| IntroductionAlveolar echinococcsis causes some hot clinic problems for the confliction between the parasite infection immune and transplantation immune. The objective is to decide if antigen B of hydatid cyst fluid induces the immune tolerance and how it works. First the humeral and cellular reaction difference between the hydatidosis patients and healthy control were tested based on the data from serum IgG, IgA, IgM, IgE and Th1/Th2 cytokines, then the secondary infected animal model of Alveolar echinococcsis was set up to apply the liver transplantation among 4 groups to observe the pathological changes and mRNA expression. AgB was produced in the lab by 3 different methods to get the crude natural cyst fluid antigen B, partially purified antigen B and the recombinant antigen B. After the comparison the recombinant antigen B was cultured with lymph cells from the hydatid patients spleens to see the Th1/Th2 cytokines level after the stimulation of antigen B. Also the recombinant antigen B is added to the Jurkat cell to see if the apoptosis happenedto illustrate the mechanism of immune tolerance. ScFv was expressed in COS-7 cells and was used as an anti- protoscolex drugs to observe the changes in morphology and quantity. Antigen B and it's specific ScFv could be used as effective molecular tools to detect the special immune tolerance and its possible mechanism, so that it can be used as a target to control the immune reaction and a potential drug to prevent the recurrence.Material and MethodsFirst the immune situation of the hydatid patients were evaluated by testing both the humeral and cellular such as the total IgG, IgM, IgE, IgA, IgM and specific IgG which was tested by self-made hydatid antigens such as protoscolex, cyst fluid antigen B and Em2. Cytokines detection included the panel of Th1 and Th2 (sIL-2 R, IL-2, 4, 8, 10 and IFN-Y).Then the secondary infected alveolar echinococcsis animal model was set up by injecting the liver with fresh Em tissue into the abdomen, the infected rats were divided into 4 groups to do liver transplantation, the number of surviving days is the most important index to show the difference. Pathological study on the routine HE stain of the liver tissue was studied to observe the infection and infiltration of lymph cells, CD30, specific mAb against T cells, which was labeled by the fluorescent stain FITC was used to illustrated the T cells distribution.The natural antigen B was got by high speed centrifuging, partially purified antigen B by affinity chromatography and recombinant antigen B by gene cloning. The recombinant antigen B was co-cultured with lymph cells from hydatid patients, IFN-r and IL-4 in the supernatant were tested to show the polarity change of TH1/TH2. the recombiant antigen B was also added to the media of Jurkat cell line to observe if antigen B can induce the cell apoptosis. DNA ladder, comet assay, and TUNUL were adopted to display apoptosis from different profile and phage. Laserconfocal microscope was used to observe intracellular Ca2+concentration to show if there is a difference with or without antigen B co-culturing.On the former work- a phage displayed human source gene monoclonal antibody specific against antigen B, the gene was expressed and transfectioned COS-cell, the ScFv was added in different dosage to the protoscolex, the dead ratio was calculated and morphology changes was recorded also.Results64 hydatid patients serum antibodies showed that IgE, IgM and IgG were statistically higher than those in the healthy control, IgA was not significantly different from control. Among all the cytokines tested, IL-2 and sIL-2R decrease significantly which means Thl is not active. IL-4 and IL-8 increase significantly. Th2 was more active and the host was characteristic with immune inhibition.Secondary infection of AE was set up by injecting the living Em tissue in the abdomen. The group with AE infection showed the longer surviving days than the group without AE infection, the difference is statistically significant. mRAN expression also showed the Th2 type cytokines IL-4 are high in the immune tolerant group and the polarity from Thl to Th2 contributed to the longer surviving days. The lymph cells in the tolerant liver showed the decreased number which indicate the parasite origin antigen changed the host's T lymph cell and made the antigen specific T cells null to the stimulation so that immune tolerance was attained.3 self-made antigen B show different components: natural cyst fluid was more sensitive but serious cross reaction with other disease, partially purified antigen B was purified by the immune affinity to get rid of the big molecular unspecific components, and the major proteins were 12KD to 20KD. The recombinant antigen B was the solo band 12KD protein on SDS-PAGE and Westen-blot. Under UV it showed a smooth and symmetric single-peak curve.Anti-antigenB ScFv was subcloned and expressed in COS-7, SDS-PAGE and Western-blot showed the good immune activity with antigen B. it served as an anti-proscolex effect when cocultrued with AgB. But the effect was dosage depend and time course. It can be potentially used as a molecular drug to avoid the recurrence of AE in the long term follow up after the liver transplantation.ConclustionsThe AE infection causes the host Th2 dominant immune inhabitation which is good for the liver transplantation for the immune tolerance can be induced. The mechanism is maybe the parasite specific T cells decrease because of the apoptosis and less inflammatory cells infiltrate the new liver. But the immune tolerance also increase the risk of recurrence, ScFv can be used as an anti-protoscolex molecular tool. |
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