Clinical Efficacy And Mechanism Of Autologous Whole Blood Injection Combined With Antihistamines In Treatment Of ASST-positive Chronic Spontaneous Urticaria

Background:Chronic spontaneous urticaria(CSU)is a common and distressing skin disease characterized by the occurrence of wheals(with or without angioedema)for>6weeks due to known or unknown causes.Several mechanisms have been known as contributing to the pathogenesis in CSU,including heredity,chronic infections,coagulation and autoimmunity.Autoimmunity is thought to be one of the most important and frequent cause in CSU.The functional IgG autoantibodies against IgE or Fc?RI?and the IgE autoantibodies to autoantigen,are thought to play a key role in the pathogenesis of CSU by directly or indirectly activating mast cells and basophils.CSU related to these functional IgG or IgE autoantibodies was defined as autoimmune chronic urticaria(ACU).Compared with ASST-negative CSU patients,patients with ASST-positive CSU usually have more severe symptoms,more antihistamines needed to control symptoms,and longer disease durations.Nonsedating H1-antihistamines are the main therapy to CSU,however,almost half of CSU patients would resistant to H1-antihistamines with licensed doses.In recent years,several reports have demonstrated the clinical improvement of refractory chronic urticaria after injection of autologous whole blood(AWBI)or autologous serum.Although their immunological mechanism of action remains unclear,it is speculated that AWBI could desensitize CSU patients to serous histamine-releasing factors including autoantibodies.Purpose:to evaluate the clinical efficacy of AWBI combined with antihistamines for the treatment of patients with ASST positive CSU,to evaluate its effect on the expression of plasma IgG anti-Fc?RI,total IgE,D-dimmer,Fc?RI and CD63 on basophils and Treg cells and to understand the possible mechanism of the treatment.Methods:Eighty patients with ASST-positive chronic spontaneous urticaria were collected and randomly divided into AWBI group(n=40)and control group(n=40).Second-generation H1-antihistamines were given to all the patients.Patients in the AWBI group were also injected with autologous whole blood once a week for 12 times.Weekly urticaria activity score(UAS7)and dermatology life quality index(DLQI)in both group were evaluated at the baseline,4 week,8 week and the end of the treatment.The levels of plasma IgG-anti-Fc?RI,total IgE and D-dimmer were measured in patients with AWBI before and after treatment by ELISA or immunoturbidimetric assay.Fc?RI and CD63expression on the blood basophils from patients with AWBI were assessed at the baseline,4week,8 week and the end of the treatment by flow cytometry.Peripheral blood CD4~+CD25~+CD127~-Treg cells expression in patients with AWBI was also evaluate before and after treatment by flow cytometry.In addition,healthy volunteers(n=25)were collected as healthy control group.In order to explore the therapeutic mechanism of AWBI,correlation analysis was made for analyzing the correlation between the above laboratory indicators and UAS7.Results:1.Before the treatment,no significant differences in UAS7 and DLQI scores were observed between the AWBI groups and control group(P>0.05).After 8 weeks of treatment,statistical differences were observed for the decline of both the scores in two groups(all P<0.001),and UAS7 and DLQI scores were significantly lower in AWBI group than in control group(all P<0.01).After treatment,the total effective rate of AWBI group was 75%(30/40),which was significantly higher than that of the control group by 45%(18/40)(P<0.05).2.The baseline levels of plasma IgG anti-Fc?RI,total IgE and D-dimer in ASST-positive CSU patients were all significantly higher than those in healthy controls(both P<0.05)or normal reference values.After treatment,the levels of IgG anti-Fc?RI and D-dimers in patients with AWBI were significantly lower than those before treatment(both P<0.05),but no significant change was oberved in total IgE.Both the baseline levels of total IgE and D-dimmer were positively correlated with the baseline UAS7 and UAS7variation.(all P<0.05).3.Baseline basophil Fc?RI expression in ASST-positive CSU patients was significantly higher than that in healthy controls(P<0.001),which was positively correla ted with total IgE levels(r=0.637,P<0.001).A statistically significant reduction in basophils Fc?RI and CD63 expression in patients with AWBI was abserved at week 4 and week 8(all P<0.01).The changes in Fc?RI expression from baseline to week4,from week4 to week8,and from week 8 to week 12 were positively correlated with the changes in CD63expression induced by ASST-positive serum.Baseline Fc?RI/CD63 expressions were also positively correlated with UAS7 varation(all P<0.05).4.The percentage of CD4~+CD25~+CD127~-Treg cells in peripheral blood was lower in ASST-positive CSU patients than that in healthy controls(P<0.05).After treatment,the percentage of Treg cells in patients with AWBI was significantly higher than that at baseline(P<0.05).5.Patients with good improvement in UAS7 after treatment(UAS7 varition314)were grouped as“responders”,while patients with lesser improvement were grouped as“non-responders”(UAS7 varition<14).Baseline levels of total IgE,D-dimer,basophils Fc?RI and CD63 expression in responders were significantly higher than those in non-responders(all P<0.05).After treatment,the levels of plasma IgG anti-Fc?RI,D-dimer in responders were significantly lower than those before treatment(P<0.05).No significant change in the levels of plasma IgG anti-Fc?RI and D dimer was oberved in non-sponders before and after treatment.A statistically significant reduction in basophils Fc?RI and CD63expression in responders was abserved during treatment.After treatment,the percentage of Treg cells in the responders,but not non-responders,was significantly higher than that before treatment.6.Comparison of indexes in Responders and Non-responders indicated that the cutoff value of total IgE was 51 UI/mL,in which yielded an ROC of 0.756(P<0.05),with a higher sensitivity(75%)and specificity(69.23%);the cutoff value of D-dimmer was 0.545mg/L,in which yielded an ROC of 0.756(P<0.05),with a higher sensitivity(76.47%)and specificity(92.31%).Conclusion1.Autologous whole blood injection(AWBI)combined with antihistamines could improve the clinical symptoms and the life quality of patients with ASST-positive CSU.2.Our study indicates that autoimmune and coagulation indeed play an key role in the pathogenesis of CSU.AWBI combined with antihistamines could down-regulate levels of plasma IgG anti-Fc?RI,D-dimer in patients with ASST+CSU.The levels of baseline total IgE and D-dimer hold promise as a clinically useful immunological markers predicting response to AWBI combined with antihistamines.3.The abnormal high expression of basophils Fc?RI correlates with an overexpression of plasma total IgE;high expression of Fc?RI and IgE may aggravate urticaria.Dual inhibition of basophils Fc?RI and CD63 expression is one of the most important cellular molecular mechanisms for AWBI combined with antihistamines treating urticaria.CSU patients with high expression of basophils Fc?RI and CD63 are more likely to benefit from AWBI combined with antihistamines.4.Treg cells may be involved in the pathogenesis of CSU.Restoring the normal expression of Treg cells may be one of the mechanisms for AWBI combined with antihistamines successfully treating ASST-positive CSU.5.Our study found that there were different inflammatory cytokines/cells expression patterns between responders and non-responders.As compared to non-responders,responders had higher baseline expression of plasma IgE,D dimer,Fc?RI and CD63 on basophils;after treatment,responders show a marked reduction in expression of plasma IgE,D-dimer,basophils Fc?RI and CD63,a elevated expression in expression of Treg cells,compared to non-responders.