The Study On The Relationship Between Anti-alpha-1,4-D-Polygalacturonic Acid Antibody And Non Nephritis Henoch-Sch(?)nlein Purpura In Children

Objective:To study the relationship between anti-alpha-1,4-D-polygalacturonic acid (PGA, the main ingredient of pectin) antibody and non-nephritis HSP in children, especially the anti-PGA IgA antibody, and clarify the contribution and possible mechanisms of anti-PGA antibody in the development of HSP.Methods:The sera of neonates, children with urticaria in acute phase, acute respiratory infection, and surgical patients including children hernia surgery, circumcision, polydactyly, non-nephritis HSP children in acute period were collected to test anti-PGA IgA and IgG antibody by enzyme-linked immunosorbent assay. CD3~+, CD3~+CD4+and CD3~+CD8+T cells, CD3-CD16+CD56+NK cells, CD3CD19+and CD19+CD23+B cells, CD4+CXCR5+and CD4+CXCR5+ICOS+follicular helper T cells (Tfh) were analyzed by flow cytometry. Humoral immunity studied by detecting C3, C4, IgA, IgG and IgM in Children with HSP. The intolerance against 14 food antigen were also studied and divided into four ranks according IgG levels, and their humoral immunity were compared with that of diarrhea children in gastroenterology department who were positive for one more antigens in 14 food specific IgG antibodies. The relationships between anti-PGA antibody, cellular and humoral immunity were then analyzed. In addition, Tfh cell activation factor Bcl-6 gene was analyzed by real-time fluorescence relative quantitative RT-PCR assay(RQ-PCR). For evaluating the glycosylation changes about IgA, C1GalT1, ST6GalNAc-II, and Cosmc, the chaperone in the maintenance of C1GalT1, were also tested by RQ-PCR.Results:(1) Immune dysfunction existed in children with HSP. For the cellular immunity in HSP compared with age-matched normal people, CD3~+, CD3~+CD4+T cells and CD3-CD16+CD56+NK cells decreased significantly in HSP patients (p <0.001), while the CD3~+CD8+T cells, CD3-CD19+and CD19+CD23+B cells were significantly increased (p<0.001). Serum IgA, IgG, IgM and complement C4 increased markedly (p<0.001), while C3 had no significant difference (p=0.104). (2) Anti-PGA antibody was specifically elevated in children with HSP. Compared with urticaria, acute respiratory infections, surgical patients and neonates, anti-PGA IgA antibody was increased dramatically in HSP (p<0.001). The antibody level in neonatal group was lower than that in other groups (p<0.001), while the level in Urticaria and acute respiratory infections were higher than that in surgical patients (p<0.001). Similarly, Anti-PGA IgG antibody in HSP was significantly higher than that in other groups (p<0.001), but there were no statistical differences among other four groups. (3) Anti-PGA antibody had diagnostic value for HSP. The area under curve (AUC) of anti-PGA IgA antibodies for HSP was 0.91, and AUC of anti-PGA IgG antibody was 0.72(p0.001 for both). (4) There was no significant difference for gene expression of C1GalT1, Cosmc and ST6GalNAc-II among HSP, acute respiratory infections and surgical patients, which indicated that there was no change of O-glycosylation related enzymes for IgA in children with HSP. (5) The correlation analysis showed that anti-PGA IgA antibodies were obviously correlated with serum IgA (Pearson coefficient r= 0.310,/?<0.001), as well as C3 (Pearson coefficient r=0.125,p= 0.049). (6) Food intolerance was not the main factor that affected the immune function of children with HSP. For food intolerance,94.02% HSP patients were positive for at least one food antigen, their cellular immunity differences were not statistically significant among different positive rank groups, and for humoral immunity, only IgG was showed higher levels in positive food intolerance children than that in negative ones (p=0.009). (7) Food intolerance was not the major factor in the rise of IgA in children with HSP. Compared with diarrhea children, HSP children with positive food intolerance had higher C3, C4, IgA, IgG and IgM (p<0.001). Although IgA and IgG levels in diarrhea children were also higher than that in normal people, but the average value of IgA in HSP were increased 6.34 times of the normal population, and 0.64 times for IgG, it was much higher compared with diarrhea and normal population, which was only 0.94 times for IgA, and 0.10 times for IgG. (8) The proportion of circulating Tfh cells was significantly increased in children with HSP. Compared with acute respiratory infections and surgical patient groups, CD4+CXCR5+and CD4+CXCR5+ICOS+activated Tfh cells were increased significantly in HSP (p 0.001); The expression of Bcl-6 gene in HSP group was higher than that in other two groups, but the difference was not statistically significant.Conclusions:The expression of anti-PGA antibody significantly increased in children with HSP, and the specificity of anti-PGA IgA was also much better than anti-PGA IgG one. It was proposed that anti-PGA antibody, especially the anti-PGA IgA one, was closely related with the occurrence of HSP. Food intolerance was not the main factors leading to increased IgA and IgG antibody, the aberrant of cellular immunity, especially enhanced activation of Tfh cells which was closely related with mucosal immunity, may be the main cause for increased IgA antibody. As our present experimental evidence did not support the role of Gd-IgA1 in the pathogenesis of HSP, PGA and anti-PGA specific antibody, especially for IgA one, may play more important roles in the pathogenesis of HSP. Therefor, the detection of serum anti-PGA IgA might contribute to diagnosis, prevention and understanding the cause of HSP. Further study of anti-PGA specific antibody involving in the development of HSP is of great significance to reveal its etiology and pathogenesis.