| Background:Henoch-Sch(?)nlein purpura(HSP)is a kind of leukocytoclastic vasculitis that is characterized by an immune complex mediated type III allergic disease.Because it is characterized by dominant IgA deposition in the affected capillaries and small blood vessels,HSP was renamed IgA vasculitis according to the new vasculitis classification criteria at 2012 International Chapel Hill Consensus Conference(CHCC2012),which belongs to immune complex vasculitis under small vasculitis.It often affects skin,joint,gastrointestinal tract and kidney.Renal involvement is the most severe complication and identified as a predictor of prognosis.So far,the exact etiology and pathogenesis have not been fully understood yet,therefore it is necessary to further research the etiology and pathogenesis,especially the study on the causes may help reduce the incidence.The trigger factors of HSP are unknown,but infection,vaccination,autoimmunity,food allergen or other factors may be the catalysts.Its pathogenesis is currently believed to be multifactorial,and ascribed to comprehensive effect of immune abnormalities,environmental factors and individual heredity.Infection and food factor are considered to be the major risk factors of childhood HSP.Food allergic reactions are generally divided into Ig E mediated and non Ig E mediated reactions.Non Ig E mediated mechanism of food allergy is not entirely clear,Ig G,Ig M,IgA,T cells,other immune active cells,cytokine and complement etc can participate in the occurrence of food allergy,involving the type II,type III or IV allergic reactions.Milk and eggs are common food for children.Overwhelming majority of the researches on the correlation between food allergens and HSP were performed to explore food specific Ig E/Ig G antibodies by using food specific Ig E/Ig G antibody detection kit.The researches on correlation between food and HSP should focus on the role of food specific IgA antibodies.Therefore,it is necessary to detect the food specific IgA antibodies for illuminating the correlation between food allergy and HSP based on the pathological features of predominated IgA deposition in HSP.At present,there are no studies on the role of food specific IgA antibodies in HSP,and also no related reports on food protein antigen in blood and skin tissues of patients with HSP.This study attempted to clarify the correlation of food antigens from milk&eggs and their specific IgA antibodies with HSP in children and provide a theoretical basis for the role of food in HSP.Part One The level and clinical significance of food specific IgA antibodies in the blood of children with Henoch-Sch(?)nlein purpuraObjective:To investigate the roles of food protein specific IgA antibodies in children with HSP by examining the levels of food protein specific IgA antibodies to bovine casein(CA),bovine?-lactoglobulin(BLG),bovine serum albumin(BSA)and egg ovalbumin(VOA)in children with HSP and control groups.Methods:Peripheral blood serum samples of children who met the inclusion criteria of this study were collected at Children's Hospital of Soochow University from April 2016 to September 2017.Participants:160 patients with acute stage HSP were recruited,36 cases of whom came to reexamination and consented to blood collection in the recovery period;106 cases of elective surgery children with inguinal hernia,redundant prepuce and phimosis were selected as normal control group;the disease control groups consisted of the following 5 groups:38 children with juvenile idiopathic arthritis(JIA),24 children with systemic lupus erythematosus(SLE),17 children with IgA nephropathy(IgAN),30children with persistent or chronic diarrhea(referred to as diarrhea group),31 children with gastritis or duodenal inflammation or/and peptic ulcer with abdominal pain onset(referred to as abdominal pain group).Meanwhile,the clinical data,laboratory test data,renal biopsy pathological data of the patients with HSP and basic data of the control groups were gathered.ELISA system to detect food protein specific IgA antibody was established,then levels of above four kinds of food protein specific IgA antibodies in all groups were assayed,and its clinical significance were be analyzed by combining with the clinical and pathological index.Results:1.ELISA system to detect food protein specific IgA antibody was successfully established in the part.The differences of the parameters size of the standard curve equation in every test were very small,and R~2 of the regression equation was more than0.996,suggesting the high stability of the established ELISA system and high goodness of fit of the regression equation.The vast majority of OD values of blank wells and the neonatal negative serum in every test were less than 0.1,and no more than 0.2,indicating the effectiveness of the tests and availability of the results.2.Children in the normal control group had a basal level of food protein specific IgA antibodies in the serums.There were statistical differences in the levels of specific IgA antibodies to CA,BLG and VOA among the seven groups(all P<0.001).The levels of VOA specific IgA antibodies in HSP patients were significantly higher than those in the normal control group,the diarrhea group,the abdominal pain group,the JIA group and SLE group(all P<0.01).The levels of BLG specific IgA antibodies in HSP patients were significantly higher than those in the normal control group,the diarrhea group,the abdominal pain group and JIA group(all P<0.001).The levels of CA specific IgA antibodies in children with HSP were higher than those in the normal control group,but lower than that in IgAN group(both P<0.01).At the same time,it was found that the levels of CA specific IgA antibodies in the IgAN group were higher compared to other groups except for the SLE group(P<0.05).3.The results of the pairing tests of food protein specific IgA antibodies in 36 cases of patients with HSP in the acute and recovery phase showed that the levels of four kinds of protein specific IgA antibodies in the recovery phase decreased compared with those in the acute phase(all P<0.001).4.The levels of BLG specific IgA antibodies in children with renal-type HSP were lower than those in children with joint-type HSP(P<0.05).The levels of VOA specific IgA antibodies in children with mixed-type and renal-type HSP were lower than those in children with joint-type HSP(all P<0.05).5.Correlation analyses between food protein specific IgA antibodies and food specific Ig G antibodies showed that CA and BSA specific IgA antibodies were positively correlated with milk Ig G antibodies(both P<0.05).VOA specific IgA antibodies were positively correlated with egg white/yolk Ig G antibodies(P<0.05).6.Kidney biopsies were performed in 17 cases of children with renal-type HSP.The BSA specific IgA antibodies in these patients were negatively correlated with glomerular IgA deposition(P<0.05)and had no correlation with glomerular Fib deposition and pathological grading.Three kinds of protein specific IgA antibodies to CA,BLG and VOA were not correlated with glomerular IgA deposition,glomerular Fib deposition and pathological grading(all P>0.05).7.The result of multiple regression analysis of total serum IgA in children with HSP showed that the factors which could enter the regression model were CA specific IgA antibody,VOA specific IgA antibody,BSA specific IgA antibody and age in the order of contribution in 23 factors that could influence the level of total serum IgA.In the regression model,the sum of squares of regression was 57.605,the residual sum of squares was 55.013,and the total was 112.618,which showed that the sum of the squares of the regression accounted for more than half of the total sum of squares,indicating that more than half of the total serum IgA variability was caused by these four factors,of which the role of BSA specific IgA antibodies on the regression model was negative and the remaining three were positive.8.The results of receiver operating characteristic(ROC)curve and area under the curve(AUC)analysis suggested that CA specific IgA antibody was the most valuable when differentiating HSP from the normal control group,and AUC was 0.803,the sensitivity was 0.763,and the specificity was 0.707.Followed by BLG specific IgA antibodies,AUC was 0.707,the sensitivity increased but the specificity decreased compared with CA specific IgA antibody.When differentiating HSP from the non-rheumatic disease control group(the diarrhea group and abdominal pain group),VOA specific IgA antibody was the most valuable,AUC was 0.850,the sensitivity was 0.763,and the specificity was 0.787.Followed by BLG specific IgA antibodies,AUC was 0.764,the sensitivity increased but the specificity decreased compared with VOA specific IgA antibody.When differentiating HSP from other rheumatic disease control group(JIA and SLE),VOA specific IgA antibody was the most valuable,AUC was 0.807,the sensitivity was0.775,and the specificity was 0.726.Followed by BLG specific IgA antibodies,AUC was0.719,the sensitivity increased but the specificity decreased compared with VOA specific IgA antibody.When differentiating HSP from all control groups,VOA specific IgA antibody was the most valuable,AUC was 0.758,the sensitivity was 0.763,and the specificity was 0.630.Followed by BLG specific IgA antibodies,AUC was 0.717,the sensitivity increased but the specificity decreased compared with VOA specific IgA antibody.Conclusion:1.ELISA system for detecting the specific IgA antibodies of milk and egg protein was successfully constructed in the absence of food specific IgA detection kit on the market.The detection and analysis results of food specific IgA antibodies indicated that the levels of specific IgA antibodies to some milk and egg proteins increased,and decreased after remission,suggesting that its specific IgA antibodies are probably associated with HSP in children.Among them,VOA specific IgA antibodies are the most relevant,followed by BLG specific IgA antibodies.2.VOA specific IgA antibody was the most valuable in distinguishing HSP group from the disease control group and all the control groups,followed by the BLG specific IgA antibody.BSA specific IgA antibodies were negatively correlated with glomerular IgA deposition in children with renal-type HSP,suggesting that BSA specific IgA antibodies may play a role in renal-type HSP.Part Two The level and clinical significance of the food protein components in blood,skin and kidney tissues in children with Henoch-Sch(?)nlein purpuraObjective: To explore the roles of food protein antigens and its specific IgA antibodies in children with HSP by investigating the levels of four kinds of food protein antigens(CA,BLG,BSA and VOA)in the serums and the deposition of the above protein allergens in skin and kidney in children with HSP and the control groups.Methods: The purchased ELISA kit was used to detect the levels of food protein antigens in children's serums of each group.The research subjects in the part were selected from the first part,and the specimen collection method and preservation were the same as the first part.The blood specimens of children with HSP in the acute phase and normal control group were randomly selected from the first part,97 cases and 68 cases respectively.The samples of 36 children with HSP in the acute and recovery phase were both assayed.The disease control groups did not include the abdominal pain group,and the remainder was the same as the first part,namely 38 cases of JIA,24 cases of SLE,17 cases of IgAN and 30 cases of diarrhea.The deposition of four kinds of food protein allergens in the small blood vessels of the skin tissue was detected by the immunohistochemical staining method.The skin samples were skin wax block specimens of 12 patients with HSP at the Department of Nephrology and rheumatology at Children's Hospital of Soochow University from January 2012 to May 2017.The resected finger skin wax blocks of 6 patients with multiple fingers were collected as the normal skin control group.In the same period,the wax block specimens of other patients with skin biopsy at the Department of Nephrology and rheumatology were treated as the disease control groups,including 4 cases of allergic vasculitis(AV),3 cases of juvenile dermatomyositis(JDM),1 case of vasculitis associated with systemic JIA.The immunofluorescence method was used to detect the deposition of four kinds of food protein allergens in the glomeruli.The kidney samples were from 27 patients with HSP at the Department of Nephrology and rheumatology at Children's Hospital of Soochow University from October 2017 to January 2018.Renal biopsy samples of other disease patients were used as disease control group,including 5 cases of IgAN,5 cases of glomerular hematuria,4 cases of acute nephritis,5 cases of SLE,1 case of NS in the same period.Fresh kidney tissue was sectioned using the constant temperature cryosection machine,and then experiments were carried out immediately after slicing.Results: 1.Food protein components BSA and VOA in the serums could be detected in the vast majority of children in each group.The detection rates of BSA in each group were as following: HSP group: 98.97%,the normal control group: 91.18%,the diarrhea group: 93.33%,JIA group: 94.74%,SLE group: 100%,IgAN group: 94.18%.The detection rates of VOA in each group were as following: HSP group: 98.97%,the normal control group: 100%,the diarrhea group: 100%,JIA group: 97.37 %,SLE group: 100%,IgAN group: 100%.The levels of BSA and VOA in children with HSP were positively correlated with the specific IgA antibodies to BSA and VOA in serum(both P < 0.05).In addition,the results of paired comparison of food protein antigens in 36 cases of patients with HSP in the acute and recovery phase showed that the levels of BSA and VOA in the recovery phase were significantly lower than those in the acute phase(both P < 0.05).BSA antigens were positively correlated with BSA specific IgA antibodies in normal control group,diarrhea group and JIA group(all P < 0.05).There was no correlation between BSA antigen and its specific IgA antibodies in SLE group and IgAN group(all P > 0.05).There was no correlation between VOA and its specific IgA antibodies in each control group(all P > 0.05).2.Food protein components CA and BLG in the serums were not detected in some children in each group due to lower than the minimum detection limit of the kit.The detection rates of CA in each group were:HSP group: 62.89%,the normal control group: 35.29%,the diarrhea group: 23.33%,JIA group: 42.11%,SLE group: 95.83%,IgAN group: 35.29%.The detection rates of BLG in each group were: HSP group: 54.64%,the normal control group: 11.76%,Dia group: 46.67%,JIA group: 55.26%,SLE group: 91.67%,and IgAN group: 41.18%.The detection rates of CA and BLG in children with HSP were higher than those in the control group,but were lower than that in SLE group,and the differences were statistically significant(all P < 0.05).In addition,the CA detection rate in children with HSP was also higher than that in the diarrhea group and the IgAN group,and the differences were statistically significant(all P< 0.05).The levels of CA antigen in children with HSP were positively correlated with the specific IgA antibody to CA in peripheral blood(P < 0.05).The results of paired comparison showed that the levels of BLG antigen in 36 cases of children with HSP in the recovery phase were lower than those in the acute phase,and the difference was statistically significant(P < 0.05).There was no correlation between CA,BLG and its specific IgA antibodies in each control group(all P > 0.05).3.The results of food protein antigen test in skin tissue showed that four kinds of food protein allergens could be detected in different degrees in skin small blood vessels of children with HSP.The coloration of CA and VOA was darker,and BLG and BSA had relatively weak staining.There was no statistically significant difference in the average density of staining positive blood vessels among CA,BLG and VOA in children with HSP(all P > 0.05).The average density of staining positive blood vessels of CA,BLG and VOA was significantly greater than that of BSA positive blood vessels(all P < 0.05).The comparison of multiple groups showed that the average density of CA,BLG and VOA positive vessels in HSP children was higher than that in normal children,AV and JDM group(all P < 0.05).The mean density of BSA staining positive vessels of HSP children was higher than that of normal children and childen with JDM(both P < 0.05),but there was no statistical difference compared with children with AV(P > 0.05).4.The results of renal tissue food protein antigen test showed that the deposition rates of glomerular BSA and VOA in children with HSP were 88.89% and 81.48%,respectively,which were higher than those in children with acute nephritis(all P < 0.05),and the deposition rate of BSA was also higher than that in children with glomerular hematuria(P < 0.05).The deposition rates of glomerular CA and BLG in children with HSP were 44.44% and 74.07%,respectively,and there was no significant difference compared with those in other control groups(P > 0.05).Conclusion: The results of blood and histological examination showed that the partial protein components of milk and eggs had a good correlation with HSP.They were related to the specific IgA antibody levels and consistent with the disease conditions.It is speculated that the deposition of the protein components of food protein antigens and specific IgA antibodies in small blood vessel walls may be involved in the pathogenesis of HSP. |
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