肝片形吸虫感染宿主细胞免疫的研究进展

肝片形吸虫病是一种危害严重的人兽共患病,肝片形吸虫主要感染反刍动物及包括人类在内的哺乳动物,是一个严重威胁健康、影响经济发展的公共卫生问题。目前治疗肝片形吸虫病的药物少、疗效有限。大量研究表明,肝片形吸虫受宿主免疫应答的调控,本文对肝片形吸虫病的发病机制、肝片形吸虫主要参与诱导的几种机体细胞免疫反应及相关的研究进展进行综述.以期为肝片形吸虫病的临床治疗提供资料。     

Fasciola hepatica infection downregulates Th1 responses in mice

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low-dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL-4 and IL-5, and low levels of IFN-gamma and IL-2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high-dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL-4 defective (IL-4-/-) C57BL/6 mice infected with 5 metacercariae produced less IFN-gamma and more IL-5 compared to their wild-type C57BL/6 counterparts, suggesting that IL-4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN-gamma and IL-2 was completely suppressed in the high-dose infection and this was also observed in IL-4-/- mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL-4 and IL-5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.     

Fasciola hepatica: rapid switching of stage-specific antigen expression after infection

Early developmental stages of the trematode parasite Fasciola hepatica were collected from the peritoneal cavity and liver of mice during a ten day infection period. Using one dimensional SDS-PAGE, differences in protein expression profiles were observed in stages collected on the same day post-infection in different physiological locations and also in juvenile parasites collected from the same location on different days post-infection. Four rat monoclonal antibodies were raised against the parasite using lymph nodes draining infected tissues. Three monoclonal antibodies, FY3-1, FY3-2 and FY4-7, were generated using cells from the mesenteric lymph node of recently challenged immune rats, while FY1-6 was derived from hepatic lymph node cells of a chronically infected rat. The epitope recognized by FY3-2 appeared to be carbohydrate in nature and was present on the surface of newly excysted juveniles. Immunoblots revealed that the antigens recognized by FY3-1, FY3-2 and FY4-7 were only expressed for two days after infection. In contrast, FY1-6 recognized epitopes expressed across all developmental stages screened. The rapid changes in protein and antigen expression observed during the early stages of infection may assist the parasite to evade the host immune response.     

Does Fasciola hepatica infection modify the response of acute hepatitis C virus infection to IFN-α treatment?

Immunologic response to acute hepatitis C is mainly a Th1 response, whereas fasciolopsiasis is associated with a diverse T-cell response. Interferon-alpha has immunomodulatory effects and enhances Th1 immune response. Fasciola infection could theoretically interfere with the Th1 immune response, even when acquired after an initial response to interferon-alpha treatment for acute hepatitis C virus (HCV) infection. We report here the case of a male patient who acquired Fasciola hepatica infection after an initial response to IFN-alpha therapy with a favorable outcome     

肝片形吸虫囊蚴抵抗力观察

目的观察肝片形吸虫囊蚴经加热、干燥、食用调料等处理后的存活情况。方法将肝片形吸虫囊蚴经1)加热:微波高火30s、60s;沸水10、30s和1、2、3min。2)干燥:室温下自然干燥24、40h。3)食源调料:蒜汁、酱油、醋以及3种等量混合液浸泡10、20、30min;38°白酒及饱和盐水各浸泡10、20、30min。4)4℃下保存10个月。结果 2种加热方式处理后囊蚴全部死亡;放置于玻片上的囊蚴干燥24h,死亡4只,附着于叶片上的囊蚴干燥40h,死亡2只;在蒜汁、酱油、醋中浸泡10、20、30min,囊蚴全部存活;在3种调料的等量混合液、饱和盐水及38°白酒中浸泡10、20、30min后,均有部分存活,另有一部分囊蚴结构清晰但虫体不动,但也无明显死囊蚴的特征;在4℃下保存10个月,仍有存活的囊蚴。结论常用的调料对囊蚴不起作用,自然干燥1~2d不能完全杀灭囊蚴,在低温潮湿环境中可较长时间保持活力。沸水加热10s或微波高火30s即可杀死囊蚴。     

Cell mediated immunity to liver fluke antigens during experimental Fasciola hepatica infection of cattle

Summary Cell mediated immunity (CMI) to Fasciola hepatica antigens was detected by lymphocyte proliferation and interleukin-2 (IL-2) production tests in cattle during the first 4 weeks following liver fluke infection. From the fifth week of infection onwards peripheral blood lymphocytes were unresponsive to fluke antigens by these in vitro tests. Investigations into the cause of this unresponsiveness found no evidence to suggest a selective loss of the IL-2 producing lymphocyte sub-population or that macrophages were responsible for the suppression or that antigen responsive cells were being sequestered in the spleen and mesenteric lymph nodes. Tests carried out on culture supernatants demonstrated the production during this unresponsive period of factors capable of suppressing in vitro responses to PHA. Although further tests failed to show antigen specific suppressor factors the presence of MHC restricted suppressor factors could not be ruled out. The early and transient appearance of CMI during F. hepatica infection of cattle indicates that delayed type hypersensitivity is unlikely to be important in protective immunity in cattle.     

Fasciola hepatica in the rat: immune responses associated with the development of resistance to infection

F. hepatica infections were established in rats and immune responses were monitored during primary and challenge infections. Antibody levels peaked at 3 weeks post-primary infection and at 6 days post-challenge infection. No significant correlation was found between antibody titre and number of flukes recovered at autopsy. Immunoblotting revealed a limited number of immunogenic polypeptides. When antibodies from these reactive bands were eluted and tested by IFA they all gave identical binding patterns: on juvenile fluke sections tegumental syncytium, tegumental cells and gut cells were labelled, while on adult sections the same antibodies labelled gut cells, reproductive tissue, excretory ducts and flame cells. This suggested that these tissues shared a common epitope or range of epitopes. A pronounced eosinophilia was observed throughout the infection period studied and infected liver sections showed massive cellular infiltration. Histochemical and immunocytochemical investigation of infected liver revealed the presence of large numbers of eosinophils, neutrophils, lymphocytes and phagocytes. The implications of these findings, to an understanding of concomitant immunity in the rat are discussed.     

Vaccination against Fasciola hepatica infection using a Schistosoma mansoni defined recombinant antigen,Sm14

Fasciola hepatica is the causative agent of fasciolosis in many areas in America, Europe, Africa, Asia and Australia. There is an urgent need for improved methods to control the parasite's transmission. We describe the use of an experimental vaccine based on a recombinant antigen cloned from another parasite, Schistosoma mansoni (Sm14), that induces high levels of cross protection in mice against both S. mansoni and F. hepatica. Sheep and mice vaccinated with Sm14 were significantly protected against challenge infection with metacercariae of Fasciola hepatica and were completely free of the histopathological hepatic damage related to liver fluke infection. The vaccine will provide a valuable new tool to aid in transmission control of this economically important disease.     

The immune response of regional lymph nodes during the early stages of Fasciola hepatica infection in cattle

In this study we examined regional immune responses to Fasciola hepatica infection in the natural ruminant host. Naïve cattle and those pre‐exposed to a drug‐abbreviated infection were subsequently challenged and lymph nodes extracted at slaughter. In vitro proliferation and cytokine production by mononuclear cells isolated from hepatic and mesenteric lymph nodes were measured after culture with whole fluke antigen (WFA). Hepatic lymph node cells had a significantly greater response to parasite antigen than mesenteric lymph node cells (P < 0·02), although there was no difference in the magnitude of the proliferative response between naïve and pre‐exposed challenged cattle. Mononuclear cells from hepatic lymph nodes produced interferon gamma, interleukin 2 and interleukin 4 after culture with parasite antigen, indicative of a mixed, T helper type 0, response. Comparison of the hepatic node response to a variety of F. hepatica antigens showed that proliferation was lower after culture with cathepsin‐L, than with a high molecular weight fraction, WFA or excretory–secretory antigen. Cell culture supernatant fluid from unstimulated hepatic lymph node cells showed an IgG1 response to antigens of 48, 52–70, 82, 96 and 120–190 kDa on Western blot in pre‐exposed, but not naïve, challenged animals.     

A study of the antigens produced by adult Fasciola hepatica maintained in vitro

Summary Metabolic antigen of adult Fasciola hepatica was prepared by concentrating the culture medium in which 100 adult flukes had been maintained in vitro for 7 days. Serological reactions between this antigen and sera from fluke-infected rabbits, rats and sheep were studied using immunodiffusion in agar and enzyme linked immunosorbent assay. The used medium contained a number of antigenic components and their various reactions with the sera from these different hosts were investigated.     

Fasciola hepatica: immunoprecipitation analysis of biosynthetically labelled antigens using sera from infected sheep

The antigenicity of the biosynthetically labelled somatic and excretory/secretory proteins of adult Fasciola hepatica was investigated over 20 weeks of an infection with F. hepatica in sheep. The antibody response was initially detected by ELISA 2 weeks after infection, and was sustained at this level for the remainder of the infection. Immunoprecipitation analysis indicated that a large number of proteins were recognized by the sheep, with several dominant antigens occurring in the 29-31 kilodalton molecular weight range. No differences were found between the antigens recognized by sheep in the early or late stages of infection suggesting that there are similarities between the antigens of the immature and mature forms of the parasite.     

The serum antibody response of infected rats, rabbits, lambs and calves to Fasciola hepatica adult antigen fractions separated by preparative flatbed iso-electrofocussing

Electrofocusing of F. hepatica adult antigen in granulated gels separated the material into 22 arbitrary fractions. Polyacrylamide gel analysis demonstrated groups of proteins with similar iso-electric points in 19 of the fractions. A microplate ELISA detected antigens in all 22 fractions and was used to test the serum antibody response in infected rats, rabbits, lambs and calves to these antigen fractions. The results indicated that rat and calf sera gave a much stronger response than rabbit and lamb sera to the antigens which separated above pH 7.0. It is possible that the greater efficiency of the rat and bovine immune systems in combating re-infection with F. hepatica may be related to this response.     

Fasciola hepatica cathepsin L cysteine proteinase suppresses Bordetella pertussis-specific interferon-γ production in vivo

We have previously demonstrated that Fasciola hepatica infection significantly reduced Bordetella pertussis-specific interferon (IFN)-gamma production in mice coinfected with B. pertussis or immunized with a pertussis whole cell vaccine (Pw). In the present study, we have identified parasite molecules capable of mimicking this suppressive effect of F. hepatica. Parenteral injection of mice with culture medium in which adult F. hepatica were maintained (excretory/secretory, ES, products) suppressed B. pertussis-specific IFN-gamma production in mice immunized with Pw. The suppressive effect of ES was abrogated by coinjecting ES with the cysteine proteinase inhibitor, Z-Phe-Ala-diazomethylketone. Furthermore, purified cathepsin L proteinase (FheCL), a major component of ES products, was capable of suppressing IFN-gamma production. The suppressive effect of FheCL was attenuated in interleukin (IL)-4 defective (IL-4-/-) mice. Therefore, FheCL released by F. hepatica is involved in the suppression of Th1 immune responses and this suppression may be dependent upon IL-4.     

肝片吸虫GST真核表达载体构建及重组蛋白活性分析

目的构建肝片吸虫谷胱甘肽S-转移酶（GST）的真核表达载体,研究重组蛋白的免疫原性。方法以构建好的重组质粒pET30a-FhGST为模板,利用PCR技术扩增肝片吸虫谷胱甘肽S-转移酶基因（GST）,连接真核表达载体pEGFP-N1,构建重组质粒pEGFP-GST,转染Hela细胞,荧光显微镜下观察绿色荧光,Western blotting检测重组蛋白表达情况。结果重组质粒pEGFP-GST在Hela细胞中获得了表达,Western blotting结果表明真核表达质粒表达的重组蛋白能与自然感染肝片吸虫的山羊阳性血清发生特异性反应。结论肝片吸虫GST真核表达载体构建成功,真核表达产物可与自然感染的山羊阳性血清发生特异性反应,具有生物学活性,可做为分子疫苗的候选进行进一步的研究。     

Neutralization of the activity of a Fasciola hepatica cathepsin L proteinase by anti-cathepsin L antibodies

Fasciola hepatica secretes a cathepsin L proteinase that is suggested to play an in vivo role in immunoprotection since the enzyme can cleave host immunoglobulin. In the present report, rabbit anti-cathepsin L IgG was shown to bind to the cathepsin L enzyme and inhibit its ability to cleave IgG molecules. Cathepsin L can prevent the antibody-mediated attachment of eosinophils to newly excysted juveniles in in vitro assays; however, if anti-cathepsin L IgG are mixed with the cathepsin L prior to the addition of the enzyme to the assay, eosinophils attach to the newly excysted juveniles. Thus it is possible to prepare antibodies that can bind and disrupt the biological activity of the F. hepatica cathepsin L.     

肝片吸虫组织蛋白酶L1基因cDNA序列的克隆与分析

目的　研制肝片吸虫的候选DNA疫苗。　方法　根据国际发表的相关序列设计引物,在引物端加酶切位点,应用RTPCR方法。　结果　成功地将肝片吸虫组织蛋白酶L1(FheCL1)基因cDNA序列克隆进真核表达载体pcDNA3.1中。对所克隆的FheCL1基因序列及其推导的氨基酸序列进行分析,发现其与已发表序列相比,核苷酸序列有4.3%的差异;推导的氨基酸序列有6.9%的差异。两者蛋白质二级结构主要有3个区域的区别,磷酸化位点有10个不同,但共有一由20个疏水氨基酸残基组成的保守区域。　结论　实验构建的pcDNA3.1FheCL1重组质粒是一种新型的抗肝片吸虫DNA疫苗候选重组质粒;肝片吸虫可能存在不同的亚种,两者的FheCL1基因编码的第1～20个氨基酸残基可能组成膜螺旋。     

Type I hypersensitivity reactions in intestinal mucosae from rats infected with Fasciola hepatica

Type I hypersensitivity reactions in the intestinal tract of sensitized animals may contribute to resistance to reinfection with Fasciola hepatica. Colonic mucosae isolated from previously infected rats were voltage clamped in Ussing chambers. Antigen was prepared as a crude homogenate from adult liver fluke. Assay of serum antibodies against fluke antigen confirmed sensitization. Antigen challenge evoked a rapid onset, transient inward current in sensitized but not in control preparations. Chloride secretion accounted for at least part of the response since the loop diuretic bumetanide reduced the effect of antigen by 61%. Anti-rat IgE mimicked the response to antigen and desensitized tissues to subsequent antigen challenge. Local synthesis of eicosanoids may mediate the response to antigen since the cyclo-oxygenase inhibitor piroxicam reduced the response by 76%. In contrast, mepyramine which is a histamine receptor antagonist did not alter the ion transport response evoked by antigen. Tetrodotoxin reduced the response to antigen by 53% implicating intrinsic neurons within the lamina propria as effector cells in the responses of this tissue to antigen. We propose that antigen stimulation of electrogenic chloride movement and consequent fluid secretion in vivo may contribute to a local effector mechanism in prevention of reinfection of previously sensitized hosts.     

Killing of newly excysted juveniles of Fasciola hepatica in sensitized rats

Newly excysted juvenile Fasciola hepatica were injected intraperitoneally into previously sensitized rats. They were recovered at various intervals post-injection and examined by light and electron microscopy. The challenge flukes were rapidly coated with peritoneal cells which, in the early stages, were mainly eosinophils. The eosinophils adhered closely to the flukes and degranulated on to their surface releasing cytochemically detectable peroxidase. Vacuoles formed in the tegument of the flukes beneath the adherent eosinophils and these increased in size until they spanned the width of the tegument, thus destroying its continuity. From 4 h post-injection some of the flukes had lost their teguments and were surrounded by phagocytic cells, particularly neutrophils; at this stage they were judged to be dead. During the first five minutes post-injection degranulating mast cells were associated with the challenge flukes. Their role in eosinophil chemotaxis is discussed as are the possible mechanisms of eosinophil adherence and degranulation. Using an anti-C₃ fluorescein conjugate, it was demonstrated that C₃ was not bound to the surface of challenge flukes either in vitro or in vitro in immune serum.     

肝片吸虫谷胱甘肽S-转移酶基因的克隆表达及活性分析

目的克隆和表达肝片吸虫谷胱甘肽S-转移酶基因(FhGST)以研究重组蛋白免疫学特性。方法参考Gen-Bank上发表的FhGST基因序列设计一对特异性引物,利用RT-PCR法扩增FhGST基因,将扩增产物克隆入pET-30a(+),构建重组表达载体。经PCR、双酶切和序列测定鉴定后转入E.coliBL21(DE3)宿主菌,经IPTG诱导表达,SDS-PAGE和Western blotting对该重组蛋白进行分析和鉴定。结果成功获得肝片吸虫GST基因全长为657bp的编码序列,SDS-PAGE结果表明重组蛋白分子量为31.3kDa,以包涵体形式表达。Western blotting结果证实重组蛋白能被其免疫的SD大鼠血清识别,表明其具有免疫原性;并且能与感染肝片吸虫的绒山羊血清发生反应,表明重组蛋白具有免疫反应性。结论成功的克隆了肝片吸虫谷胱甘肽S-转移酶基因,实现了在原核表达系统中高效表达重组蛋白,并具有良好的免疫学活性。     

T cell subset involvement in immune responses to Fasciola hepatica infection in cattle

The lymphocyte response to F. hepatica during a primary infection in cattle was analysed to define the role of T cell subsets in the immune response. Blood lymphocytes were isolated from eight cattle infected with F. hepatica via trickle infection over a ten-day period and from two non-infected controls. CD4+, CD8+ and gamma delta + T cells were depleted from whole lymphocyte populations by magnetic bead depletion. Lymphocytes from infected animals demonstrated a transient, but marked elevation in responsiveness to F. hepatica antigen between weeks 3 and 8 post-infection. Responses were attenuated by depletion of CD4+ and CD8+ T cells during this period. Depletion of gamma delta + T cells attenuated antigen responses at one time point only, and at an earlier stage post-infection than when alpha beta + T cells were depleted. Responses to antigen correlated positively with both hepatic fluke burden and with the degree of hepatic damage. This suggests that the cellular immune response was not protective. Antigen responses in gamma delta + T cell-depleted populations were also associated with post-mortem fluke burden and with hepatic damage. This suggests that gamma delta + T cells are involved in down regulating alpha beta + lymphocytes which may have a role in a non-protective or immunopathological immune responses.