Effect Analysis And Mechanism Study Of Lymphoplasmapheresis In Severe Myasthenia Gravis

Background:Lymphoplasmapheresis(LPE)is a new immunotherapy method developed on the basis of traditional plasma exchange(PE)combined with lymphocyte removal technology.Compared with PE,it not only removes soluble pathological substances circulating in plasma,such as autoantibodies,inflammatory factors,and adhesion molecules,but also removes immunocompetent cells,such as sensitized T cells and B cells,thus leading to more effective and durable control of the pathological immune response.At present,LPE has been successfully applied for the treatment of some refractory severe autoimmune diseases,but its application value in the treatment of severe myasthenia gravis(MG)is still unclear.Research purposes:To evaluate the efficacy and safety of LPE in severe MG,and provide evidence and theoretical support for its application in MG treatment.Methods:(1)A total of 123 patients with impending myasthenic crisis(IMC)who received LPE treatment in the Department of Neurology,Xiangya Hospital,Central South University were included in a retrospective analysis.The efficacy was judged by evaluating the changes in the quantitative myasthenia gravis score(QMGS)before and after treatment,and a reduction of ≥ 3 points in QMGS after treatment was taken as the threshold for effective treatment.Data on treatment-related adverse reactions were collected for safety evaluation.(2)75 and 82 IMC patients treated with PE and intravenous immunoglobulin(IVIG)at Xiangya Hospital,Xiangya Second Hospital,and Changsha First Hospital during the same period were included in the comparative analysis of efficacy with LPE.The propensity score matching(PSM)was used to balance the differences in baseline information between groups.(3)A total of 121 patients with myasthenic crisis(MC)who received LPE,PE or IVIG at Xiangya Hospital,Xiangya Second Hospital,and Changsha First Hospital were included in the comparative analysis of efficacy,including 47 patients in the LPE treatment group,39 patients in the PE treatment group,and 35 patients in the IVIG treatment group.The duration of mechanical ventilation(MV),intensive care unit(ICU)stay,and total hospital stay were used for efficacy evaluation in MC.(4)Multiple machine learning algorithms were applied to construct the optimal LPE efficacy prediction model,combined with logistic regression analysis,to find important variables affecting the prognosis of LPE treatment.(5)Peripheral blood mononuclear cells(PBMCs)were extracted from patients before and after LPE and transcriptome sequencing was performed.The effects of LPE on immune activity in MG patients were investigated by Gene-Ontology(GO)enrichment analysis,Kyoto Encyclopedia of Genes and Gnomes(KEGG)enrichment analysis,Gene Set Enrichment Analysis(GSEA),and Gene Set Variation Analysis(GSVA).(6)The effects of LPE on lymphocyte subsets and inflammatory cytokines in peripheral blood of MG patients were assessed by flow cytometry and Luminex liquid-phase microarray.The titers of acetylcholine receptor autoantibody(ACh R-Ab)before and after treatment were measured by enzyme-linked immunosorbent assay to understand the effect of LPE on autoantibody levels in MG patients.Results:(1)Efficacy and safety of LPE in impending myasthenic crisis1)LPE significantly improved the symptoms of patients with IMC.The effective rate was 75.6%(93/123)and the incidence of crisis was 2.4%(3/123).The mean QMGS of the patients decreased from 23.40 ± 4.25 points at baseline to 17.93 ± 5.61 points after treatment,with a decrease of5.47 ± 4.16 points(P < 0.001).The ocular muscles,limb muscles,bulbar muscles,and respiratory muscles involved in MG responded well to LPE treatment with significant improvement in scores(P < 0.001).During the60-day follow-up period,the patients’ QMGS showed a gradual improvement trend.2)In the traditional PE treatment group(62 patients after PSM),patients received 232 replacements with a mean of 3.74.The effective rate was 67.7%(42/62)and the incidence of crisis was 6.5%(4/62).The mean QMGS before treatment was 22.98 ± 4.03 points,and the mean QMGS after treatment was 18.34 ± 5.03 points,a decrease of 4.68 ± 4.04 points,a significant improvement(P < 0.001).In the LPE treatment group(62patients after PSM),patients received 117 replacements with a mean of1.89.The effective rate was 79.0%(49/62)and the incidence of crisis was1.6%(1/62).The mean QMGS before treatment was 23.19 ± 4.11 points,and the mean QMGS after treatment was 16.94 ± 5.78 points,a decrease of 6.26 ± 4.39 points(P < 0.001).Compared with patients receiving PE,patients in the LPE group experienced fewer replacements,but had a more significant improvement in scores(6.26 ± 4.39 vs.4.68 ± 4.04 points,P =0.039).Compared with the traditional PE treatment group,the LPE treatment group has higher effective rate(79.0% vs.67.7%)and lower crisis incidence(1.6% vs.6.5%),but there were no statistically significant differences between them(P > 0.05).3)No significant difference was found in baseline characteristics between the LPE treatment group(123 patients)and the IVIG treatment group(82 patients).In the IVIG group,the effective rate was 62.2%(51/82)and the incidence of crisis was 7.3%(6/82).The mean QMGS before treatment was 23.55 ± 4.43 points,and the mean QMGS after treatment was 19.38 ± 5.25 points,a decrease of 4.17 ± 3.34 points,a significant improvement(P < 0.001).Compared with IVIG,LPE had a better therapeutic performance(effective rate,75.6% vs.62.2%,P = 0.04;score improvement,5.47 ± 4.16 vs.4.17 ± 3.34 points,P = 0.014).In addition,compared with IVIG treatment group,LPE treatment group had a lower crisis rate(2.4% vs.7.3%),but no statistically significant difference was found(P > 0.05).4)LPE was well tolerated in the treatment of IMC.Among 123 patients,16(13%)experienced treatment-related adverse reactions,with allergic symptoms and citrate reaction being common.No serious adverse events leading to death were observed.(2)Efficacy and safety of LPE in myasthenic crisis1)Patients in the traditional PE treatment group had a mean MV duration of 11.88 ± 4.75 days,a mean ICU stay of 16.27 ± 6.13 days,and a mean total hospital stay of 24.81 ± 7.24 days.In the LPE treatment group,patients had a mean MV duration of 8.50 ± 4.69 days,a mean ICU stay of11.88 ± 5.78 days,and a mean total hospital stay of 22.73 ± 7.79 days.Compared with the PE group,the patients receiving LPE had a shorter MV duration(P = 0.013)and ICU stay(P = 0.011).There was no significant difference in the total length of stay between the two groups(P = 0.32).2)Patients in the IVIG-treated group had a mean MV duration of13.68 ± 3.33 days,a mean ICU stay of 18.73 ± 4.83 days,and a mean total hospital stay of 27.35 ± 7.14 days.Compared with patients treated with LPE,the IVIG-treated group had significantly longer MV duration(P <0.001)and ICU stay(P < 0.001),and its total hospital stay was also significantly prolonged(P = 0.03).3)The safety profile of LPE in the treatment of MC was good.Adverse events occurred in 12 of 106 replacements in 47 patients,accounting for 11.3%.No serious adverse events were observed,with allergic and citrate reactions being common.(3)The machine learning model for predicting LPE efficacy andfactors affecting the efficacy of LPE1)With treatment effectiveness or ineffectiveness as classes and 37 features as predictors,models were constructed using 13 machine learning algorithms,including lasso and elastic-net regularized generalized linear model(GLMnet),generalize linear model(GLM),partial least square regression model(PLS),linear discriminant analysis model(LDA),random forest(RF),e Xtreme Gradient Boosting Tree(XGBTree),e Xtreme Gradient Boosting Linear(XGBLinear),support vector machine with radial basis function kernel(svm Radial)and linear kernel(svm Linear),neural networks(NNET),gradient boosting machine(GBM),C5.0decision tree(C5.0),and k-Nearest Neighbor(KNN).Among them,the GLMnet had the best combined performance in terms of accuracy(0.83),sensitivity(0.78),and specificity(0.86),and was determined as the optimal model.2)In the ranking of variable importance,co-infection was found to be the most important characteristic affecting the outcome of LPE therapy.In the univariate logistic regression,age ≥ 50 years old(OR 0.383,P = 0.027)and co-infection(OR 0.274,P = 0.004)were the unfavorable factors affecting the efficacy of LPE.Further multivariate logistic regression suggested that co-infection was an independent risk factor for poor treatment outcome(OR 0.31,P = 0.01).(4)The mechanism of LPE in the treatment of severe MG1)The results of enrichment analysis and GSVA suggested that LPE significantly affected the immune activity of MG patients.The activity of T-and B-cell-related inflammation and MG-associated immune signaling pathways(IL-17 signaling pathway,chemokine signaling pathway,NF-κB signaling pathway,and TNF signaling pathway)was significantly reduced after LPE.The abundance of Th17 cells decreased significantly compared with that before treatment,while the abundance of Th2 and Treg cells increased significantly.2)The results of flow cytometry showed that the frequencies of Th17 and activated Th1 cells decreased after LPE treatment,and the frequencies of Treg and Th2 cells increased.In addition,the frequency of plasmablast was significantly reduced after treatment.The results of Luminex liquidphase microarray indicated that the levels of Th1-related cytokines(IL-1β,TNF-α,IFN-γ,IL-12p70,and IL-2)and Th17-related cytokines(IL-6 and IL-17)were significantly decreased after treatment,and the levels of Th2-related cytokines(IL-4 and IL-5)and Treg-related cytokine(IL-10)were significantly increased.3)The levels of lymphocytes,complement C4,and Ig G in MG patients decreased after LPE treatment.The titer of ACh R-Ab also decreased significantly,from 32.45 ± 9.66 nmol/L before treatment to18.78 ± 7.78 nmol/L after treatment(P < 0.001).Conclusions:(1)LPE is a safe and effective immunotherapy for patients with severe MG.Compared with the current first-line treatment options PE and IVIG,it achieves better therapeutic effect than traditional PE with fewer replacements,and is also more advantageous in terms of efficacy compared to IVIG.(2)Infection is an independent risk factor affecting the prognosis of LPE treatment.(3)In the treatment of severe MG,the direct clearance of circulating soluble immunopathogenic factors such as autoantibodies and inflammatory cytokines and correction of Th1/Th2/Th17/Treg cell imbalance are the action mechanisms of LPE.