| BackgroudHenoch-Sch(?)nlein purpura (HSP) is one of the most common systemic small vessel vasculitis affecting children. The characteristic pathological feature of HSP vasculitis is a deposition of IgA-containing immune complexes in vessel walls of affected organs and in the kidney mesangium. HSP is characterized clinically by nonthrombocytopenic purpuric, arthrocele or arthralgia, gastrointestinal symptoms and renal disease, intussusception and central nervous system injury are rare. HSP is considered to be self-limiting. The prognosis is related to the children's age and degree of renal involvement. Some patients may develop into chronic renal failure. However, the etiology and pathogenesis of HSP remains unknown:the basic scene comes across an abnormal inflammatory process deriving from immune reactions to various antigenic stimuli, which might be bacterial, viral, or parasitic agents, in a genetically prone individual. Then, a peculiar immune complex deposition in the vascular walls and overproduction of different proinflammatory molecules elicit different clinical signs. HSP is diagnosed based on symptoms and signs and histopathological findings because there is no gold standard disease biomarker for HSP. There are still many problems to be further studied and solved. So, it is a challenge for clinical and scientific researchers to find biomarker for HSP and predictor for the involvement of kidneys in children with HSP.Midkine (MK) is a heparin-binding growth factor belongs to midkine family which is composed of MK and pleotrophin (PTN), firstly defined in the early differentiation stage in embryonal life. In healthy adults, MK protein is detected at very low level. Recent studies found that MK concentration was significantly increased in patients with malignant tumors (such as Wilms tumor, gastric cancer, pancreatic cancer, hepatocellular carcinoma, esophageal cancer, breast cancer, leukemia), ischaemia, tissue repair, inflammatory and autoimmunity disease (such as Crohn's disease, rheumatoid arthritis, chronic nephritis, multiple sclerosis).MK also has many biological activities associated with the above diseases. Such as promoting tumor growth, survival, migration and tumor angiogenesis, promoting tissue infiltration with inflammatory cells, inducing the expression of proinflammatory cytokines, extracellular matrix components and metalloproteinases. Because MK is a soluble and secreted cytokine, it can be readily quantitated in serum, so making it a minimally invasive biomarker for predicting, monitoring and managing illness, such as malignant tumors, autoimmune diseases, infectious diseases and chronic inflammatory diseases.This study was based on the expression of MK in peripheral blood of children with HSP. To investigate the changes of immune function and cytokine levels at early onset of HSP. Lay a theoretical foundation for further clarifying the pathogenesis of HSP and finding the specific diagnostic criteria of HSP.ObjectiveThe purpose of our study was to investigate the expression of MK in peripheral serum of children with HSP. For the purpose of correlation analysis of other cytokine and MK, IL-2, IL-4, IL-6, IL-10, TNF, IFN-?,IL-17A, IgA, IgM, IgE, IgG, CD4+ and CD8+were also detected. We also aimed to evaluate whether or not MK can be used as a novel biomarker for HSP and a predictor for renal involvement in children with HSP. The biological parameters such as complement, creatinine and urea were detected and analyzed statistically in order to find the early predictive indexes of renal damage in HSP patients.MethodsA total of 203 children with a diagnosis of HSP, who presented to Department of Rheumatology and Clinical Immunology of Qilu Children's Hospital of Shandong University in 2015, were screened, of whom 92 eligible patients were enrolled in this study. As well as 60 age-and sex-matched healthy controls without any infection or any other systemic diseases were enrolled in this study.Blood samples were collected from the healthy children and the patients before medical treatment in a fasting state. Serum MK concentration was detected by Enzyme-Linked Immunosorbent Assay. IL-2, IL-4, IL-6, IL-10, TNF, IFN-?, and IL-17A were conducted by cytometric bead array (CBA) technology. Urine micro albumin,24 hour urine protein quantity, Urinary protein/creatinine ratio, complement C3, complement C4, T lymphocyte subsets, IgA, IgM, IgG and IgE were measured in the patients. Combined with clinical laboratory tests, we preliminary analysis the role of MK in the development of HSP in children.The data were analyzed using SPSS version 23.0 statistical software (IBM). Two-tailed p<0.05 was considered statistically significant. According to the characteristics of the continuous variables, t test, analysis of variance (ANOVA), Kruskal Wallis test and Mann Whitney U test were used. Categorical variables were compared using x2test.Correlation analysis was conducted with Spearman's rank correlation test. A Bonferroni correction for multiple correlations was adopted with significance set for a p-value less than 0.0029 (0.05 divided by 17). The diagnostic ability of MK was evaluated by means of receiver operating characteristics (ROC) curve analysis. The area under the curve (AUC) was calculated. A perfect test was indicated by AUC of 1.0; an unvalued test was indicated by AUC of 0.5. AUC between 0.5 and 0.7 indicated low diagnostic value, AUC between 0.7 and 0.9 indicated good diagnostic value, AUC between 0.9 and 1.0 indicated better diagnostic value. The sensitivity, specificity, cutoff concentration and confidence interval (CI) were also described.Results1. Ninety-two HSP patients were included in the study, with 56(60.87%) boys, 36(39.13%) girls ranging in age from 1 to 13 years. And 42(70%) boys,18(30%) girls ranging in age from 2 to 13 years were included in the normal control. Among the ninety-two HSP patients, thirty-six patients developed renal involvement (HSPN) in six months, with 24(66.67%) boys,12(33.33%) girls. And 56 patients have no renal involvement (HSP/no-nephritis), with 32(57.14%) boys,24(42.86%) girls. The mean ages of the children in HSP/no-nephritis, HSPN, and NC groups were 6.46±2.12, 7.22±1.83, and 7.01±1.79 years, respectively. No statistical differences in age and gender were observed among the three groups (all P>0.05).2. Median concentration of circulating MK was significantly elevated in HSP patients as compared to controls (291.74 (248.56-396.42) pg/ml versus 217.3 (198.98-243.65) pg/ml, P<0.05), both in HSP without renal involvement and HSPN. MK elevation in HSPN group was higher than in HSP without renal involvement group (326.58 (266.58-459.25) pg/ml versus 280.72 (233.67-384.36) pg/ml, P<0.05).3. Compared to the healthy control, the concentrations of serum IL-4, IL-6, IL-17A, IFN-y and TNF were significantly increased, whereas the serum level of IL-10 decreased in HSP patients. We also found there was no obvious difference in the level of serum IL-2 between the patients with HSP and the control. But there were no significant differences in cytokine levels between HSP patients without renal involvement and HSPN patients.4. Statistical analysis of the results of the detection indexes of immune function in children with HSP in acute stage.(1) T lymphocyte subsets analysis:compared with healthy control group, the levels of CD4+and CD4+/CD8+in children with HSP were significantly decreased (P <0.01), while the levels of CD8+had no significant difference (P> 0.05). There were no significant differences in CD4+, CD8+and CD4+/CD8+levels between HSP patients without renal involvement and HSPN patients (all P> 0.05).(2) Immunoglobulin analysis:compared with healthy control group, the levels of IgA, IgM and IgE in children with HSP were significantly increased (P<0.01), while the levels of IgG had no significant difference (P>0.05). There were no significant differences in IgA, IgM and IgE levels between HSP patients without renal involvement and HSPN patients (all P>0.05), but the level of IgG was lower in the HSPN group than in the HSP patients without renal involvement group (P=0.04).(3) Other laboratory indexes:compared with healthy control group, the levels of D dimer in children with HSP were significantly increased (P=0.002). Fibrinogen was also significantly increased (P< 0.01), while the levels of complement C3, C4, hematuria nitrogen and creatinine had no significant difference (all P>0.05). There were no significant differences in D dimer, complement C3, C4, hematuria nitrogen and creatinine levels between HSP patients without renal involvement and HSPN patients (all P>0.05). The fibrinogen of the HSPN group was higher than that of the HSP without renal involvement group OP=0.02).5. MK positively correlated with IL-4, IL-6, IL17A, IgA and IgE. In HSPN group, MK positively correlated with serum creatinine, urinary micro albumin,24 hour urinary protein and urinary protein/creatinine ratio.6. The areas under the ROC curve (AUCROc) of MK was 0.902, with the 95% confidence interval (CI) was 0.841-0.963 (P<0.001). The cutoff concentration of MK was 295.58pg/ml. The sensitivity and specificity of serum MK concentration in predicting HSPN were 80.6% and 88.3% respectively.Conclusions1. Circulating midkine level was elevated in children with HSP, expecially in children with HSPN.2. This study confirmed that there is an abnormal expression of inflammatory cytokines in children with HSP. It is also demonstrate that there exist immune and coagulation disorders in children with HSP.3. Elevated MK positively correlated with IL-4, IL-6, IL17A, IgA and IgE. In HSPN subgroup, MK positively correlated with serum creatinine, urinary albumin,24 hour urinary protein and urinary protein/creatinine ratio.4. MK seems to be involved in the development of HSP. Measurement of serum levels of MK is helpful in confirming the diagnosis of HSP and predicting HSPN. |
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