日本血吸虫卵胚胎发育的组织化学研究

血吸虫病的病变和发病机理是由于虫卵在人体组织中引起肉芽肿所致。目前认为发育成熟含毛蚴的血吸虫卵分泌可溶性抗原物质,它致敏寄主后,使淋巴细胞、巨噬细胞和嗜酸粒细胞集聚在虫卵周围形成了肉芽肿,并证明血吸虫卵肉芽肿是属于迟发型超敏免疫反应(Warren,1972)。Warren等(1975)还认为日本血吸虫卵和曼氏血吸虫卵肉芽肿形成的有关免疫因素及发病机理可能有所不同。通过组织化学的途径对组织内日本血吸虫卵胚胎发育的研究,不仅有助于探讨血吸虫卵发育的生理及寄主组织和虫卵之间的相互关系,而且对血吸虫病的病原诊断方面亦有一定的实际意义。为此,作者对沉积于组织内的虫卵从受精卵细胞至形成毛蚴的胚胎发育全过程进行了一般组织学及组织化学的观察。     

日本血吸虫中国株次黄嘌呤鸟嘌呤磷酸核糖转移酶的克隆、表达及其免疫保护性研究

根据基因库中日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (HGPRT) EST (BU803192) 以及日本血吸虫成虫cDNA文库载体λgt11多克隆位点邻近核苷酸序列设计引物,以日本血吸虫成虫cDNA文库为模板,采用锚式PCR对SjHGPRT基因不完整的3′端和5′端进行扩增、测序,用电子软件拼接,获得SjHGPRT全长cDNA (1 270 bp),经序列分析,推断该片段含有编码SjHGPRT基因的完整阅读框,其编码基因与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SmHGPRT) 全长编码基因碱基一致性为82%,其理论推导的氨基酸组成与曼氏血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶的一致性约为83%. 将其编码基因克隆到表达载体pQE30上,在大肠杆菌M15中获得准确、高效表达,表达产物分子质量约为28 ku. 用日本血吸虫成虫抗原免疫血清对表达产物进行蛋白质印迹检测,在预测位置上出现明显的识别条带. 重组蛋白动物免疫保护性结果显示：在虫荷、每克肝卵、每克粪卵和雌子宫内卵数方面,疫苗组与对照组比较差异均具有显著性 (P < 0.05,P < 0.01). 结果表明,日本血吸虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 (SjHGPRT) 全长cDNA成功克隆并在大肠菌中得到表达,表达产物具有良好的抗原性和动物免疫保护效果,是一种潜在的具有部分免疫保护性的抗血吸虫病疫苗候选分子.     

血吸虫病肝纤维化形成的作用机制

血吸虫病(schistosomiasis)是世界上严重危害人类健康的人兽共患寄生虫病之一。在中国主要流行的是日本血吸虫病,其致病机制是由于血吸虫卵沉积于宿主肝内,形成虫卵肉芽肿,导致肝纤维化。血吸虫病肝纤维化免疫过程中,肝星状细胞、T细胞、B细胞及多种细胞因子的调节起着重要作用。现就血吸虫病肝纤维化形成机制的相关研究进展进行了综述。     

血吸虫病感染特征及其免疫病理机制的研究进展

血吸虫病是由血吸虫病裂体吸虫属血吸虫引起的一种寄生虫病,分布范围广,广泛分布在热带及亚热带的贫困偏远地区,其在公共卫生方面的重要性仅次于疟疾,该病已经造成了大量的残疾和死亡。据世界卫生组织(WHO)血吸虫病专家委员会1984年统计,全球感染血吸虫人口已达2亿。血吸虫种类繁多,寄生于人体的有6类,其中埃及血吸虫、曼氏血吸虫和日本血吸虫是主要的致病种类。血吸虫卵是血吸虫病的主要致病因子,寄生于宿主组织中的虫卵使宿主发生免疫病理反应,导致泌尿生殖系统(埃及血吸虫)炎症和梗阻性疾病、肠道疾病、肝脾炎症和肝纤维化(曼氏血吸虫和日本血吸虫)的发生。没有得到有效控制的早期血吸虫病发展到晚期,可引起包括上消化道出血、肝性脑病(日本血吸虫和曼氏血吸虫)、膀胱鳞状细胞癌(埃及血吸虫)和不孕不育症在内的严重并发症,主要对血吸虫病流行病学、临床表现、临床分期、并发症、致病机制及其免疫病理机制作一综述。     

扶正化瘀胶囊对鼠血吸虫肝纤维化的作用及机制探讨

目的:研究扶正化瘀胶囊对实验性血吸虫病肝纤维化的影响,探讨其相关作用机制。方法:70只昆明小鼠分为正常对照组(A)、模型组(B)、吡喹酮组(C)、INF-γ组(D)、扶正化瘀胶囊低剂量组(E)、中剂量组(F)、高剂量组(G),采用日本血吸虫尾蚴30条/只攻击感染小鼠建立血吸虫性肝纤维化模型。第6周用吡喹酮驱虫治疗后予扶正化瘀胶囊干预,第14周末观察血清透明质酸、层粘连蛋白、III型前胶原、IV型胶原水平,HE染色观察肝脏病理改变及虫卵结节变化,免疫组化检测PDGF-BB和I、III型胶原的表达。结果:扶正化瘀胶囊各剂量组肝脏病理损伤减轻,虫卵肉芽肿面积和数量明显减小;血清HA、LN、PC-III、C-IV水平显著下降(P<0.05);肝组织I、III型胶原和PDGF-BB表达明显低于模型组(P<0.05)。结论:扶正化瘀胶囊具有抗血吸虫性肝纤维化作用,降低肝组织PDGF-BB的表达可能是其抗纤维化作用机制之一。     

血吸虫的组织化学研究进展(一)碳水化合物、蛋白质、核酸、脂类及无机成分的组织化学研究

在人们急需对血吸虫作全面、深入地了解,以便对血吸虫病提出切实可行的防治对策的今天,应用组织化学(简称组化)方法研究血吸虫,正日益受到国内外学者的重视。通过这方面的研究,不仅能在一定程度上阐明血吸虫的代谢状态和生理功能,而且有助于了解抗血吸虫药物作用的机制;有助于对有关血吸虫病免疫学机理的探讨。同时,组化研究资料将对血吸虫生活史各期的体外培养起指导作用,亦可为寻找抗血吸虫新药与血吸虫疫苗提供线索。     

MICA/B基因多态性与血吸虫病相关性研究

本研究采用PCR-SSP与PCR-SBT方法对正常健康对照组与血吸虫病感染组、血吸虫病性重度肝纤维化病人组和轻度肝纤维化病人组中MICA/B基因进行分型,并比较各组基因的多态性。结果在血吸虫感染组与健康对照组中共发现13种MICA等位基因和5种MICA-STR基因型,MICA*012:01(11.58%vs 5.83%)、MI-CA*017(2.11%vs 0.00%)及MICA*027(3.16%vs 0.97%)在对照人群组较血吸虫病人组中分布频率较高,但Pc值显示没有统计学意义(Pc>0.05)。MICA-STR型别分析显示,MICA-STR与血吸虫病易感没有相关性,但MICA*A5基因型的分布频率在重度肝纤维化组显著高于轻度肝纤维化组(45.10%vs 26.92%,Pc<0.05)。在血吸虫病人组中一共检出10种MICB等位基因。在本研究人群中未发现与日本血吸虫感染显著相关的MICB等位基因。同时MICB等位基因多态性在重度纤维化组、轻度纤维化组、以及正常对照组相互之间均无显著的相关性。研究显示在血吸虫病人组中,MICA和MICB具有连锁不平衡,其中单倍型MICB*008-MICA*002:01和MICB*014-MICA*045在血吸虫病人组中显示具有显著的连锁不平衡。     

苦荞类黄酮糖基转移酶FtUFGT1的重组表达与酶学性质分析

黄酮类化合物是苦荞重要的功能性成分,其糖基化修饰可改变生物体内黄酮类化合物的稳定性、可溶性及生物活性。该研究基于苦荞转录组数据并以苦荞叶片中提取的总RNA为材料,利用RT PCR克隆了苦荞类黄酮糖基转移酶(UDP glycose:flavonoidglycosyltransferase,UFGT)基因FtUFGT1,采用无缝克隆方式构建其重组表达载体并转化大肠杆菌Rosetta(DE3)感受态,采用GST resin纯化重组表达的蛋白,采用高效液相色谱(HPLC)技术检测分析纯化后FtUFGT1的酶学性质。结果表明：(1)成功克隆的FtUFGT1编码区为1 413 bp,其编码470个氨基酸,并成功构建了FtUFGT1的重组表达载体pGEX 6p 1 FtUFGT1。(2)经转化苦荞FtUFGT1基因在大肠杆菌Rosetta(DE3)中得到可溶性的表达,并通过GST亲和层析纯化得到高纯度的苦荞FtUFGT1蛋白。(3)HPLC分析显示,以槲皮素为底物,苦荞FtUFGT1可催化异槲皮素的合成,比活力为9.174 U/mg;重组FtUFGT1的最适温度为30 ℃,最适pH为7.0,5%(V/V)的甲醇和0.5%(V/V)的Triton X 100可以显著抑制其活性。研究结果为深入揭示FtUFGT1的生物学功能及体外催化黄酮类衍生物的合成奠定了基础。     

纤维堆囊菌活性次级代谢产物的研究进展*

粘细菌中的纤维堆囊菌（Sorangium　cellulosum）在近20年的新型天然化合物的筛选中以其产生的化合物种类新、结构多样、作用机理特殊等而崭露头角,已经报道的200多种化合物及衍生物在抗细菌、抗肿瘤、抗病原真核生物中有很强的作用,是很好的天然药物筛选资源,具有极大的应用价值。本文将对10种作用机理具有典型意义的次级代谢产物的生物活性做一概述。     

蛇含委陵菜的化学成分及抗炎活性研究

为了研究蛇含委陵菜(Potentilla kleiniana)的化学成分及其抗炎活性,该文利用D-101大孔树脂、硅胶及Toyopearl HW-40F等色谱技术对蛇含委陵菜60%乙醇提取物进行分离纯化,通过NMR和HR-ESI-MS波谱数据鉴定化合物的结构,采用小鼠巨噬细胞(RAW 264.7)体外炎症模型评价化合物的抗炎活性。结果表明:(1)从蛇含委陵菜中分离得到15个化合物,分别鉴定为2-(heptadecanoyloxy)propane-1,3-diyl distearate(1)、9,12,13-三羟基-10,15-十八碳二烯酸(2)、9,12,13-三羟基-10,15-十八碳二烯酸甲酯(3)、2,2''-氧代双(1,4-二叔丁苯)(4)、大黄素(5)、大黄酚(6)、(6R,9R)-3-酮-α-紫罗兰醇-9-O-β-D-吡喃葡萄糖苷(7)、新穿心莲内酯(8)、甲基-α-D-呋喃果糖苷(9)、1-O-β-D-吡喃果糖-α-D-吡喃阿洛糖苷(10)、对香豆酸(11)、cesternosides A(12)、koaburaside(13)、荭草素(14)、异荭草素(15),均首次从委陵菜属植物中分离得到。(2)抗炎实验结果显示,化合物1-3、8、11-15具有一定NO释放抑制活性,其中化合物8在 25 μmol·L^-1浓度下抑制率为72.5%。该研究丰富了蛇含委陵菜的植物化学信息,明确了脂肪酸衍生物、酚性成分及二萜类成分是其抗炎活性成分,为蛇含委陵菜的进一步开发利用提供了理论依据。     

Schistosoma mansoni: angiotensin converting enzyme activity in mice under the influence of praziquantel and/or captopril.

Murine schistosomiasis is usually associated with hepatic granulomatous lesions together with high serum and granuloma angiotensin converting enzyme (ACE) activity. Praziquantel (PRZ) which is known to reduce granuloma size was studied to show whether this effect is related to changes in ACE activity. Furthermore, captopril was studied to show whether by inhibiting ACE activity, the drug could also affect granuloma size. PRZ, captopril, and their combination led to significant reduction in liver granuloma. However, in normal mice, captopril was shown to increase rather than decrease serum ACE. The decrease in ACE activity by PRZ was correlated with its curative effect in infected mice. However, in experimentally induced pulmonary granulomata, the drug reduced granuloma size without affecting ACE activity of either serum or granuloma. It may be concluded that reduction in ACE activity may be beneficial as far as diminution of granuloma size is concerned and irrespective of whether there is an active infection or not. The possible use of Captopril as an antihypertensive in bilharzial infections associated with hypertension would probably not adversely affect the granulomatous lesions.     

Potato apyrase reduces granulomatous area and increases presence of multinucleated giant cells in murine schistosomiasis

Granulomas are inflammatory tissue responses directed to a set of antigens. Trapped Schistosoma mansoni eggs promote productive granulomas in the tissues, and they are the main damage caused by schistosomiasis. Some S. mansoni antigenic proteins may have a direct involvement in the resolution of the granulomatous response. The ATP diphosphohydrolases isoforms of this parasite are immunogenic, expressed in all phases of the parasite life cycle and secreted by eggs and adult worms. Potato apyrase is a vegetable protein that cross-reactive with parasite ATP diphosphohydrolases isoforms. In this study, the vegetable protein was purified, before being inoculated in C57BL/6 mice that were later infected with cercariae. Sixty days after infection, adult worms were recovered, antibodies and cytokines were measured, and morphological granuloma alterations evaluated. Immunization of the animals induced significant levels of IgG and IgG1 antibodies and IFN-γ, IL-10 and IL-5 cytokines, but not IL-13, suggesting that potato apyrase is an immunoregulatory protein. Supporting this hypothesis, it was found that liver damage associated with schistosomiasis was mitigated, reducing the size of the areas affected by granuloma to 35% and increasing the presence of multinucleated giant cells in this environment. In conclusion, potato apyrase was found to be effective immunomodulatory antigen for murine schistosomiasis.     

In vitro delayed hypersensitivity granuloma formation: development of an antigen-coated bead model

Previously, we have described an in vitro model of granulomatous hypersensitivity around Schistosoma mansoni eggs in both the murine model of schistosomiasis and in human schistosomiasis. These studies describe a new model of in vitro granuloma formation that complexes soluble egg antigen from S. mansoni eggs, a partially purified protein derivative of Mycobacterium tuberculosis (PPD), or bovine serum albumin to carrier beads. Ultrastructural and morphologic evaluations demonstrate that there are initial macrophage interactions, followed by the recruitment of antigen-specific T cells that interact with and recruit macrophages, lymphocytes, granulocytes, and fibroblasts. Finally, there is a stage of granulomatous organization involving fibroblast proliferation and collagen deposition. The in vitro reactivity, defined by a quantitative granuloma index, correlates with in vivo granulomas around S. mansoni eggs in the livers of infected cell donor animals. In vitro granuloma formation against PPD-coated beads correlated with delayed cutaneous hypersensitivity against PPD, which was judged by footpad swelling. The reactions demonstrate antigenic specificity and were intrinsically modulated in a manner that is analogous to that previously shown with the in vitro egg granuloma model. This model of in vitro granuloma formation promises to be a useful tool for elucidating mechanisms of cellular immunity and regulation.     

Fibroblast stimulation in schistosomiasis. V. Egg granuloma macrophages spontaneously secrete a fibroblast-stimulating factor

Isolated intact egg granulomas from the liver of Schistosoma mansoni-infected mice have been previously shown to elaborate factors in vitro that can stimulate fibroblasts for biological functions that are of potential importance in the pathogenesis of hepatic fibrosis in schistosomiasis. We report here that cell cultures obtained from monodispersed granuloma cell suspensions, and specifically enriched for macrophages (95% to 100%) spontaneously elaborated fibroblast proliferation-stimulating activity in vitro. These cells possessed functional and phenotyptic characteristics of activated macrophages. In contrast, control peritoneal macrophages from uninfected mice lacked such phenotypic characteristics, and did not spontaneously elaborate fibrogenic activity in vitro. The granuloma macrophage activity was present, pre-formed within the isolated cells, and was continuously elaborated during 72 hr of incubation. By gel infiltration chromatography (Sephacryl S-200 sf), fibroblast-stimulating activity was identified in two pooled fractions, one with estimated molecular radius (Mr) of 46 kd to 57 kd and the other with Mr of 10 kd to 16 kd. Preparative isoelectric focusing in granular gel of crude macrophage culture supernatants identified peak activity in fractions with pI approximately 5. Two different serine esterase inhibitors had no effect on the ability of crude granuloma macrophage supernatants to stimulate fibroblast proliferation. Whereas crude and chromatographed fractions of granuloma macrophage supernatant were active for fibroblasts, they had minimal or no interleukin 1 (IL 1) activity when tested in a thymocyte proliferation assay. In contrast, resident peritoneal macrophages from the same infected mice spontaneously secreted substantial IL 1 and fibroblast-stimulating activity in vitro. We conclude that egg granuloma macrophages are activated in vivo to secrete fibrogenic molecules functionally distinct from IL 1, which might contribute to the pathogenesis of hepatic fibrosis in schistosomiasis.     

Evaluation of the role of angiogenic factors in the pathogenesis of schistosomiasis

Schistosomiasis is one disease produced by helminths, which affect many people in tropical areas. Granuloma formation is the main mechanism involved in the pathogenesis of this disease. Experimental studies have demonstrated angiogenesis (blood vessels formation from pre-existing vessels) in the initial phase of granuloma formation. In the present work, VEGF (vascular endothelial growth factor) levels were analyzed in sera from people diagnosed with different helminthic infections. Patients with schistosomiasis and filariasis had significantly high VEGF levels in compared with healthy people and patients diagnosed with hookworms. In addition, the effects of angiogenesis inhibition using anti-angiogenic factors (endostatin) were evaluated in a schistosomiasis murine model. A lesion decrease was observed in mice infected with Schistosoma mansoni and treated with endostatin. Finally, mechanisms of angiogenesis induction were studied and observed that cercariae antigens stimulated the angiogenic factors by host alveolar macrophages.     

Tolerization of mice to Schistosoma mansoni egg antigens causes elevated type 1 and diminished type 2 cytokine responses and increased mortality in acute infection

The granuloma that surrounds the Schistosoma mansoni egg is the cause of pathology in murine schistosomiasis, and its formation is driven by egg Ag-stimulated type 1 and type 2 cytokines. To determine the role of egg-driven immune responses during schistosome infection we rendered CBA/Ca mice unresponsive to schistosome eggs by combined cyclophosphamide treatment and thymectomy. In the early acute stages of schistosome infection, egg-tolerized mice suffered high mortalities. Granuloma size and deposition of collagen in the liver were significantly reduced in egg-tolerized mice. Similarly, limited granuloma responses were detected in the intestines of these mice, and this was associated with a >90% reduction in egg excretion. Histologically, egg-tolerized mice had exacerbated hepatocyte damage, with extensive microvesicular steatosis. Elevated plasma transaminase levels confirmed the damage to hepatocytes. Infected egg-tolerized mice had impaired proliferation responses to egg Ag but intact responses to worm Ag. Tolerized mice had diminished Ab responses to egg Ag and had a type 1 cytokine isotype pattern to worm Ag, with elevated IgG2a and diminished IgG1 and IgE. Egg-tolerized mice failed to down-regulate type 1 cytokines that are normally elicited during early schistosome infection. Hepatic granuloma cells from egg-tolerized mice were also type 1 cytokine dominated, with elevated frequencies of Tc1/Th1 and reduced Tc2/Th2 cells. This study demonstrates that mice tolerized to schistosome eggs have elevated type 1 cytokine responses with diminished type 2 responses and reduced anti-egg Ab during schistosome infection, and these effects are detrimental to the host.     

Differential immunoregulation of granulomatous inflammation, portal hypertension, and hepatic fibrosis in murine schistosomiasis mansoni

The present study investigates the immunoregulation of hepatic fibrosis in experimental murine schistosomiasis. Disease parameters measured were portal pressure, hepatic granuloma area, hepatic interstitial collagen, and glycosaminoglycans. C57BL/6 mice were infected with 25 Schistosoma mansoni cercariae and administered splenocytes or serum derived from uninfected mice or chronically infect syngeneic mice at 6 and 7 wk of infection. Immunologically mediated modulation was noted in animals receiving splenocytes derived from chronically infected mice. Both a reduction in portal pressure and hepatic granuloma areas were noted. Hepatic collagen content but not glycosaminoglycan content was reduced by the administration of either lymphoid cells or serum from chronically infected mice. The isotypic profile of hepatic interstitial collagens was modulated by both the administration of serum or lymphoid cells. Augmented levels of type III collagen was noted on administration of serum derived from chronically infected mice, whereas type I collagen levels were relatively elevated on administration of splenocytes. The data indicate that immunomodulation of inflammation and hepatic fibrosis can occur in murine schistosomiasis but that fibrotic events and inflammatory processes are independently modulated.     

Granuloma formation and killing functions of granuloma in congenitally athymic nude mice infected with Blastomyces dermatitidis and Paracoccidioides brasiliensis

We did this experiment to clarify the mechanism of granuloma formation and the killing functions of granuloma in nude mice against Blastomyces dermatitidis and Paracoccidioides brasiliensis infections. B. dermatitidis A-295 and P. brasiliensis B-1183 were the cultures used. Congenitally athymic nude (nu/ nu) mice and their heterozygous (nu/ +) littermates of BALB/ c background were the test animals. From culture A-295, 0.1% and 1% cell suspensions (wet weight) were prepared and from culture B-1183 0.2% and 2% cells suspensions were prepared. Ten nu/ + and 10 nu/ nu mice were allotted to each of four cell suspensions. For experimental blastomycosis each mouse was inoculated intravenously with 0.2 ml of the cell suspension of A-295 and for experimental paracoccidioidomycosis, with 0.15 ml of the cell suspension of B-1183. Two mice from each of the four groups were killed at 5, 8, 12, 18 and 25 days after inoculation, and histopathologic sections, stained with H&E or by PAS, were prepared from various internal organs.In the nu/ nu mice inoculated with B. dermatitidis A-295 granuloma was formed in the brain tissue after the 12th day. However, mononuclear cells, which formed the granuloma, did not kill the fungal cells, and the fungal cells continued to multiply in the granuloma. On the other hand, in the heart, kidney and fat tissue, their histopathological findings after the 18th day were clumps of fungal cells with slight cell reactions. In these organs the exertion of cell-mediated immunity was necessary for granuloma formation against the fungal infection.In the nu/ nu mice infected with P. brasiliensis B-1183, granuloma appeared in the brain and kidney after the 18th day and fungal cells continued to multiply within the granuloma as well as in those inoculated with culture A-295.These results show that the exertion of cell-mediated immunity plays an important role as the defense mechanisms of hosts against these fungal infections. However, PMNs also play an important role in the mouse's defense mechanisms against these fungal infections.We assume that the defense mechanisms of immunocompetent mice against B. dermatitidis or P. brasiliensis infection consist chiefly of two steps: in the first step phagocytosis by PMNs occurs and in the second step cell-mediated immunity enters into play.     

Elevated serum levels of TNF-alpha, sTNF-RI and sTNF-RII in murine schistosomiasis correlate with schistosome oviposition and circumoval granuloma formation

Levels of circulating tumour necrosis factor (TNF-alpha) and its soluble receptors are elevated in chronic human schistosomiasis. However, the kinetics of TNF-alpha production and release of its soluble receptors have not been studied in humans or animals. Here we report on increased levels of TNF-alpha and its soluble receptors in murine schistosomiasis, beginning with schistosome oviposition and circumoval granuloma formation. TNF-alpha, sTNF-RI and sTNF-RII were measured in sera of mice infected with Schistosoma mansoni each week for 10 weeks postinfection. TNF-alpha levels increased gradually in all mice during the first 3 weeks. From 6th week postinfection, TNF-alpha levels in infected mice increased steadily, whereas those of uninfected mice remained essentially unchanged. sTNF-RI levels fluctuated in all mice during the first 3 weeks, and increased in infected mice during the following 5 weeks. sTNF-RII levels were similar in all mice for the first 4 weeks but increased in infected mice throughout the remainder of the experimental period. These data may be helpful in understanding pathogenesis in schistosomiasis as TNF-alpha plays a crucial role in circumoval granuloma formation and adversely affects schistosome fecundity.