Experimental Study On Coronary Arteritis Inducted By Lactobacillus Casei Cell Wall Extract In Mice-an Animal Model Of Kawasaki Disease

BackgroundKawasaki disease (KD) is a kind of common seen autoimmune vasculitic syndrome in children, the syndrome which complicated with coronary aneurysm(CAA) and coronary stenosis is responsible for sudden death of adolescence and myocardial infarction, recent study demonstrated Kawasaki disease which was suffered from is a novel risk factor for adult ischemic heart disease. Kawasaki disease has become the main cause for acquired heart disease in children instead of rheumatic fever. Although the Kawasaki disease's mortality and coronary complications have been significantly declined by extensive application of the combination of intravenous gammaglobulin (IVIG) injection and Aspirin, but recently, we found the IVIG treatment cases which developed drug tolerance had augmented tendency. But the etiology and mechanism are not clear yet, it is presumed at present, that the features of Kawasaki disease are consistent with infectious disease and autoimmune disease. Bacterium superantigen is the crucial factor inducing Kawasaki disease, the abnormal immune activation is mediated by bacterium or virus toxin superantigen. But the samples for investigating Kawasaki disease are difficult to be obtained, there is lack of accepted Kawasaki disease animal model applying at the study of angio-inflammatory injury and coronary aneurysm at present both at home and abroad. Lactobacillus casei cell wall extract (LCWE) ip into juvenile mouse was performed to observe it whether or not can induce coronary arteritis in mice, which aims at discussing the possiblity of establishing novel Kawasaki disease animal model; investigating the relationship of LCWE superantigen activation with angio-immunology injury and coronaritis, and its mechanism. Combine with objectively evaluating IVIG therapy and curative effect for the prophylaxis of Kawasaki disease coronary affection,to investigate IVIG therapeutic effect correlated factors.Part â…  Establish an animal model of coronary arteritis induced by Lactobacillus casei cell wall extractObjectiveTo establish an animal model of coronary arteritis induced by Lactobacillus casei cell wall extract (LCWE). LCWE-induced coronary arteritis in mice mirrors Kawasaki disease in children.MethodsLCWE was preparated and diluted to 1 mg/ ml. Then it was used for immunization of genetically predisposed mice to establish animal model of coronary arteritis. There were 50 mice as experimental group and 50 mice as control group. Mice (4 weeks old) were respectively injected i.p. with 500 μl of PBS containing 0.5 mg LCWE or PBS alone on day 0, and 10 mice in each group were sacrificed on days 3, 5,10 and 28. Blood was collected and Cardiac tissues were removed and embedded in the embedding medium OCT.ResultsAfter the injection with LCWE ,the ratio which BALB/ c mice developed histological coronary arteritis was about 92.5 % with pathological features of being characteristic of many neutrophil and a few mononuclear cell infiltrations, severe medial lesions with smooth muscle cell loss and inflammatory cell infiltration, and adventitial lesions with invasion of some inflammatory cells in the ostium of coronary arteries and main coronary artery at 1³ day. After LCWE treatment lesions were found at the ostium of the coronary artery showing mild intimal thickening with prominant macrophage and slight neutrophil infiltration at 4 weeks. EM microscopy showed different degrees of swelling, necrosis and abscission of endothelial cells, aggregation of chromatain, degeneration of smooth muscle cell, swelling of mitochondria and augmentation of endoplasmic reticulum in the coronary artery of experimental group. Experimental group significantly improved the absolute number of CD4+and CD8+ T cells (P<0.05). The absolute number of CD4⁺and CD8⁺ T cells reached the peak of (8.23±1.32)×10⁹/Land (6.19±1.08)×10⁹/L at 72 hours.ConclusionCoronary arteritis and myocarditis can been induced by immunization of mice with LCWE injected i.p. single, pathological changes were typical, the morbidity meets the requirement of research. Lactobacillus casei cell wall extract- induced coronary arteritis in mice mirrors Kawasaki disease in children.Part â…¡ Superantigenic activity of LCWE and superantigen-mediated mechanism on induction of coronary arteritis in miceObjectiveTo study the specificity of the LCWE-mediated activation of TCR v_β and the cell proliferation features of TCR v_β2, 4, 6 positive T cell activated by LCWE; to observethe dynamics of CD69 expression and the function of CD69 in mice T cell activated byLCWE in vitro and in vivo; to explore the relationship of superantigenic activity ofLCWE with vascular immunologic injury and with coronary arteritis in mice, and theirmechanisms.Materials and Methodsâ‘ BALB/C mice (n=122, aged from 4-6 weeks) were randomly divided into experimental and control group. Mice model of coronary arteritis was established by using LCWE based on the protocols mentioned above.â‘¡The mice were injected ip with superantigen LCWE, muticlone stimulator Con A, and PBS, respectively. The dynamics of the superantigen-mediated TCR v_β2, 4, 6 positive T cell proliferation and the expression percentage as well as dynamical changes of CD4⁺CD69⁺T cell were observed by using a flow cytometer.â‘¢Mononulear cells in the mice spleen were stimulated by LCWE or Con A in vitro to observe the specificity of the binding of LCWE with mice TCR v_β. Positive rate of CD69 expression and dynamic changes in CD4⁺T cell were further examined after treated by LCWE (5, 10, 15, 20, 25, 40 ng/ml), SEB (2.5μg/ml), Con A (1mg/ml, 20μl, final concentration 10μg/ml), or PBS.â‘£Serum levels of IL-1β, IL-6, and TNF-α were detected by ELISA.⑤Using corresponding models, the relationship of cell proliferation of TCR Vβ6⁺CD4⁺T cell with the activated CD69 expression in T cell and with the changes of serum TNF-α level was observed.â‘¥Using Logistic regression model, the correlation between the occurrence of coronaiy arteritis and the proliferation of TCR Vβ⁺ T cell, T cell activation, or TNF-α production was analyzed.Resultsâ‘ The ip injection of LCWE stimulated the proliferation of T cell that expressed TCR Vβ2, 4, 6, compared with pre-injection, P<0.001 and ip injection of Con A, P<0.05. â‘¡The proliferation of T cell that expressed TCR Vβ family regressed very soon. After the administration of LCWE, the percentage of CD4⁺ TCR Vβ6⁺ T cell reached the peak at 3d (11.11±1.08) %, but declined to the basal line at 5d and the percentage reduced to 0.98 % at 10d. The tendency of cell percentage of TCR v_β2 CD4⁺ T cell and TCR v_β4 CD4⁺ T cell was nearly the same.â‘¢Superantigenic activity was highly related to coronary arteritis in mice. The occurrence of coronary arteritis was related to the proliferation of TCR Vβ⁺ T cell, T cell activation, and TNF-α production, respectively (Wald x² =12.473, P<0.001; 11.912, P<0.05; and 3.469, P<0.05, respectively. Compared with non coronary arteritis group, coronary arteritis group indicated a significant higher TCR Vβ⁺ T cell rate, t=3.469, P<0.001; a significant lymphocyte activation, t=4.143, P<0.001; and a higher serum level of TNF-α, t=5.978, P<0.001.â‘£The cell proliferation of TCR V_β6⁺ T cell was closely correlated with the T cell activation (increase of CD69) and with the TNF-α production, r=0.884, P<0.05; r=0. 846, P<0.001, respectively. The cell proliferation of CD69⁺ T cell was closely correlated with the the TNF-α production, r=0.947, P<0.05.⑤The dynamic changes of LCWE mediated CD69⁺ T cell expression in vivo indicated that, after LCWE injection, the CD4⁺CD69⁺T cell rate in mononuclear cells of BALB/C mice peripheral blood and spleen increased, compared with control group, P<0.001. In mice spleen, mononuclear cells started to increase at 3h (19.32 %) after LCWE injection, peaked at 12h (38.8 %), followed by continuous decrease, at 24 h (9.89 %). The tendency in peripheral blood mononuclear cells was similar to that in spleen mononuclear cells. Spleen mononuclear cells, however, showed a higher CD4⁺CD69⁺T cell rate than peripheral blood mononuclear cells, P<0.001. After transient high expression, CD69 had a quick decrease rate and large amplitude in vivo. When in vitro, the CD69 expression in CD4⁺T cell increased by LCWE injection, with a larger amplitude, P>0.05.Conclusion â‘ Superantigen is responsible for severe immunologic injury and coronary arteritis induced by LCWE in mice.â‘¡LCWE directly induces the activation of earlier T cell, indicating the obvious increase of CD4⁺CD69⁺T cell.â‘¢LCWE induces the over-production of TNF-α, an inflammatory cytokine that may be a crucial reason in the onset of coronary arteritis, and therefore the block of the activity of TNF-α my be a drug target in the treatment of Kawasaki Disease.Part â…¢ Effect of leukotriene B4 receptor in immunity vasculitis ObjectiveTo observe the expression of BLT1 mRNA and BLT2 mRNA in the heart and liver, as well as the expression of BLT2 in the CD4⁺T cells of spleen in mice with coronary arteritis; to explore the effect of LTB-BLT1 and LTB-BLT2 pathway on mice coronary arteritis.Materials and methodsRT-PCR was performed. Total RNA was extracted from obtained tissue. cDNA was synthesized under the guidance of Olig(dT). Primers for BLT1 and BLT2 were designed to amplified target BLT1 mRNA and BLT2 mRNA through PCR. The expression levels of BLT1 and β-actin mRNA as well as BLT2 and β-actin mRNA were visualized in a gel image system. BLT2 expression in CD4⁺T cell of spleen mononuclear cells was detected by indirect fluorescent labeling FCM. Serum levels of IL-1β, IL-6, and TNF-α were detected by ELISA.Results The amplified BLT1 mRNA is 430bp and BLT2 mRNA is 331bp. Expression of BLT1 mRNA and BLT2 mRNA of model mice started to rise at 6h after the injection of LCWE, peaked at 12h and kept for the third day, and then declined gradually. Expression of BLT1 mRNA and BLT2 mRNA of model mice fell back to the basal line at the fifth day. The mRNA expression of BLT1 and BLT2 in the heart and liver was compared intra-group at different checkpoint of time, respectively. 12 h after the administration, the OD ratio of BLT1 mRNA and that of BLT2 mRNA were significantly increased, P<0.05. By FCM, expression of BLT2⁺ CD4⁺T cell in mice spleen at 1d after the administration reached the peak. The reach time was later in the spleen than that in the heart and the liver. 3 d after the injection, serum TNF-α, IL-1, and IL-6 increased compared with control group, P<0.001.ConclusionOver-expression of BLT1 and BLT2 in liver, heart and spleen in process of coronary arteritis induced by LCWE. LCWE can increase the secretion of inflammatory mediators such as TNF-α, IL-1β, IL-6 in mice. So the pathways of LTB-BLT1 and LTB-BLT2 may be involved in the onset of mice coronary arteritis.Part â…£ Evaluation of the efficacy of intravenous gammaglobulin in the prevention and treatment of coronary artery lesion in Kawasaki diseaseObjectiveTo evaluate the efficacy of intravenous gammaglobulin (IVIG) in the prevention and treatment of coronary artery lesion (CAL) in Kawasaki disease (KD) and the related factors influencing the IVIG efficacy.Methods314 children with KD were reviewed retrospectively in a contrast study.ResultsThe incidence rate of CAL was 34.3% in IVIG+ ASA group and 56.0% in ASA group, p<0.001 in comparison of the two groups. The incidence of CAL was reduced in the group in which 2.0g/kg or 1.0g/kg IVIG was administered. CAL occurred less frequently when IVIG was administered at 3-10 day of the course, p<0.05. About 22.2% of the KD treated with IVIG still developed CAL. About 13.4% of the CAL treated with IVIG was not recovered at the 12^th month of the course. The drug-resistant cases accounted for 10.5% in this group. ConclusionIVIG treatment can remarkably shorten the course of patients with KD and decrease the incidence of coronary artery events, but the efficacy of IVIG in the prevention and treatment of KD disease is not as expected by people, therefore, reevaluation of the practical efficacy of IVIG is required.