Experimental Study On The Treatment Of EAE With Adenovirus-mediated CTLA-4Ig

Objective Multiple sclerosis (MS) is a common chronic, multifocal, inflammatory, demyelinating disease of the central nervous system. Its major mechanism involves T-cell mediated autoimmune response. Its pathologic characteristics are infiltration of lymphocyte and macrophage and local demyelinating lesions of the white substance of the central nervous system. This disease frequently occurs in the youth 20-30 years of age. At present, because the exact pathogenesis of MS remains unclear, effective therapeutic measures are unavailable. The disease is one of the major causes of disability in youth. In this study, the animal model of MS, i.e., CTLA-4 was used to block the initial stage of immune response, i.e., experimental autoimmune encephalomyelitis (EAE) was established using Wistar rats. AdCTLA-4Ig was reconstructed by combining the gene of a fusion protein of CTLA-4 and adenoviral vectors. AdCTLA-4Ig was injected into animal body to observe its effects in blocking immune response and, thus, providing an experimental basis for the treatment of MS with CTLA-4Ig.MethodsMS animal model was established. Adenovirus-mediated CTLA-4Ig was administered systemically or locally. The therapeutic effect of AdCTLA-4Ig on EAE was observed in contrast with that of glycocorticosteroid therapy.The experiment was composed of two sections: Section one was establishment and evaluation of Wistar rat model of EAE. Homogenate of white substance of brain and spinal cord of guinea pig was mixed with complete Freund's adjuvant. The EAE animal model was established by injecting the admixture into rat palms. Part 2 was the observation on the treatment of EAE with AdCTLA-4Ig. AdCTLA-4Ig was administered via lateral cerebral ventricles or intra-peritoneally respectively to block the pathological process of EAE, which was compared with the treatment using hexadecadrol. Observation on incidence, BAEP(brain stem auditory evoked potential),SEP (somatosensory evoked potentials), pathological and immunohistochemical examinations were conducted to assess thetherapeutic effects. Main Resultsl.The results of model establishment: All rats in the model group fell ill. Their onsets were sudden, and the time of onset was 11.9±0.88 days. The incidence rate was 100%. Clinical scores were 3.4±1.07. The pathological manifestations included significant inflammatory cell infiltration of cerebrospinal tissues, formation of vessel gloves but no apparent amyelination zone on myelin staining. Immunohistochemical examination found there were a large number of activated T cells in brain and spinal cord. No rats fell ill in the control group.2. Comparison of effects of different treatments2.1 Comparison of AdCTLA-4Ig and hexadecadrol treatments: Compared with the model group, the incidence rates and clinical scores decreased and the latency periods extended in the treated rats. The differences were not obvious between the AdCTLA-4Ig and the hexadecadrol groups.BAEP examination found that III-V interpeak latency of the model group was prolonged significantly compared with the normal group; and that III-V interpeak latency of the treatment groups was not prolonged significantly compared with the model group; but the differences were significant between the AdCTLA-4Ig and the hexadecadrol groups.SEP examination: P40 wave latency significantly prolonged in the model group compared with that of the normal group. P40 wave latency did not prolong significantly in the treatment groups compared with that of the model group. The differences in P40 wave latency were significant between the AdCTLA-4Ig and the hexadecadrol groups.Immunohistochemical examination: Filtration of brain and spinal cord of a large number of activated T cells was found in the model group. The filtration significantly decreased in the treatment groups. The number of activated T cells was significantly lower in the AdCTLA-4Ig group compared with the hexadecadrol group.2.2 Comparison of intraperitoneal administration of AdCTLA-4Ig and administration of AdCTLA-4Ig via cerebral ventricle: The incidence rates and clinical scores were lower in the ventricle-administered group than the intraperitoneal administration group, but the difference has no statistical significance. BAEP examination revealed that various administration methods did not significantly affect III-V interpeak latency. SEPexamination showed that P40 wave latency was significantly different between various administration methods. Immunohistochemical examination showed that the decrease in activated T cells of the ventricle-administered group was significantly apparent than the intraperitoneal administration group Conclusions1. Homogenate of white substance of brain and spinal cord of guinea pig plus complete Freund's adjuvant can successfully induce rat EAE model.2. Both AdCTLA-4Ig and hexadecadrol have therapeutic effects on the onset of EAE in rats, significantly lowering the incidence rate and decreasing damage to the nervous tissue.3. AdCTLA-4Ig is obviously superior to hexadecadrol in the therapeutic effects on EAE, suggesting that CTLA-4 can block the whole immune response more effectively by inhibiting its initial stage and that the dosage of CTLA-4Ig transgenic therapy is low but with persistent effects.4. Cerebral ventricular administration of AdCTLA-4Ig produces better therapeutic effects than intra-peritoneal administration.