致,现报道如下.     

新生隐球菌的基因结构分析

综述了近年来克隆和鉴定的新生隐球菌基因，并对其基因结构进行了分析，新生隐球菌基因的克隆，有助于研究新生隐球菌的分类地位，种系发生来源以及进行流行病学调查，更可为发展抗真菌治疗药物提供全新的靶目标。     

播散性隐球菌病

报告1例免疫功能正常的播散性隐球菌病.患儿女,11岁.全身包括肺、淋巴结、骨髓、肝脾、脑及皮肤等多器官受累,表现为痤疮样皮损.患儿临床症状进展缓慢,全身中毒症状轻微,嗜酸性粒细胞升高.皮损、淋巴结组织病理检查示组织细胞内、外大量隐球菌酵母细胞,PAS染色阳性.皮损、骨髓、淋巴液真菌培养阳性,菌种鉴定为新生隐球菌,经PCR扩增测序为新生隐球菌grubii变种.     

CAP64荚膜相关基因在新生隐球菌分型中的意义

新生隐球菌的多聚糖荚膜成分是新生隐球菌血清型分型的物质基础^[1],而CAP64荚膜相关基因是新生隐球菌荚膜合成的必需基因之一^[2],我们根据新生隐球菌血清型DCAP64荚膜基因的序列设计引物,将设计的引物扩增5个标准血清型新生隐球菌,已分型和未分型新生隐球菌临床分离株,以及其他的致病真菌,以寻找引物的型特异性。     

一步法提取隐球菌DNA

我们在对隐球菌临床和环境分离株进行基因分型的过程中，将隐球菌ＤＮＡ提取方法进行了改革，建立了一个简便、快速、高产的方法，现介绍如下。一、材料和方法（一）菌株：选择４株新生隐球菌（血清Ａ、Ｂ、Ｃ和Ｄ型），其中９４５７５２（Ａ型）由意大利米兰大学卫生与预...     

新生隐球菌不同变种所致小鼠原发性皮肤感染的比较

目的：探讨新生隐球菌不同变种在原发性小鼠新生隐球菌皮肤感染中的作用。方法：按照我们建立的原发性皮肤隐球菌感染模型的方法，将新生隐球菌新生变种标准野生株B3501与格特变种标准株ATCC32609分别皮内接种于免疫抑制与非抑制的BALB／c小鼠，皮损真菌培养与组织病理检查确证感染。观察2种隐球菌感染的病程，比较皮损形成与消退的平均时间。结果：2种变种的新生隐球菌皮下接种于BALB／c小鼠后，可以在免疫抑制与非抑制的BALB／c小鼠皮肤上产生丘疹、结节、溃疡、传染性软疣样皮损，皮损可以自愈，真菌培养与病理确证为隐球菌感染。2种菌株只在免疫正常小鼠的皮损形成时间上存在差异。结论：新生变种与格特变种的新生隐球菌均可以造成BALB／c小鼠相似的皮肤感染。推测2种变种对原发性皮肤感染的致病力可能无差异，新生变种发病较多可能与其分布有关。     

C57BL/6小鼠感染新生隐球菌对大脑皮质BDNF和bFGF表达的影响

目的：观察C57BL／6小鼠感染新生隐球菌后对大脑皮质BDNF和bFGF表达的影响，并探讨BDNF和bFGF在新生隐球菌感染过程中的作用及其意义。方法：48只C57BL／6雌性小鼠随机分为对照组、环磷酰胺组和试验组，免疫组化法检测大脑皮质BDNF和bFGF的表达。结果：尾静脉注入新生隐球菌后6h大脑皮质查见新生隐球菌，2h大脑皮质BDNF的表达明显高于对照组和环磷酰胺组（P〈0．01），0.5h大脑皮质bFGF的表达明显高于对照组和环磷酰胺组（P〈0.01）。结论：新生隐球菌感染后大脑皮质BDNF和bFGF表达增高，提示BDNF和bFGF对新生隐球菌性脑膜炎具有修复作用，有望成为潜在的防治新生隐球菌性脑膜炎的药物。     

PCR-RFLP鉴别念珠菌、曲霉和隐球菌的探讨

为快速鉴别３类医学上重要真菌－念珠菌、曲霉和隐球菌,应用聚合酶链反应－限制性片段长度多态（ＰＣＲ－ＲＦＬＰ）的分子生物学说法方法对白念珠菌、克柔念珠菌、季地蒙念珠菌、近平滑珠菌、热带念珠菌、乳念珠菌、新生隐球菌格梯变种、新生隐球菌新生变种、罗伦隐球菌格梯变种、新生隐球菌新生变种、罗伦隐球菌１、工霉、黄曲霉和杂色曲霉进行体外研究。新生隐新生变种、罗伦隐球菌、曲霉隐球菌放增出３１０ｂｐ经ＰＣＲ含珠菌、     

新生隐球菌磷脂酶的研究进展

新生隐球菌是重要的条件致病菌 ,它主要感染艾滋病患者和其他免疫功能受损的人群。磷脂酶作为新生隐球菌的一种潜在的毒力因子日益受到重视。综述近年来对新生隐球菌磷脂酶的检测及其性质、可能的致病机制和分子水平的研究进展     

荚膜在小鼠原发性皮肤新生隐球菌感染中的作用

目的探讨新生隐球菌多糖荚膜在小鼠原发性皮肤新生隐球菌感染中的作用。方法按照笔者建立的原发性皮肤隐球菌感染模型构建方法,将新生隐球菌标准野生株B3501与荚膜缺陷株cap64分别皮内接种于免疫抑制与非抑制的BALB/c小鼠,皮损真菌培养与组织病理检查确证感染。观察2种隐球菌感染的病程,比较皮损形成与消退的时间。结果野生株与荚膜缺陷株新生隐球菌皮下接种于BALB/c小鼠后,均可以在免疫抑制与非抑制的BALB/c小鼠皮肤上产生结节、丘疹、溃疡、传染性软疣样皮损,皮损可以自愈,真菌培养与病理确证为隐球菌感染。2种菌株感染的病程差异无显著性意义。结论野生株与荚膜缺陷株新生隐球菌均可以造成BALB/c小鼠相似的皮肤感染。荚膜可能不是小鼠皮肤隐球菌感染的主要毒力因子。     

国产氟康唑体外和体内抗真菌作用研究

国产氧康唑体外对２５株常见致病真菌以及体内对小鼠感染白念珠菌和新型隐球菌的抗菌作用与酮康唑和两性霉素Ｂ进行了比较。结果表明氟康唑在体外最小抑菌浓度（ＭＩＣ）不如其他两药。而口服氟康唑对感染小鼠的保护作用明显优于酮康唑和两性霉素Ｂ，口服治疗白念珠菌感染小鼠的５０％动物的有效浓度（ＥＢ５０）分别为０．８５ｍｇ／ｋｇ和３９．９８ｍｇ／ｋｇ，口服治疗新型隐球菌感染小鼠的ＥＤ５０分别为３．５４ｍｇ／ｋｇ和８１．６９ｍｇ／ｋｇ。     

Toll‐like receptor 4 attenuates a murine model of atopic dermatitis through inhibition of langerin‐positive DCs migration

Atopic dermatitis (AD) is a common chronic inflammatory skin disease that is often associated with skin barrier dysfunction leading to a higher frequency of bacterial and viral skin infections. Toll‐like receptor (TLR) 4 on resident skin cells was involved in sensing pathogens and eliciting pathogen‐specific innate and adaptive immune responses. Previous studies have demonstrated that TLR4 was linked to AD severity in context of pathogen infection. However, the immune regulatory role of TLR4 in AD remains to be defined. We here investigated the immune regulatory function of TLR4 in AD induced by repeated epicutaneous application of a hapten, 2,4‐dinitrochlorobenzene (DNCB). Our results showed that TLR4‐deficient (TLR4^?/?) mice exhibited more severe AD symptoms than WT mice after DNCB challenge. The DNCB‐treated TLR4^?/? mice also displayed higher expression levels of inflammatory cytokines and stronger Th2 response than WT counterparts. Moreover, the skin expression of thymic stromal lymphopoietin (TSLP), an important potential contributor to allergic inflammation, was significantly elevated in TLR4^?/? mice compared with that in WT mice upon DNCB administration. Furthermore, we demonstrated that the migration of langerin‐positive dendritic cells (DCs) into draining lymph nodes was enhanced in TLR4^?/? mice following DNCB challenge, which is partially dependent on the production of pro‐inflammatory cytokine TNF‐α. Together, these results determined that TLR4 affected the hapten‐induced skin inflammation in the absence of exogenous pathogen infection, suggesting that TLR4 not only regulates infection but also may serve as a modulator of the immune response during AD development.     

Beneficial effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on chronic dermatitis

Chronic dermatitis, such as contact dermatitis (CD) or atopic dermatitis (AD), is a longstanding inflammatory skin disease with cutaneous damage such as erosion, ulceration, and lichenification due to itch-induced scratching. The resultant lesion can be considered to be a kind of wound. The tissue inhibitor metalloproteases-2 (TIMP-2) accelerates wound healing by enhancing the proliferation and migration of epidermal keratinocytes and dermal fibroblasts; it is also a physiologic inhibitor of matrix metalloproteinases. The aim of this study was to test the effect of TIMP-2 on chronic dermatitis. NC/Kuj mice were sensitized with Dermatophagoides farinae (Df) extract. Eczema was induced by repeated applications of this mite allergen to the skin of 20 sensitized mice that were maintained under specific pathogen-free conditions. One group of 10 mice was then treated with topical TIMP-2 solution (0.1 ml, 0.5%) for 28 days, and the other with vehicle alone and the effects of TIMP-2 were evaluated macro- and microscopically. The effect on skin barrier function was estimated by measuring transepidermal water loss (TEWL). Scoring of gross skin findings showed that TIMP-2 significantly reduced the severity of eczema (P<0.05) on days 12-28. Histological examination revealed that TIMP-2 treated mice manifested lower degrees of hyperkeratosis, acanthosis, and spongiosis in the epidermis and fewer inflammatory cells in the dermis than vehicle-treated mice. There were significant reductions in the epidermal thickness and dermal inflammatory cells in the TIMP-2 treated animals (P<0.01); their TEWL was significantly decreased on day 28 (P<0.05). Our results suggest that NC/Kuj mice with Df extract-induced chronic eczema may be a useful model for investigating chronic dermatitis, and that TIMP-2 may be a good agent for treating chronic dermatitis as well as chronic ulcers.     

新生隐球菌荚膜毒力作用的研究进展

新生隐球菌是重要的机会致病真菌,其胞外包裹的荚膜被公认为隐球菌关键的毒力因子.近年来,研究初步证明荚膜有抗吞噬细胞吞噬作用.另外,荚膜在新生隐球菌感染扩散过程中以及诱导宿主细胞凋亡中均发挥了关键的作用.概述荚膜的抗吞噬作用及荚膜在感染扩散中的作用等,对这些机制的探讨将有助于理解新生隐球菌的致病机制,同时为治疗新生隐球菌病提供新的靶位点.

Abstract：

Cryptococcus neoformans is an important conditional pathogenic fungus and capsule is widely acknowledged to be a key virulence factor of it. It has been proved that capsule posseses antiphagocytosis acitivity, and plays an essential role in the dissemination of C. neoformans infection and induction of apoptosis in host cells. The review describes the roles of capsule in antiphagocytic process and dissemination of C. neoformans infection, which will help to better understand the pathogenic mechanism of cryptococcosis and to provide new therapeutic targets for cryptococcosis.     

Erythema migrans: a reassessment of diagnostic criteria for early cutaneous manifestations of borreliosis with particular emphasis on clonality investigations

BACKGROUND: Controversy exists about the relationship of borrelia infection with B-cell lymphomas because B-cell clonality has been identified in infiltrates that contained borrelia-specific DNA. Systematic clinicopathological, immunophenotypical and molecular pathological studies of early borreliosis are lacking. OBJECTIVES: (i) To clarify whether clonal B-cell populations are present already in early borreliosis of the skin (erythema migrans); (ii) to re-evaluate clinicopathological, immunophenotypical and molecular pathological criteria for diagnosis of erythema migrans. METHODS: Study of 34 patients with erythema migrans confirmed by polymerase chain reaction (PCR). Infiltrates were analysed by histopathological and immunohistochemical methods and multiplex PCR for clonal IgH rearrangements. RESULTS: Erythema migrans shows a broad spectrum of changes including sparse infiltrates of T lymphocytes, dense interstitial granulomatous infiltrates (CD68+), and pseudolymphomatous patterns with germinal centre formation. There were accompanying epidermal changes in 59% of patients, and plasma cells were an inconsistent finding. B cells were few when infiltrates were sparse, but increased disproportionately when infiltrates were dense. IgH rearrangement studies revealed one pseudo-oligoclonal, three pseudoclonal and three clonal infiltrates. Pseudoclonality was encountered when infiltrates contained only few B lymphocytes. CONCLUSIONS: Infiltrates in erythema migrans are dominated by T cells followed by CD68+ histiocytes and B lymphocytes. Plasma cells are an inconsistent finding. Pseudoclonality of IgH rearrangement is a result of infiltrates being sparse in B lymphocytes and represents a pitfall in molecular pathological diagnosis that can only be avoided by duplicate or triplicate tests. Incidental B-cell clonality may be encountered in patients with unequivocal erythema migrans and should not be interpreted as a malignant lymphomatous process induced by borrelia.     

The VEGF-C/VEGFR3 signaling pathway contributes to resolving chronic skin inflammation by activating lymphatic vessel function

BackgroundThe functions of lymphatic vessels are to drain the protein-rich lymph from the extracellular space, to maintain normal tissue pressure, and to mediate the immune response, particularly in inflammatory conditions.ObjectiveTo evaluate the function of the vascular endothelial growth factor (VEGF)-C/VEGF receptor (VEGFR)-3 signaling pathway in chronic skin inflammation.MethodsWe used adenovirus-mediated VEGF-C or VEGFR3-immunoglobulin (Ig) production and investigated the effects of VEGF-C/VEGFR3 signaling on the resolution of inflammation using the experimental chronic contact hypersensitivity (CHS) reaction mouse model.ResultsVEGF-C gene transfer promoted significant reduction of ear swelling and ear weight in CHS reaction-induced skin inflammation. Although, there was no significant difference in the number of lymphatic vessels, the number of infiltrating CD11b-positive inflammatory cells was significantly reduced in the VEGF-C group, which suggested that VEGF-C upregulated the drainage of interstitial fluid and inflammatory cells via lymphatic vessels. Furthermore, blockade of VEGFR3 expression resulted in a significant delay in the recovery from CHS reaction-induced skin inflammation. Lymphatic vessel size was enlarged and a significant increase of infiltrating CD11b inflammatory cells was observed in mice with VEGFR3-Ig gene transfer compared to control mice. These results suggested that blockade of VEGFR3 inhibited the drainage function of the lymphatic system.ConclusionThis study provides evidence that VEGF-C/VEGFR3 signaling plays an important role in the resolution of skin inflammation; the regulation of lymphatic function may have a great therapeutic potential in inflammatory skin diseases.     

Pathological findings in cumulative irritation induced by SLS and croton oil in hairless mice

It is known that the pathological features of acute irritant contact dermatitis are specific according to the irritant. However, in chronic irritant contact dermatitis, it is not clear whether specific patterns exist. To investigate whether the specific pathology of acute irritant contact dermatitis is sustained in chronic irritant contact dermatitis, sodium lauryl sulfate (SLS) and croton oil were applied 3x a week for 2 weeks on the dorsal skin of hairless mice using Finn Chambers. The pathologic changes induced by irritants at various concentrations were evaluated using H&E and Luna's staining, as well as immunohistochemistry for 5-bromo-2-deoxyuridine (BrdU), keratin 6 and loricrin. Our results showed that epidermal hyperplasia and inflammatory infiltration were relatively marked in the groups treated with higher concentrations of irritants. These features were more prominent in the 1% croton oil treated group than in the 0.25% SLS treated group. However, lower concentrations of irritants resulted in very similar histological changes, characterized by epidermal hyperplasia with minimal inflammatory infiltration, irrespective of the chemical. Our results suggest that the histological responses to irritants vary with concentration in cumulative irritation, as in acute irritation, but repetitive mild irritation may evoke common histological changes, characterized by epidermal hyperplasia with minimal inflammatory infiltration, irrespective of the chemical used.