| 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.
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