阿苯达唑联合甘草酸二铵治疗广州管圆线虫感染小鼠体液和细胞免疫机制的研究
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摘要
广州管圆线虫(Angiostrongylus cantonensis)隶属于圆线虫目、后圆线虫科、后圆线虫亚科、管圆线虫属,由我国学者陈心陶于1933年发现。其在中间宿主、转续宿主、终末宿主和自然界中循环迁徙的过程就是其发育、繁衍的过程。人摄食生的或未完全熟的中间宿主,或被污染的水和食物可致血管圆线虫病(angiostrongliasis)。感染后虫体迁徙途经的乙状结肠、肝、心、肺、脑等器官均可能受到损伤,甚至脾、肾等虫体并未接触的部位都有可能因为免疫反应受损。患者临床表现多样,主要侵犯中枢神经系统,导致嗜酸粒细胞增多性脑膜炎、脑膜脑炎、脑(脊膜)脊髓炎、脑(脊)膜神经根炎等,也可以累及其他部位和器官,如肺部、眼部、乙状结肠等,只是发病例数极少。广州管圆线虫病全球散发,目前已报告3000多个病例,主要在东南亚及太平洋各岛屿流行,中国大陆、美洲大陆及加勒比海各岛屿有少量报道。我国以广西、海南、福建、台湾为主的南方7个省份为主要流行区域。
多种淡水螺作为中间宿主在更多地方的养殖和销售,使得广州管圆线虫病成为我国最具潜在危险的食源性寄生虫病,研究认为广州管圆线虫幼虫入侵中枢神经系统是其发育的关键阶段,其入侵宿主造成损伤的机制与多种因素有关。除虫体迁徙引起组织机械性损伤、虫体占位导致组织挤压损伤及颅内高压、虫体分泌的代谢物质或死亡虫体释放的物质对神经细胞的毒性等外,机体对虫体的防御、杀伤机制大多与免疫系统有关。据文献报道,广州管圆线虫感染后,嗜酸粒细胞迅速升高；多克隆B细胞激活迅速发生,产生大量IgE、IgM、IgG、IgA；体内CD4+、CD8+T细胞增多；外周血IL-4、IL-5、IL-10、IL-13等Th2类细胞因子明显升高,而IL-2、IFN-γ、TNF-α等Thl类细胞因子则无明显变化,Th1/Th2细胞因子比例失衡。免疫网络具有复杂性和多重性,T淋巴细胞在一定条件下又可以相互转化,因此仅仅归结为Th1/Th2细胞因子失衡,或许简化了广州管圆线虫病的发病机制,除了Th1、Th2淋巴细胞参与外,其它亚群T淋巴细胞也很有可能参与了发病。那么与Th1、Th2产生于同一前体细胞,且功能上有密切关系的CD4+CD25+ Treg亚群是否也参与了广州管圆线虫病的发病呢?它们在发病及治疗过程中有何变化?这些问题目前还未见报道。
T细胞重要组成部分——CD4+CD25+ T辅助细胞,又称天然调节细胞(regulatory T cells, Treg),由胸腺T细胞或外周naive T细胞发育而来,被某些特异抗原或特定因素激活后,主要通过细胞接触机制和分泌抑制性细胞因子IL-10和TGF-β等方式发挥对CD4+/CD8+ T细胞的非特异性抑制效应,在炎症反应中避免过强的免疫应答造成的机体组织损伤,但如果抑制过度则可能减弱机体抗感染作用,导致感染迁延不愈。该细胞亚群具有的免疫抑制和免疫无反应两大特征,在感染免疫、肿瘤免疫、免疫病理、移植物耐受、阻止自身免疫反应和维持机体免疫平衡等方面的作用日益受到关注,成为目前研究最为深入的调节性T细胞。近年来发现多种寄生虫感染过程中都有Treg的参与,目前尚无其在广州管圆线虫病发病中作用的相关文献报道。
阿苯达唑联合地塞米松是目前我国用用最广泛的广州管圆线虫病治疗方法。由于对长期大量使用地塞米松可能带来的不良反应的担心,多项研究提出多种替代治疗方法,但未见与地塞米松疗效的比较研究。甘草酸又称甘草甜素,是甘草中的主要活性成分。其中的甾体部分与肾上腺皮质激素结构相似,因此具有较强的抗炎、抗过敏、解毒等糖皮质激素样作用,而对下丘脑、垂体、肾上腺轴无明显影响,故无明显糖皮质激素的不良反应。近年来发现它还具有抗病毒、抗肿瘤、抑制MMP-9及免疫调节等作用。甘草酸可诱导干扰素产生、活化T细胞和自然杀伤细胞、纠正Th1/Th2失衡,从而发挥其免疫调节作用。甘草酸二铵是甘草酸的二铵盐,具有更稳定的结构和更强的生物活性,据其功能推测可以使用在广州管圆线虫病的治疗中,或许能代替糖皮质激素的使用,以减少不良反应的发生。但其是否真能发挥作用?疗效如何?发挥治疗作用的机制具体有哪些?与嗜酸粒细胞、IgE、Th1/Th2平衡、CD4+CD25+ Treg细胞是否有关?这些问题尚不明确。
本项目拟通过对不同感染时间广州管圆线虫病小鼠模型相关免疫指标的检测,了解免疫反应在其发病机制中的作用及具体机制；通过对不同方式治疗小鼠免疫指标的检测及比较,了解治疗机制,进一步认识免疫发病机制在广州管圆线虫病发病中所占的地位。本项目选取了广州管圆线虫病这一我国最具威胁的食源性传染病的发病机制进行深入研究,并拟通过对发病机制的进一步认识,找出更有效的治疗方法,具有实际应用价值。具体步骤如下：(1)观察并详细记录广州管圆线虫感染小鼠模型发病不同阶段的基础状况、临床表现、预后及生存时间。(2)比较小鼠感染后发病不同阶段外周血、脑脊液中嗜酸粒细胞比例、IgE、IgA、IgM、IgG、IL-5、eotaxin水平。(3)比较小鼠感染后发病不同阶段外周血Treg亚群数量及其中Foxp3表达。从比较结果中认识体液免疫、嗜酸粒细胞、Th2炎症反应、Treg细胞和组织病理变化在广州管圆线虫病发病中的作用。(4)分别以阿苯达唑联合地塞米松(AD)和阿苯达唑联合甘草酸二铵(AG)治疗感染小鼠,观察以上指标,探讨甘草酸二铵的治疗作用和可能机制。
通过实验优化了用以获取广州管圆线虫的人工消化法,最优实验流程为：人工消化液中含2g胃蛋白酶,37℃消化2小时后,过滤去除组织碎屑,以PBS洗涤滤液3次,自然沉淀后收集沉渣中幼虫。以50条广州管圆线虫感染期幼虫成功建立广州管圆线虫病小鼠模型。初步观察到E50模型小鼠约自13dpi开始逐渐出现毛发湿润、脊柱后凸、偏瘫和打转等体征,持续并到约16dpi全部死亡,确定生存时间、体征和体重作为疗效观察指标。以外周血嗜酸粒细胞和IgE的观察确定10和15dpi两个观察时间点；通过多个脏器的病理改变的观察,确认脑部是做显著病理损伤的部位,出现脑膜炎、蛛网膜下腔出血、炎症,嗜酸粒细胞广泛浸润,适宜用于疗效观察。实验观察感染和AD、AG治疗小鼠生存时间、体重和外貌体征,发现AD治疗能够治愈部分小鼠,使其生存时间延长、体重恢复、体征缓解,但对其余大多小鼠并不能改变死亡预后,只能稍延长生存时间,并不能改善体重减低和体征出现；AG疗效优于AD治疗,能够治愈全部小鼠,使其生存时间延长、体重恢复、完全不出现脊柱后凸、偏瘫和打转等严重体征,毛发湿润延后出现但能很快恢复。观察感染小鼠和AD、AG治疗小鼠10和15dpi时体征、嗜酸粒细胞比例、IgE、IgA、IgM、IgG、IL-5、eotaxin、IFN-γ、IFN-γ/ IL-5、CD4+CD25+、CD4+Foxp3+和CD4+CD25+Foxp3+细胞比例、颅内虫数、脑部病理变化,发现：(1)小鼠感染后外周血和脑脊液嗜酸粒细胞比例升高,AD和AG治疗都能抑制外周血和脑脊液嗜酸粒细胞,但AD停药后脑脊液嗜酸粒细胞比例反跳；(2)小鼠感染后血清和脑脊液IgE、IgA、IgM、IgG升高,AD能抑制血清和脑脊液IgE、脑脊液IgA、血清IgM和IgG,停药后均反跳；AG治疗与AD一样可以抑制脑脊液IgG,甚至能够更好抑制血清和脑脊液IgE和脑脊液IgA,但对脑脊液IgM无影响；(3)小鼠感染后血清和脑脊液IL-5和eotaxin升高,AD治疗能够抑制血清、脑脊液中的IL-5和eotaxin,停药无反跳,AG治疗不能抑制血清IL-5和eotaxin,甚至使得水平高于感染组,但能较AD更好抑制脑脊液IL-5和eotaxin;(4)小鼠感染后血清和脑脊液IFN-γ和IFN-γ/IL-5降低,AD治疗能够恢复血清、脑脊液中的IFN-γ和IFN-γ/IL-5,停药后仍继续恢复,AG治疗能更好恢复血清、脑脊液IFN-γ和IFN-γ/IL-5;(5)小鼠感染后外周血CD4+CD25+细胞比例升高、CD4+Foxp3+和CD4+CD25+Foxp3+细胞比例无变化,AD治疗与AG治疗一样能抑制这三种细胞的比例；(6)上述指标中与体征出现和加重关系最为密切的是脑脊液嗜酸粒细胞比例、血清和脑脊液IgE水平、外周血CD4+CD25+细胞比例,嗜酸粒细胞、IgE、IgG、eotaxin相互间关系密切,IL-5与eotaxin关系密切,IFN-γ和IFN-γ/IL-5与嗜酸粒细胞比例、IgE、IgG、IL-5和eotaxin负相关,外周血CD4+CD25+细胞比例与IgE关系密切,CD4+Foxp3+和CD4+CD25+Foxp3+细胞比例间关系密切；(7)感染小鼠脑部病理变化多样,以脑膜水肿,嗜酸粒及其他炎症细胞浸润和蛛网膜下腔嗜酸粒细胞浸润最为典型。AD能缓解部分小鼠的脑部病理损伤,其余则轻度改善；AG能够更好改善全部小鼠脑部病理损伤。
总结以上结果,推测广州管圆线虫病体液与细胞免疫发病机制如下：广州管圆线虫感染后虫体代谢分泌抗原及被杀死后释放的组织抗原激活抗原提呈细胞——淋巴系树突状细胞DC2,两者共同作用选择性活化Th2细胞。活化的Th2细胞介导体液免疫分泌免疫球蛋白的同时,分泌特异细胞因子,趋化嗜酸粒细胞进入虫体入侵组织。在免疫球蛋白的协助和活化作用下,嗜酸粒细胞发生脱颗粒,杀伤虫体。过强的Th2应答和嗜酸粒细胞分泌性毒素除杀伤虫体外,还可能造成宿主组织损伤。幼虫在进化过程中产生了抑制宿主免疫反应从而保护自身延长寄生时间的能力,虫体某些抗原激活DC2的同时,另一些进化了的分泌性抗原或通过死虫释放的抗原激活了另一群树突状细胞DCreg。在外周血特殊细胞因子的共同作用下,DCreg激活Treg细胞,Treg细胞发挥广谱免疫抑制作用,使Th2应答被控制在适度的程度,免疫球蛋白的合成和细胞因子的释放都被抑制,嗜酸粒细胞的趋化和杀伤作用就被适度抑制。另外,虫体某些抗原在体液免疫整体被抑制的同时,能够激活一些非特异性B细胞克隆,产生大量非特异性IgE,以封闭嗜酸粒细胞上的IgE受体,进一步抑制嗜酸粒细胞杀虫作用。这些抑制作用的结果就是宿主对虫体的杀伤作用减低,但又不是完全无应答,这样就使幼虫不被过多杀死,宿主也不会由于杀伤能力太弱致虫数过多消耗过大而死亡；同时免疫反应对宿主机体造成的损伤减轻,这样就不会由于针对虫体的免疫反应过于强烈导致宿主死亡。最终目的是使得宿主在带虫的同时能够更长时间、更好的存活,有利于虫体长期寄生。AD和AG都对广州管圆线虫病有治疗作用,AG疗效更好。阿苯达唑杀死幼虫,活虫抗原诱发的DC的激活减少,待已激活的DC代谢减少后体液免疫、Th2应答和Treg活化均减轻,但用药后死亡虫体却可能释放抗原物质引发不同的免疫应答,这一应答除包括激活Th2应答、体液免疫甚至Treg应答外,似乎并不抑制Th1应答。地塞米松这时发挥免疫抑制作用,除Th2应答以外,地塞米松还可以直接抑制嗜酸粒细胞、体液免疫、Thl应答和DC,或许还可能通过增多Treg来进一步抑制Th2应答,同时恢复血脑屏障功能。但这一抑制作用使得免疫损伤减轻的同时,不利于残存活虫的杀伤,且地塞米松直接诱导免疫细胞凋亡发挥抑制作用,并不清除抗原,故7天疗程结束后残存活虫分泌抗原和尚未完全清除的死虫抗原仍能引发免疫反应,导致嗜酸粒细胞、免疫球蛋白和Treg反跳。可能由于Treg反跳后的抑制作用,Th2应答并没有反跳,但少了地塞米松的抑制,Th1进一步回升,Th1/Th2因此回升。虽然具体机制不明,但甘草酸二铵与阿苯达唑联合治疗可能具有更好的杀虫作用,待已活化的DC代谢清除后,活虫分泌抗原引发的嗜酸粒细胞、体液免疫、Th2和Treg应答都将更轻微,但死虫抗原诱发的免疫反应却可能更早、更强烈或时间更长。而甘草酸二铵是通过增加Thl,改善Th1/Th2平衡来抑制Th2应答的,故作用缓慢而持久,10dpi时虽有作用但还不足以完全抑制Th2, Th1已经逐渐恢复。停药后在残余抗原物质的刺激下,Thl进一步回升,Treg也有反跳,使Th2逐渐降低,Th1/Th2恢复。两种治疗对Treg的作用没有差异,具体作用机制还有待进一步实验验证。甘草酸二铵或许能够更好恢复血脑屏障功能,使得脑脊液中各项免疫指标都有较好恢复。总之甘草酸二铵联合阿苯达唑用于广州管圆线虫病的治疗时疗效好于地塞米松联合阿苯达唑,其作用机制可能与更好的杀虫作用和血脑屏障功能恢复和免疫抑制有关。
本研究进一步深化了对广州管圆线虫非适宜宿主免疫发病机制的认识；找到一种更好的治疗方式,并探讨了它的治疗机制,为临床治疗提供了依据。
Angiostrongylus cantonensis (A. cantonensis) was found by Chen in 1933. The process of natural migration cycle in its intermediate host, transferring host, and final host is the process of development and reproduction. People are infected by eating raw or uncooked intermediate host, or contaminated water and food, and caused angiostrongliasis. After infected, the sigmoid colon, liver, heart, lung, brain and other organs of host which are passed by parasite migration are damaged. Even other organs, which are not passed by parasite migration, like spleen, kidneys, are damaged by immune response. Mainly effect of A. cantonensis is on the central nervous system, resulting in eosinophilia meningitis, meningoencephalitis, brain (meninges) myelitis, brain (spinal) ramitis. Now more than 3,000 cases angiostrongliasis have been reported all over the world. Mainly popular of it in Southeast Asia and the Pacific islands, and China, America and the Caribbean islands have a few. The main endemic region in China is Guangxi, Hainan, Fujian, Taiwan-based Southern 7 provinces.
A variety of freshwater snails as intermediate hosts are farmed and marketed in more places. Angiostrongliasis become the most potentially dangerous food-borne parasitic disease. Research suggests that the invading the central nervous system of A. cantonensis larvae is the critical stage of development. The mechanisms of invasion and damage host are complex. In addition to mechanical damage tissue caused by parasite migration, occupying intracranial hypertension and tissue injury, and neurotoxicity of larvae metabolites or toxic substances released by death parasite, the killing mechanism is mostly associated with the immune system. According to the literature, after A. cantonensis infection, eosinophils increased rapidly; polyclonal B cell activation occured rapidly, resulting in a large number of IgE, IgM, IgG, IgA; CD4+, CD8+ T cells increased; IL-4, IL-5, IL-10, IL-13 and other Th2 cytokines in peripheral blood were significantly increased, while IL-2, IFN-α, TNF-γand other Th1 cytokines were not changed significantly, caursing Th1/Th2 imbalance. Complexity and multiplicity of immune network and translation to each other of different T lymphocytes populations under certain conditions make the conclusion of Th1/Th2 imbalance is not the only immune pathogenesis of Angiostrongyliasis. Other T cell populations are also likely involved in the disease.
CD4+ CD25+ Treg develop from the thymus or peripheral naive T cells, activated by some specific antigen, then play a role on the CD4+/CD8+ T cells in non-specific inhibitory effect through cell contact mechanism and the secretion of inhibitory cytokines IL-10 and TGF-β. The effect of Treg is to avoid the body tissue injury causes by excessive immune response in the inflammatory response; however, if it is curbed excessively, it will make the body weaken to against infection. This cell subsets, with characteristics of immune suppression and immune anergy, act on infection immunity, tumor immunity, immunopathology, graft tolerance and autoimmune response and the maintenance of immune balance. In recent years, Treg are found involved in the course of in a variety of parasitic infections. There is no report about the role which Treg plays on the course of A. cantonensis infection.
The combination therapy of Albendazole and dexamethasone is widely used in A. cantonensis in China. Because of worrying about the adverse reactions, a number of studies proposed a variety of alternative therapies, but no comparison with the effect of dexamethasone. Glycyrrhizic acid, also known as glycyrrhizin (GL), is the main active ingredient in licorice. GL has the steroid, with similar structure like adrenal cortex hormones, so it has glucocorticoid-like effect, but no glucocorticoid-like adverse reactions. It has the effect of anti-inflammatory, anti-allergy, detoxification, anti-virus, anti-tumor, inhibition of MMP-9 and immune regulation and so on. It also can induce interferon, activated T cells and natural killer cells, correct Thl/Th2 imbalance, so play a role in immune regulation. It is speculated that it can be used in A. cantonensis, and might be able to replace the use of corticosteroids to reduce the incidence of adverse reactions. But can it really work? How effective? What are the mechanisms? Does eosinophils, IgE, Thl/Th2, CD4+ CD25+ Treg cells are related? These problems are not clear.
The project is planned to understand the role of immune response in A. cantonensis pathogenesis by detection of immune parameters in different time on course of infection and treatment. The project discussed the pathogenesis and found a more effective treatment of A. cantonensis, with practical of applications. Steps are as follows:(1)observing and detailed recording based conditions, clinical manifestations, prognosis and survival time of A. cantonensis-infected mice at different stages of disease. (2)Comparing levels eosinophils, IgE, IgA, IgM, IgG, IL-5, eotaxin in peripheral blood and CSF in different stages of infected mice. (3)Comparing the percentage of Treg subsets and expression of Foxp3 in it. Understanding the effect of eosinophils, humoral immunity, Th2 and Treg cells in the immune pathogenesis of A. cantonensis. (4)treating infected mice by combination therapy of albendazole+ dexamethasone and albendazole+ diammonium glycyrrhizinate respectively, discussed the effect and possible mechanisms.
The artificial digestion for A. cantonensis was optimized as follows:snail was deshelled, homogenized, digested by artificial digestive fluid containing 2g of pepsin, 37℃,2 hours, filtrated to remove tissue debris. The filtrate was washed 3 times with PBS and deposited for collection larvae. Mice modeles of angiostrongyliasis were established successfully by infection of 50 A. cantonensis larvae. The signs like hair wet, kyphosis, hemiplegic, and turned around in E50 mice began from 13dpi till died at aboutl6dpi. The survival time, signs and weight can be used as the indicators of efficacy. Make eosinophils percentage and IgE level in peripheral blood on 10 and 15dpi as the indicator of immune response. the brain is the site with the most serious pathological damage, includ meningitis, subarachnoid hemorrhage, inflammation, extensive infiltration of eosinophils. AD therapy can cure some mice, to prolonged survival, recover weight, relieve signs, but most of the rest mice can not be changed of the prognosis of death, only slightly prolonged survival time, improved weight reduced signs. AG therapy is more effective than AD, it can cure all the mice, prolong survival time, recover weight and relive signs.
Observating of eosinophils, IgE, IgA, IgM, IgG, IL-5, eotaxin, IFN-γ, IFN-γ/IL-5, CD4+ CD25+, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ cell percentage, number of intracranial worms, and the brain pathology in infected and AD, AG treated mice on 10 and 15dpi, we get these result as follow. (1) Eosinophils percentage in peripheral blood and CSF of infected mice was significantly higher. AD and AG therapy could significantly inhibit eosinophils, but it rebound after AD withdraw. (2)IgE, IgA, IgM, IgG in serum and CSF of infected mice increased significantly. AD could significantly inhibit IgE in serum and CSF, IgA in CSF, IgM and IgG in serum, but rebound after AD withdraw; AG could inhibit IgG in CSF as AD, inhibit IgE in serum and CSF and IgA in CSF more than AD, but could not inhibit IgM in CSF. (3) IL-5 and eotaxin in serum and CSF of infected mice were significantly increased. AD could significantly inhibit them in serum and CSF; AG could inhibit them in CSF, but not in serum. (4)IFN-γand IFN-γ/IL-5 in serum and CSF of infected mice were significantly decreased. AD therapy could significantly restore them, AG is more effective. (5)CD4+ CD25+ cells in peripheral blood of infected mice was significantly increased, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ cells was no significant change. AD and AG could significantly inhibite these three cells. Indicators, more closely related to beginning and sustaining of signs, are eosinophils in CSF, IgE levels in serum and CSF, CD4+ CD25+ cell percentage in peripheral blood. (6)Eosinophils cells, IgE, IgG and eotaxin are closely related to each others. IFN-γand IFN-γ/IL-5 are negative correlated with eosinophils, IgE, IgG, IL-5 and eotaxin. CD4+ CD25+ cells in peripheral blood were closely related with IgE, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ cell percentage were closely related to each other. (7) In variety of pathological changes, meningeal edema and subarachnoid eosinophil infiltration are the most typical. AD mouse can relieve the pathological damage of some mice, the rest are slightly improved; AG are better able to improve the brain pathological injury in all the mice.
Summary, the immune pathogenesis of A. cantonensis can be described as follows: after infection of A. cantonensis, antigen activated antigen presenting cells-dendritic cells (DC2). Antigen and DC2 selectively activate Th2 cells. Activated Th2 cell mediate humoral immune to secrete immunoglobulin, at the same time, secrete specific cytokines and chemokines, sent eosinophils into inflammation tissue. Eosinophils release granules to kill worm with the assistance of the immunoglobulin. Too strong Th2 response and toxins secreted by eosinophils kill worm, and cause host tissue damage. In the evolutionary process, Larvae produce the ability of inhibition host immune responses to protect themselves and extend the parasitic time. Some parasites antigen activated DC2, while others secreted antigen activated another group of dendritic cells (DCreg). With the action of special cytokines in the peripheral blood, DCreg activated Treg cells, to play an appropriate immunosuppressive effects, so that Th2 response is controlled at moderate levels, immunoglobulin producting and cytokine releasing were inhibited, and eosinophil chemotaxis and killing effect were inhibited moderately. In addition, certain antigens of the parasites could enable some non-specific B cell clones, resulting in a large number of non-specific IgE, to block IgE receptor on eosinophil and inhibit eosinophil to kill worm. The result is killing effect on parasite of host was reduced. The number of worms will not be too large to result in excessive consumption. The damage caused by immune response of host was reduced significantly, so host would not dead because of excessive immune respond. The ultimate goal is to make the host can be survive longer and better, it conducive to long-term parasitic worms. AD and AG have a therapeutic effect on A. cantonensis both, AG is more effectively.
AD therapy can kill worms, inhibit eosinophil, Th2 immune respond, and Treg, and recover Thl immune respond, but eosinophil, humoral immunity are, and Treg rebound after withdraw. Albendazole kills larvae, DC2 and DCreg activation induced by worm antigen were reduced, humoral immunity, Th2 and Treg responses are reduced, but the death worm antigens might lead to different immune response, including activation of humoral immune response, Th2 response, and Treg, but not to inhibit the Th1 response. Then dexamethasone plays inhibiting role on immune respond. In addition to Th2 response, dexamethasone can also directly inhibit eosinophil, humoral immunity, Treg, and restore blood-brain barrier function, but this action inhibit immune injury, while not conducive to kill worm. Dexamethasone play a direct inhibitory effect by inducting apoptosis of immune cells, not eliminate antigen and inhibiting DC, so the secretion of live worm and not yet fully clear antigen of dead worm can still trigger an immune response after withdraw of 7 days AD therapy. Th2 response did not rebound may be due to rebound Treg, but without inhibition of dexamethasone, Thl recover more, and then Th1/Th2 recovered more. Although the specific mechanism is unknown, AG therapy may have better killing effect. Until the DC has been cleared, eosinophils, humoral immunity, Th2 and Treg responds activated by live worm secrete antigen would be more modest, but the immune response induced dead worm antigen is likely earlier, more intense or longer. The diammonium glycyrrhizinate suppress Th2 response by increasing Th1 and improving Th1/Th2 balance, so the effect is slow and persistent. Thl has been gradually restored, but DG is not enough to completely suppress Th2 on 10dpi. After withdraw, Th1 respond recovered, Treg rebound also, so Th1/Th2 recovered and Th2 was inhibited. Both treatments did not influence Treg, the specific mechanism remains to be further verified by experiments. Diammonium glycyrrhizinate may be better able to recover blood-brain barrier function, so the immune parameters in CSF had a better recovery. In short, AG is effectively on A. cantonensis than AD, and its mechanism may be related to a better killing effect, the blood-brain barrier function recovery and immune inhibition.
多种淡水螺作为中间宿主在更多地方的养殖和销售,使得广州管圆线虫病成为我国最具潜在危险的食源性寄生虫病,研究认为广州管圆线虫幼虫入侵中枢神经系统是其发育的关键阶段,其入侵宿主造成损伤的机制与多种因素有关。除虫体迁徙引起组织机械性损伤、虫体占位导致组织挤压损伤及颅内高压、虫体分泌的代谢物质或死亡虫体释放的物质对神经细胞的毒性等外,机体对虫体的防御、杀伤机制大多与免疫系统有关。据文献报道,广州管圆线虫感染后,嗜酸粒细胞迅速升高；多克隆B细胞激活迅速发生,产生大量IgE、IgM、IgG、IgA；体内CD4+、CD8+T细胞增多；外周血IL-4、IL-5、IL-10、IL-13等Th2类细胞因子明显升高,而IL-2、IFN-γ、TNF-α等Thl类细胞因子则无明显变化,Th1/Th2细胞因子比例失衡。免疫网络具有复杂性和多重性,T淋巴细胞在一定条件下又可以相互转化,因此仅仅归结为Th1/Th2细胞因子失衡,或许简化了广州管圆线虫病的发病机制,除了Th1、Th2淋巴细胞参与外,其它亚群T淋巴细胞也很有可能参与了发病。那么与Th1、Th2产生于同一前体细胞,且功能上有密切关系的CD4+CD25+ Treg亚群是否也参与了广州管圆线虫病的发病呢?它们在发病及治疗过程中有何变化?这些问题目前还未见报道。
T细胞重要组成部分——CD4+CD25+ T辅助细胞,又称天然调节细胞(regulatory T cells, Treg),由胸腺T细胞或外周naive T细胞发育而来,被某些特异抗原或特定因素激活后,主要通过细胞接触机制和分泌抑制性细胞因子IL-10和TGF-β等方式发挥对CD4+/CD8+ T细胞的非特异性抑制效应,在炎症反应中避免过强的免疫应答造成的机体组织损伤,但如果抑制过度则可能减弱机体抗感染作用,导致感染迁延不愈。该细胞亚群具有的免疫抑制和免疫无反应两大特征,在感染免疫、肿瘤免疫、免疫病理、移植物耐受、阻止自身免疫反应和维持机体免疫平衡等方面的作用日益受到关注,成为目前研究最为深入的调节性T细胞。近年来发现多种寄生虫感染过程中都有Treg的参与,目前尚无其在广州管圆线虫病发病中作用的相关文献报道。
阿苯达唑联合地塞米松是目前我国用用最广泛的广州管圆线虫病治疗方法。由于对长期大量使用地塞米松可能带来的不良反应的担心,多项研究提出多种替代治疗方法,但未见与地塞米松疗效的比较研究。甘草酸又称甘草甜素,是甘草中的主要活性成分。其中的甾体部分与肾上腺皮质激素结构相似,因此具有较强的抗炎、抗过敏、解毒等糖皮质激素样作用,而对下丘脑、垂体、肾上腺轴无明显影响,故无明显糖皮质激素的不良反应。近年来发现它还具有抗病毒、抗肿瘤、抑制MMP-9及免疫调节等作用。甘草酸可诱导干扰素产生、活化T细胞和自然杀伤细胞、纠正Th1/Th2失衡,从而发挥其免疫调节作用。甘草酸二铵是甘草酸的二铵盐,具有更稳定的结构和更强的生物活性,据其功能推测可以使用在广州管圆线虫病的治疗中,或许能代替糖皮质激素的使用,以减少不良反应的发生。但其是否真能发挥作用?疗效如何?发挥治疗作用的机制具体有哪些?与嗜酸粒细胞、IgE、Th1/Th2平衡、CD4+CD25+ Treg细胞是否有关?这些问题尚不明确。
本项目拟通过对不同感染时间广州管圆线虫病小鼠模型相关免疫指标的检测,了解免疫反应在其发病机制中的作用及具体机制；通过对不同方式治疗小鼠免疫指标的检测及比较,了解治疗机制,进一步认识免疫发病机制在广州管圆线虫病发病中所占的地位。本项目选取了广州管圆线虫病这一我国最具威胁的食源性传染病的发病机制进行深入研究,并拟通过对发病机制的进一步认识,找出更有效的治疗方法,具有实际应用价值。具体步骤如下：(1)观察并详细记录广州管圆线虫感染小鼠模型发病不同阶段的基础状况、临床表现、预后及生存时间。(2)比较小鼠感染后发病不同阶段外周血、脑脊液中嗜酸粒细胞比例、IgE、IgA、IgM、IgG、IL-5、eotaxin水平。(3)比较小鼠感染后发病不同阶段外周血Treg亚群数量及其中Foxp3表达。从比较结果中认识体液免疫、嗜酸粒细胞、Th2炎症反应、Treg细胞和组织病理变化在广州管圆线虫病发病中的作用。(4)分别以阿苯达唑联合地塞米松(AD)和阿苯达唑联合甘草酸二铵(AG)治疗感染小鼠,观察以上指标,探讨甘草酸二铵的治疗作用和可能机制。
通过实验优化了用以获取广州管圆线虫的人工消化法,最优实验流程为：人工消化液中含2g胃蛋白酶,37℃消化2小时后,过滤去除组织碎屑,以PBS洗涤滤液3次,自然沉淀后收集沉渣中幼虫。以50条广州管圆线虫感染期幼虫成功建立广州管圆线虫病小鼠模型。初步观察到E50模型小鼠约自13dpi开始逐渐出现毛发湿润、脊柱后凸、偏瘫和打转等体征,持续并到约16dpi全部死亡,确定生存时间、体征和体重作为疗效观察指标。以外周血嗜酸粒细胞和IgE的观察确定10和15dpi两个观察时间点；通过多个脏器的病理改变的观察,确认脑部是做显著病理损伤的部位,出现脑膜炎、蛛网膜下腔出血、炎症,嗜酸粒细胞广泛浸润,适宜用于疗效观察。实验观察感染和AD、AG治疗小鼠生存时间、体重和外貌体征,发现AD治疗能够治愈部分小鼠,使其生存时间延长、体重恢复、体征缓解,但对其余大多小鼠并不能改变死亡预后,只能稍延长生存时间,并不能改善体重减低和体征出现；AG疗效优于AD治疗,能够治愈全部小鼠,使其生存时间延长、体重恢复、完全不出现脊柱后凸、偏瘫和打转等严重体征,毛发湿润延后出现但能很快恢复。观察感染小鼠和AD、AG治疗小鼠10和15dpi时体征、嗜酸粒细胞比例、IgE、IgA、IgM、IgG、IL-5、eotaxin、IFN-γ、IFN-γ/ IL-5、CD4+CD25+、CD4+Foxp3+和CD4+CD25+Foxp3+细胞比例、颅内虫数、脑部病理变化,发现：(1)小鼠感染后外周血和脑脊液嗜酸粒细胞比例升高,AD和AG治疗都能抑制外周血和脑脊液嗜酸粒细胞,但AD停药后脑脊液嗜酸粒细胞比例反跳；(2)小鼠感染后血清和脑脊液IgE、IgA、IgM、IgG升高,AD能抑制血清和脑脊液IgE、脑脊液IgA、血清IgM和IgG,停药后均反跳；AG治疗与AD一样可以抑制脑脊液IgG,甚至能够更好抑制血清和脑脊液IgE和脑脊液IgA,但对脑脊液IgM无影响；(3)小鼠感染后血清和脑脊液IL-5和eotaxin升高,AD治疗能够抑制血清、脑脊液中的IL-5和eotaxin,停药无反跳,AG治疗不能抑制血清IL-5和eotaxin,甚至使得水平高于感染组,但能较AD更好抑制脑脊液IL-5和eotaxin;(4)小鼠感染后血清和脑脊液IFN-γ和IFN-γ/IL-5降低,AD治疗能够恢复血清、脑脊液中的IFN-γ和IFN-γ/IL-5,停药后仍继续恢复,AG治疗能更好恢复血清、脑脊液IFN-γ和IFN-γ/IL-5;(5)小鼠感染后外周血CD4+CD25+细胞比例升高、CD4+Foxp3+和CD4+CD25+Foxp3+细胞比例无变化,AD治疗与AG治疗一样能抑制这三种细胞的比例；(6)上述指标中与体征出现和加重关系最为密切的是脑脊液嗜酸粒细胞比例、血清和脑脊液IgE水平、外周血CD4+CD25+细胞比例,嗜酸粒细胞、IgE、IgG、eotaxin相互间关系密切,IL-5与eotaxin关系密切,IFN-γ和IFN-γ/IL-5与嗜酸粒细胞比例、IgE、IgG、IL-5和eotaxin负相关,外周血CD4+CD25+细胞比例与IgE关系密切,CD4+Foxp3+和CD4+CD25+Foxp3+细胞比例间关系密切；(7)感染小鼠脑部病理变化多样,以脑膜水肿,嗜酸粒及其他炎症细胞浸润和蛛网膜下腔嗜酸粒细胞浸润最为典型。AD能缓解部分小鼠的脑部病理损伤,其余则轻度改善；AG能够更好改善全部小鼠脑部病理损伤。
总结以上结果,推测广州管圆线虫病体液与细胞免疫发病机制如下：广州管圆线虫感染后虫体代谢分泌抗原及被杀死后释放的组织抗原激活抗原提呈细胞——淋巴系树突状细胞DC2,两者共同作用选择性活化Th2细胞。活化的Th2细胞介导体液免疫分泌免疫球蛋白的同时,分泌特异细胞因子,趋化嗜酸粒细胞进入虫体入侵组织。在免疫球蛋白的协助和活化作用下,嗜酸粒细胞发生脱颗粒,杀伤虫体。过强的Th2应答和嗜酸粒细胞分泌性毒素除杀伤虫体外,还可能造成宿主组织损伤。幼虫在进化过程中产生了抑制宿主免疫反应从而保护自身延长寄生时间的能力,虫体某些抗原激活DC2的同时,另一些进化了的分泌性抗原或通过死虫释放的抗原激活了另一群树突状细胞DCreg。在外周血特殊细胞因子的共同作用下,DCreg激活Treg细胞,Treg细胞发挥广谱免疫抑制作用,使Th2应答被控制在适度的程度,免疫球蛋白的合成和细胞因子的释放都被抑制,嗜酸粒细胞的趋化和杀伤作用就被适度抑制。另外,虫体某些抗原在体液免疫整体被抑制的同时,能够激活一些非特异性B细胞克隆,产生大量非特异性IgE,以封闭嗜酸粒细胞上的IgE受体,进一步抑制嗜酸粒细胞杀虫作用。这些抑制作用的结果就是宿主对虫体的杀伤作用减低,但又不是完全无应答,这样就使幼虫不被过多杀死,宿主也不会由于杀伤能力太弱致虫数过多消耗过大而死亡；同时免疫反应对宿主机体造成的损伤减轻,这样就不会由于针对虫体的免疫反应过于强烈导致宿主死亡。最终目的是使得宿主在带虫的同时能够更长时间、更好的存活,有利于虫体长期寄生。AD和AG都对广州管圆线虫病有治疗作用,AG疗效更好。阿苯达唑杀死幼虫,活虫抗原诱发的DC的激活减少,待已激活的DC代谢减少后体液免疫、Th2应答和Treg活化均减轻,但用药后死亡虫体却可能释放抗原物质引发不同的免疫应答,这一应答除包括激活Th2应答、体液免疫甚至Treg应答外,似乎并不抑制Th1应答。地塞米松这时发挥免疫抑制作用,除Th2应答以外,地塞米松还可以直接抑制嗜酸粒细胞、体液免疫、Thl应答和DC,或许还可能通过增多Treg来进一步抑制Th2应答,同时恢复血脑屏障功能。但这一抑制作用使得免疫损伤减轻的同时,不利于残存活虫的杀伤,且地塞米松直接诱导免疫细胞凋亡发挥抑制作用,并不清除抗原,故7天疗程结束后残存活虫分泌抗原和尚未完全清除的死虫抗原仍能引发免疫反应,导致嗜酸粒细胞、免疫球蛋白和Treg反跳。可能由于Treg反跳后的抑制作用,Th2应答并没有反跳,但少了地塞米松的抑制,Th1进一步回升,Th1/Th2因此回升。虽然具体机制不明,但甘草酸二铵与阿苯达唑联合治疗可能具有更好的杀虫作用,待已活化的DC代谢清除后,活虫分泌抗原引发的嗜酸粒细胞、体液免疫、Th2和Treg应答都将更轻微,但死虫抗原诱发的免疫反应却可能更早、更强烈或时间更长。而甘草酸二铵是通过增加Thl,改善Th1/Th2平衡来抑制Th2应答的,故作用缓慢而持久,10dpi时虽有作用但还不足以完全抑制Th2, Th1已经逐渐恢复。停药后在残余抗原物质的刺激下,Thl进一步回升,Treg也有反跳,使Th2逐渐降低,Th1/Th2恢复。两种治疗对Treg的作用没有差异,具体作用机制还有待进一步实验验证。甘草酸二铵或许能够更好恢复血脑屏障功能,使得脑脊液中各项免疫指标都有较好恢复。总之甘草酸二铵联合阿苯达唑用于广州管圆线虫病的治疗时疗效好于地塞米松联合阿苯达唑,其作用机制可能与更好的杀虫作用和血脑屏障功能恢复和免疫抑制有关。
本研究进一步深化了对广州管圆线虫非适宜宿主免疫发病机制的认识；找到一种更好的治疗方式,并探讨了它的治疗机制,为临床治疗提供了依据。
Angiostrongylus cantonensis (A. cantonensis) was found by Chen in 1933. The process of natural migration cycle in its intermediate host, transferring host, and final host is the process of development and reproduction. People are infected by eating raw or uncooked intermediate host, or contaminated water and food, and caused angiostrongliasis. After infected, the sigmoid colon, liver, heart, lung, brain and other organs of host which are passed by parasite migration are damaged. Even other organs, which are not passed by parasite migration, like spleen, kidneys, are damaged by immune response. Mainly effect of A. cantonensis is on the central nervous system, resulting in eosinophilia meningitis, meningoencephalitis, brain (meninges) myelitis, brain (spinal) ramitis. Now more than 3,000 cases angiostrongliasis have been reported all over the world. Mainly popular of it in Southeast Asia and the Pacific islands, and China, America and the Caribbean islands have a few. The main endemic region in China is Guangxi, Hainan, Fujian, Taiwan-based Southern 7 provinces.
A variety of freshwater snails as intermediate hosts are farmed and marketed in more places. Angiostrongliasis become the most potentially dangerous food-borne parasitic disease. Research suggests that the invading the central nervous system of A. cantonensis larvae is the critical stage of development. The mechanisms of invasion and damage host are complex. In addition to mechanical damage tissue caused by parasite migration, occupying intracranial hypertension and tissue injury, and neurotoxicity of larvae metabolites or toxic substances released by death parasite, the killing mechanism is mostly associated with the immune system. According to the literature, after A. cantonensis infection, eosinophils increased rapidly; polyclonal B cell activation occured rapidly, resulting in a large number of IgE, IgM, IgG, IgA; CD4+, CD8+ T cells increased; IL-4, IL-5, IL-10, IL-13 and other Th2 cytokines in peripheral blood were significantly increased, while IL-2, IFN-α, TNF-γand other Th1 cytokines were not changed significantly, caursing Th1/Th2 imbalance. Complexity and multiplicity of immune network and translation to each other of different T lymphocytes populations under certain conditions make the conclusion of Th1/Th2 imbalance is not the only immune pathogenesis of Angiostrongyliasis. Other T cell populations are also likely involved in the disease.
CD4+ CD25+ Treg develop from the thymus or peripheral naive T cells, activated by some specific antigen, then play a role on the CD4+/CD8+ T cells in non-specific inhibitory effect through cell contact mechanism and the secretion of inhibitory cytokines IL-10 and TGF-β. The effect of Treg is to avoid the body tissue injury causes by excessive immune response in the inflammatory response; however, if it is curbed excessively, it will make the body weaken to against infection. This cell subsets, with characteristics of immune suppression and immune anergy, act on infection immunity, tumor immunity, immunopathology, graft tolerance and autoimmune response and the maintenance of immune balance. In recent years, Treg are found involved in the course of in a variety of parasitic infections. There is no report about the role which Treg plays on the course of A. cantonensis infection.
The combination therapy of Albendazole and dexamethasone is widely used in A. cantonensis in China. Because of worrying about the adverse reactions, a number of studies proposed a variety of alternative therapies, but no comparison with the effect of dexamethasone. Glycyrrhizic acid, also known as glycyrrhizin (GL), is the main active ingredient in licorice. GL has the steroid, with similar structure like adrenal cortex hormones, so it has glucocorticoid-like effect, but no glucocorticoid-like adverse reactions. It has the effect of anti-inflammatory, anti-allergy, detoxification, anti-virus, anti-tumor, inhibition of MMP-9 and immune regulation and so on. It also can induce interferon, activated T cells and natural killer cells, correct Thl/Th2 imbalance, so play a role in immune regulation. It is speculated that it can be used in A. cantonensis, and might be able to replace the use of corticosteroids to reduce the incidence of adverse reactions. But can it really work? How effective? What are the mechanisms? Does eosinophils, IgE, Thl/Th2, CD4+ CD25+ Treg cells are related? These problems are not clear.
The project is planned to understand the role of immune response in A. cantonensis pathogenesis by detection of immune parameters in different time on course of infection and treatment. The project discussed the pathogenesis and found a more effective treatment of A. cantonensis, with practical of applications. Steps are as follows:(1)observing and detailed recording based conditions, clinical manifestations, prognosis and survival time of A. cantonensis-infected mice at different stages of disease. (2)Comparing levels eosinophils, IgE, IgA, IgM, IgG, IL-5, eotaxin in peripheral blood and CSF in different stages of infected mice. (3)Comparing the percentage of Treg subsets and expression of Foxp3 in it. Understanding the effect of eosinophils, humoral immunity, Th2 and Treg cells in the immune pathogenesis of A. cantonensis. (4)treating infected mice by combination therapy of albendazole+ dexamethasone and albendazole+ diammonium glycyrrhizinate respectively, discussed the effect and possible mechanisms.
The artificial digestion for A. cantonensis was optimized as follows:snail was deshelled, homogenized, digested by artificial digestive fluid containing 2g of pepsin, 37℃,2 hours, filtrated to remove tissue debris. The filtrate was washed 3 times with PBS and deposited for collection larvae. Mice modeles of angiostrongyliasis were established successfully by infection of 50 A. cantonensis larvae. The signs like hair wet, kyphosis, hemiplegic, and turned around in E50 mice began from 13dpi till died at aboutl6dpi. The survival time, signs and weight can be used as the indicators of efficacy. Make eosinophils percentage and IgE level in peripheral blood on 10 and 15dpi as the indicator of immune response. the brain is the site with the most serious pathological damage, includ meningitis, subarachnoid hemorrhage, inflammation, extensive infiltration of eosinophils. AD therapy can cure some mice, to prolonged survival, recover weight, relieve signs, but most of the rest mice can not be changed of the prognosis of death, only slightly prolonged survival time, improved weight reduced signs. AG therapy is more effective than AD, it can cure all the mice, prolong survival time, recover weight and relive signs.
Observating of eosinophils, IgE, IgA, IgM, IgG, IL-5, eotaxin, IFN-γ, IFN-γ/IL-5, CD4+ CD25+, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ cell percentage, number of intracranial worms, and the brain pathology in infected and AD, AG treated mice on 10 and 15dpi, we get these result as follow. (1) Eosinophils percentage in peripheral blood and CSF of infected mice was significantly higher. AD and AG therapy could significantly inhibit eosinophils, but it rebound after AD withdraw. (2)IgE, IgA, IgM, IgG in serum and CSF of infected mice increased significantly. AD could significantly inhibit IgE in serum and CSF, IgA in CSF, IgM and IgG in serum, but rebound after AD withdraw; AG could inhibit IgG in CSF as AD, inhibit IgE in serum and CSF and IgA in CSF more than AD, but could not inhibit IgM in CSF. (3) IL-5 and eotaxin in serum and CSF of infected mice were significantly increased. AD could significantly inhibit them in serum and CSF; AG could inhibit them in CSF, but not in serum. (4)IFN-γand IFN-γ/IL-5 in serum and CSF of infected mice were significantly decreased. AD therapy could significantly restore them, AG is more effective. (5)CD4+ CD25+ cells in peripheral blood of infected mice was significantly increased, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ cells was no significant change. AD and AG could significantly inhibite these three cells. Indicators, more closely related to beginning and sustaining of signs, are eosinophils in CSF, IgE levels in serum and CSF, CD4+ CD25+ cell percentage in peripheral blood. (6)Eosinophils cells, IgE, IgG and eotaxin are closely related to each others. IFN-γand IFN-γ/IL-5 are negative correlated with eosinophils, IgE, IgG, IL-5 and eotaxin. CD4+ CD25+ cells in peripheral blood were closely related with IgE, CD4+ Foxp3+ and CD4+ CD25+ Foxp3+ cell percentage were closely related to each other. (7) In variety of pathological changes, meningeal edema and subarachnoid eosinophil infiltration are the most typical. AD mouse can relieve the pathological damage of some mice, the rest are slightly improved; AG are better able to improve the brain pathological injury in all the mice.
Summary, the immune pathogenesis of A. cantonensis can be described as follows: after infection of A. cantonensis, antigen activated antigen presenting cells-dendritic cells (DC2). Antigen and DC2 selectively activate Th2 cells. Activated Th2 cell mediate humoral immune to secrete immunoglobulin, at the same time, secrete specific cytokines and chemokines, sent eosinophils into inflammation tissue. Eosinophils release granules to kill worm with the assistance of the immunoglobulin. Too strong Th2 response and toxins secreted by eosinophils kill worm, and cause host tissue damage. In the evolutionary process, Larvae produce the ability of inhibition host immune responses to protect themselves and extend the parasitic time. Some parasites antigen activated DC2, while others secreted antigen activated another group of dendritic cells (DCreg). With the action of special cytokines in the peripheral blood, DCreg activated Treg cells, to play an appropriate immunosuppressive effects, so that Th2 response is controlled at moderate levels, immunoglobulin producting and cytokine releasing were inhibited, and eosinophil chemotaxis and killing effect were inhibited moderately. In addition, certain antigens of the parasites could enable some non-specific B cell clones, resulting in a large number of non-specific IgE, to block IgE receptor on eosinophil and inhibit eosinophil to kill worm. The result is killing effect on parasite of host was reduced. The number of worms will not be too large to result in excessive consumption. The damage caused by immune response of host was reduced significantly, so host would not dead because of excessive immune respond. The ultimate goal is to make the host can be survive longer and better, it conducive to long-term parasitic worms. AD and AG have a therapeutic effect on A. cantonensis both, AG is more effectively.
AD therapy can kill worms, inhibit eosinophil, Th2 immune respond, and Treg, and recover Thl immune respond, but eosinophil, humoral immunity are, and Treg rebound after withdraw. Albendazole kills larvae, DC2 and DCreg activation induced by worm antigen were reduced, humoral immunity, Th2 and Treg responses are reduced, but the death worm antigens might lead to different immune response, including activation of humoral immune response, Th2 response, and Treg, but not to inhibit the Th1 response. Then dexamethasone plays inhibiting role on immune respond. In addition to Th2 response, dexamethasone can also directly inhibit eosinophil, humoral immunity, Treg, and restore blood-brain barrier function, but this action inhibit immune injury, while not conducive to kill worm. Dexamethasone play a direct inhibitory effect by inducting apoptosis of immune cells, not eliminate antigen and inhibiting DC, so the secretion of live worm and not yet fully clear antigen of dead worm can still trigger an immune response after withdraw of 7 days AD therapy. Th2 response did not rebound may be due to rebound Treg, but without inhibition of dexamethasone, Thl recover more, and then Th1/Th2 recovered more. Although the specific mechanism is unknown, AG therapy may have better killing effect. Until the DC has been cleared, eosinophils, humoral immunity, Th2 and Treg responds activated by live worm secrete antigen would be more modest, but the immune response induced dead worm antigen is likely earlier, more intense or longer. The diammonium glycyrrhizinate suppress Th2 response by increasing Th1 and improving Th1/Th2 balance, so the effect is slow and persistent. Thl has been gradually restored, but DG is not enough to completely suppress Th2 on 10dpi. After withdraw, Th1 respond recovered, Treg rebound also, so Th1/Th2 recovered and Th2 was inhibited. Both treatments did not influence Treg, the specific mechanism remains to be further verified by experiments. Diammonium glycyrrhizinate may be better able to recover blood-brain barrier function, so the immune parameters in CSF had a better recovery. In short, AG is effectively on A. cantonensis than AD, and its mechanism may be related to a better killing effect, the blood-brain barrier function recovery and immune inhibition.
引文
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[42]. Yoshimura K, Aiba H, Hayasaki M, et al. Delayed hypersensitivity responses of guinea pig and rat to Angiostrongylus cantonensis infection[J]. Nippon Juigaku Zasshi.1976,38(6):579-593.
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[44]. Lee J D, Wang J J, Chang J H, et al. Role of T cell subpopulations in mice infected with Angiostrongylus cantonensis [J]. J Helminthol.1996,70(3): 211-214.
[45]. Humbert M, Corrigan C J, Kimmitt P, et al. Relationship between IL-4 and IL-5 mRNA expression and disease severity in atopic asthma[J]. Am J Respir Crit Care Med.1997,156(3 Pt 1):704-708.
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