Long-term Effect Analysis Of Myasthenia Gravis Patients After Thymectomy And The Research Of Inter-thymic And Peripheral Blood Regulatory B Cell And T Cell In MG Patients

Part OneLong-term efficacy and influencing factors analysis of myasthenia gravis patients after thymectomyObjectiveTo analyze the long-term efficacy and to identify relevant influencing factors in myasthenia gravis(MG) patients after thymectomy and compare the clinical features and prognosis of patients with different thymic pathology.MethodsA retrospective analysis of306consecutive patients undergoing thymectomy between1984and2011at Tongji Hospital was undertaken. The complete stable remission and general clinical remission were set as the analysis endpoints for efficacy in this study. Clinical efficacy and factors influencing outcome were assessed by Kaplan-Meier survival method and Cox proportional hazards regression analysis. In addition, the clinical data of174MG patients with thymoma (T-MG) and132MG without thymoma (NT-MG) were analyzed by analysis of variance or rank test.ResultsThere were151female and155male with a mean MG onset age of31.54years. Mean preoperative symptom duration was33.44months and the mean age at thymectomy was34.16years. Patients in MGFA class I, II, III, IV and V were116(37.91%),117(38.24%),53(17.32%),3(0.98%),17(5.56%), respectively.289patients underwent trans-sternal extended thymectomy and32underwent video-assisted thoracoscopic surgical (VATS) thymectomy. The perioperative mortality was2.94%(9/306). Complete follow-up information was obtained in297patients with a mean follow-up period of103months. Complete stable remission was achieved in81of297patients (27.27%), general clinical remission in172patients (57.91%) and marked improvement in69patients (23.23%). Overall,81.24%of patients benefited from the surgery, At the univariate analysis, age at thymectomy<40years (P=0.034), MGFA classification of I (P=0.001) and MG without thymoma (P=0.019) were determined as favorable predictors for CSR. Univariate analysis also showed preoperative MGFA classification of I (P=0.004), concomitant disease (P=0.022) and MG without thymoma (P=0.019) were favorable prognostic factors for clinical remission. Multivariate Cox regression revealed preoperative generalized type of MG (odds ratio:2.088,95%CI:1.339-3.265, P=0.001) and with concomitant disease (odds ratio:1.668,95%CI:1.072-2.549, P=0.023) were independent risk factor for postoperative clinical remission. The crude CSR rate was higher in the NT-MG group than in the T-MG group (33.33%vs22.42%P=0.036) and clinical remission rate showed similar result (64.39%vs52.72%; P=0.043). Moreover, compared with T-MG patients, more NT-MG patients benefited from thymectomy (86.36%vs76.97%, P=0.04). Meanwhile, clinical deterioration and mortality were significantly higher in T-MG patients than in the NT-MG group (clinical deterioration rate of15.76%vs6.06%, P=0.009; mortality11.49%vs3.03%, P=0.004). In addition, nine cases of perioperative mortality were T-MG patients. In the follow-up period,9cases of T-MG patients (5.45%) died of MG crisis, and in NT-MG patients only2cases (1.52%). ConclusionsExtended thymectomy is a preferred treatment for MG patients with a satisfactory long term remission rate. Preoperative generalized type of MG and patients with concomitant disease were independent risk factor for postoperative clinical remission. T-MG patients is more serious than the NT-MG patients, and the long-term prognosis is not optimistic. Therefore T-MG patients need to be optimized with preoperative assessment and perioperative preparation to reduce perioperative mortality and improve long-term survival after thymectomy. Apt caution must be taken to discontinue the pharmaceutical therapy since relapse remain a major concern after a patient gains symptom-free even who had already undergone thymectomy. Part TwoThe proportion of thymus regulatory B cells and T cell and related molecular expression in Myasthenia gravis patients Objective1. Comparison the proportion of CD5+CDld+CD19+Breg and CD4+CD25+CD127low/-Treg cells in MG patients between different type of thymic pathology.2. Comparison the expression of function-related molecules of regulatory B and T cells in MG thymus tissue.Method1. Immunohistochemistry were used to examined the distribution and expression of CD19, CD5, CD25and Foxp3in10cases of MG with hyperplasic thymus,14cases of MG with thymoma,4cases of MG with atrophic thymus and15cases of normal controls (7children and8adult).2. Flow cytometry analysis were used to determine the different frequencies of CDS+CD1d+CD19+Breg and CD4+CD25+CD127low/-Treg cells between6cases of MG with hyperplasic thymic,12cases of MG with thymoma, five cases of MG with atrophic thymus, and13normal controls (seven cases of children and six cases of adult).3. Real time PCR analysis were used to determine the function-associated molecules relative transcript levels of regulatory B cells and T cells in10cases of MG with hyperplasic thymic,16cases of MG with thymoma and17normal controls.Result1. CD19was scattered in a small amount of thymic medulla in atrophic normal controls, while expressed in a large amount of thymic medulla in normal children thymus. CD19was widely distributed in MG hyperplasic thymic and significantly increased in expression quantity, primarily expressed in lymphoid follicles, especially around the germinal centers and medullary thymic corpuscles. CD5was widely distributed in the range of normal controls, the same was seen in the MG hyperplasic thymic, uniquely expressed in bright areas of germinal centers, and was also visible in lymphoid follicles marginal zone. Expression of CD19and CD5in thymoma MG patients was associated with pathology classification, B1thymoma patients accompanied by the formation of lymphoid follicles showed significantly increase in CD19and CD5expression, but CD19and CD5expression in type A、AB and B3was rare. CD5and CD19expression in MG atrophic thymus was similar to the normal controls.MG patients showed similar histological distribution of CD25and FoxP3with normal controls, but the expression of CD25in MG hyperplasic thymus and children normal thymus were significantly higher than that of the normal adults atrophic thymus, MG atrophic thymus and normal adults thymus showed no differences in CD25expression, while CD25expression was significantly lower in MG patients with thymoma. Expression of FoxP3in MG thymus was significantly lower than that of the normal control, especially thymoma patients with a minimal amount.2. The proportion of thymus CD5+CD1d+CD19+Breg cells in MG patients was significantly lower than the healthy controls (p=0.013), the proportion of Breg cells in MG hyperplasic thymus was significantly higher than that of the MG atrophic thymus and MG thymoma patients (p<0.05), the percentage of CD4+CD25+CD127low/-Treg cells in overall MG patients showed no significant difference between healthy controls, also no differences was seen between MG hyperplasic thymus,atrophic thymus and MG thymoma patients.3. The relative transcript levels of various types of regulatory B cell function-associated molecules in MG hyperplasic thymic were significantly increased, but only CD5relative transcript levels in MG patients with thymoma was significantly lower than that of the controls, others indicators showed no significant differences between the two groups. According to regulatory T cells functional-related molecules, the FoxP3and AIRE relative transcript levels in MG patients were significantly lower than that of the normal controls, but CTLA-4, GITR and ICAM transcript levels in MG hyperplasic thymic were significantly higher than the normal controls, and CTLA-4and GITR in MG thymoma patients was significantly lower than the normal controls.ConclusionThe proportion of thymus CD5+CD1d+CD19+Breg cells in MG patients is significantly lower than of the healthy controls, and the expression of Breg related molecular show apparent differences between MG with thymoma and hyperplasic thymic, indicate that Breg cells involve in the pathogenesis of MG. No significant change is seen in the thymus Treg cells proportion of MG patients but FoxP3and AIRE expression are significantly reduced, thus the control function of Treg cells are seriously damaged. Breg cells and Treg cells number and/or function change are seen in MG patients, may be play a role in the pathogenesis of MG. Part ThreeThymectomy for myasthenia gravis patients with change of the proportion of peripheral blood regulatory B cells and T cellsObjectiveCompare the relationship between the proportional change of regulatory B cells (CD5+CD1d+CD19+Breg) and T cells (CD4+CD25+CD127low/-Treg) in the peripheral blood and serum AChR-Ab titers of MG patients with surgical treatment, and evaluate these cells whether can become a treatment and prognosis immunological index of MG patients.MethodWe chose MG outpatient and inpatient in wuhan Tongji hospital from October2012to July2013for the study, the proportion of CD5+CDld+CD19+Breg cells and CD4+CD25+CD127low/-Treg cells in the peripheral blood of69MG and10normal controls were analyzed by the flow cytometry, while serum AChR-Ab titers of these patients were detected by ELISA method.Result1. The proportion of CD5+CD1d+CD19+Breg cells in the peripheral blood of overall MG patients was significantly lower than that of the healthy controls (P=0.013), compared with preoperative MG paients, the percentage of CD5+CD1d+CD19+Breg cells in the postoperative patients was not increased obviously, and postoperative aggravating or recurrence patients also showed no obvious differences with stable remission patients.2. The proportion of CD4+CD25+T cells in the peripheral blood of overall MG patients showed no differences with healthy controls, but preoperative MG patients had higher proportion of CD4+CD25+T cells than that of the healthy controls(P=0.038). The proportion of CD4+CD25+CD127low/-Treg cells in the peripheral blood of overall MG patients showed no significant differences between healthy controls, although CD4+CD25+CD127low/-Treg cells in postoperative MG patients tended to increase obviously, there was no significant differences between healthy controls; and postoperative aggravating or recurrence patients also showed no difference between stable remission patients. There was no significant correlation of CD5+CD1d+CD19+Breg cells and CD4+CD25+CD127low/-Treg cells in MG patients.3. Serum AChR-Ab titers was not significantly decreased in postoperative MG, but AChR-Ab titers of thymoma patients were significantly higher than those of the non-tumor patients (P<0.001), AChR-Ab titers in aggravating or recurrence patients were significantly higher than that of the stable remission patients (P<0.001).ConclusionThe proportion of CD5+CD1d+CD19+Breg cells in the peripheral blood is significantly reduced in MG patients, and increase after thymectomy is not obvious; the proportion of CD4+CD25+CD127low/-Treg cells is not significantly different from healthy controls, but postoperative MG show higher trend of CD4+CD25+CD127low/-Treg cells proportion. Serum AChR-Ab titers are not significant decrease in MG patients after thymectomy. Regulatory B cells may participated in the MGdevelopment, but regulatory B cells MG can’t be a reliable prognostic indicators for MG patients, thymectomy may not be affected by these two cell subsets to exert its therapeutic effect.