Application of serum interleukin, anti-cardiolipin antibody and anti-endothelial cell antibody detection in children with Kawasaki disease / 中国医师进修杂志

Objective To explore the significance of serum levels of interleukin(IL)-33,21,17, 6,the positive rates of anti-cardiolipin antibody(ACA)-IgG,ACA-IgM and anti endothelial cell antibody (AECA)-IgM in diagnosis of Kawasaki disease and prediction of coronary artery lesions. Methods Seventy children with Kawasaki disease were selected as Kawasaki disease group,and the children were divided into abnormal ultrasonic group(11 cases)and normal ultrasonic group(59 cases)according to the result of cardiac ultrasound. Fifty children with upper respiratory tract infection or bronchitis were selected as control group.The serum levels of IL-33,21,17,6 and positive rates of ACA-IgG,ACA-IgM, AECA-IgM were detected.Results The serum levels of IL-33,21,17 and 6 in Kawasaki disease acute stage were significantly higher than those in control group:(127.43 ± 10.87)ng/L vs.(69.67 ± 6.38)ng/L, (130.43 ± 11.22) ng/L vs. (87.56 ± 7.76) ng/L, (1 243.38 ± 612.08) ng/L vs. (397.26 ± 182.16) ng/L, (438.35 ± 101.78)ng/L vs.(213.74 ± 104.52)ng/L;the positive rates of ACA-IgG,ACA-IgM and AECA-IgM were significantly higher than those in control group: 37.1%(26/70)vs.8.0%(4/50),32.9%(23/70) vs.6.0%(3/50)and 34.3%(24/70)vs.8.0%(4/50),and there were statistical differences(P<0.01).The acute stage serum levels of IL-33, 21, 17 and 6 in abnormal ultrasonic group were significantly higher than those in normal ultrasonic group:(135.92 ± 11.56)ng/L vs.(123.48 ± 10.14)ng/L,(138.29 ± 11.86) ng/L vs.(128.08 ± 10.94)ng/L,(2 042.47 ± 968.43)ng/L vs.(1 096.59 ± 502.82)ng/L,(495.58 ± 103.04) ng/L vs. (402.67 ± 98.26) ng/L; the positive rates of ACA-IgG, ACA-IgM and AECA-IgM were significantly higher than those in normal ultrasonic group:7/11 vs.32.2%(19/59),8/11 vs.25.4%(15/59) and 7/11 vs. 28.8% (17/59), and there were statistical differences (P < 0.01 or <0.05). In Kawasaki disease, the acute stage serum levels of IL-33, 21, 17 and 6 were significantly higher than those in subacute stage:(127.43 ± 10.87)ng/L vs.(94.48 ± 8.56)ng/L,(130.43 ± 11.22)ng/L vs.(91.78 ± 8.03) ng/L, (1 243.38 ± 612.08) ng/L vs. (527.12 ± 236.94) ng/L and (438.35 ± 101.78) ng/L vs. (308.41 ± 144.09)ng/L,and there were statistical differences(P<0.01).Conclusions IL-33,21,17,6 and ACA-IgG, ACA-IgM, AECA-IgM participates in the process of Kawasaki disease vasculitis and coronary artery damage,which can assist the early diagnosis of Kawasaki disease and predict the coronary artery lesions.

Prokaryotic expression of recombinant human α enolase and the prevalence of anti-α enolase antibody in connective tissue diseases / 中华风湿病学杂志

Objective In our previous work, the prevalence of anti-endothelial cell antibodies(AECA) in patients with systemic vasculitis and other autoimmune diseases was analyzed. AECA against a 47 000 endothelial cell antigen was found in patients of a variety of systemic vasculitis and systemic lupus erythematosus (SLE). It was suggested to be α-enolase by the combination of immunoblotting and proteomics methods. The aim of this work is to demonstrate that α-enolase is one of the targets of AECA, and to detect the prevalence of anti-α-enolase antibody in sera of patients with autoimmune disorders including systemic vasculitis. Methods The CDS of human Enol gene was amplified by polymerase chain reaction (PCR), with template of human placenta λzap express Cdna library. The product was then recombined with expression vector. After expression and purification from E.coli, the recombinant protein was analyzed by mass spee-trometry. The prevalence of anti-α-enolase antibody in patients with autoimmune disorders including systemic vasculitis was tested by Western blot and enzyme-linked immunosorbent assay (ELISA). Results The CDS of human Enol gene was subcloned to the expression vector. Recombinant human α-enolase was expressed and purified in E.coli. The recombinant protein was demonstrated to be his-tagged human a-enolase by mass spectrometry. Results of Dot-Blot revealed that the prevalence of anti-α-enolase antibody was 76.7% in systemic vasculitis [including 74.0% in Behcet's disease (BD), 81.5% in Takayasu artefitis (TA), 62.5% in Wegener's granulomatosus (WG), 92.3% in microscopic polyangitis (MPA) and 80.0% in Churg-Stranss syndrome (CSS)], 78.3% in SLE, 63.6% in Sjogren's syndrome (SS) and 78.9% in rheumatoid arthritis(RA). No positive signals were detected in sera of normal controls or patients with polymyositis/ dermatomyositis (PM/DM). There was no statistical significance among positive rates of anti-α-enolase antibody in systemic vasculitis, SLE, SS or RA patients. The prevalence of positive signals at the most extensive level (+++～++++) was 51.7% in patients with systemic vasculitis, 33.3% in SLE, 42.9% in SS and 20.0% in RA. There was statistical significant difference between RA and systemic vasculitis. Conclusion The identification of human α-enolase as one of the targets of AECA and its prevalence in a variety of autoimmune disorders will shed some light on the understanding of the pathogenesis of vascular injury in autoimmune diseases.

Anti-endothelial cell antibodies and central nervous system involvement in Behçet's disease

INTRODUCTION: Previous studies have detected the presence of anti-endothelial cell antibodies (AECA) in patients with Behçet's disease (BD). However, no real evidence exists whether these antibodies exert any influence on clinical presentation and/or activity of this disease. OBJECTIVES: To determine the frequency of AECA in patients with BD and analyze possible clinical associations. METHODS: 50 patients with BD who fulfilled diagnostic criteria were selected. Thirty-seven patients were females, and 13 were males; the mean age was 44 ± 9 years with a mean follow-up time of 10 ± 7.5 years. AECA were assayed by ELISA using ECV-304 cells as the antigenic substrate. The prevalence of AECA was determined, and their possible relationships with present and past clinical features were investigated. RESULTS: AECA were detected in the sera of 38 percent of the patients (IgG in 13, IgM in four, and IgG plus IgM in two). An association was observed between AECA and a previous history of central nervous system involvement (OR= 5.4, p= 0.03). This association was more evident for IgG-AECA (OR= 6.0, p= 0.02). A trend of an increased risk of aneurysms was also observed in patients with IgG-AECA (OR= 2.58, p= 0.77). None of the other clinical characteristics showed a relevant association with these antibodies. CONCLUSION: Our data suggest that IgG-AECA may be a marker of more severe lesions in patients with BD based on the higher frequency of previous central nervous system manifestations in patients who presently display circulating AECA.

INTRODUÇÃO: Estudos anteriores detectaram a presence de anticorpos anti-célula endotelial (AACE) em pacientes com doença de Behçet, porém não há nenhuma evidência se a presença destes anticorpos exerce alguma influência na apresentação clínica ou atividade da doença. OBJETIVOS: Determinar a freqüência de AACE em pacientes com doença de Behçet e analisar possíveis associações clínicas. MÉTODOS: Foram selecionados 50 pacientes que preencheram corretamente os critérios diagnósticos para a doença de Behçet. Trinta e sete pacientes eram do sexo feminino e 13 do sexo masculino, média de idade de 44 ± 9 anos e tempo médio de seguimento de 10 ± 7,5 anos. O AACE foram analisados por ELISA utilizando células ECV-304 como substrato antigênico. A prevalência de AACE foi determinada e foram investigadas possíveis relações com características clínicas atuais e pregressas. RESULTADOS: Os AACE foram detectados no soro de 38 por cento dos pacientes (13 na forma IgG, 4 IgM e 2 nas formas IgG e IgM). Observamos uma associação entre o AACE e história pregressa de envolvimento de sistema nervoso central (OR=5,4; p=0,03). Esta associação era mais evidente para o AACE na forma IgG (OR=6,0; p=0,02). Observamos também uma tendência de risco aumentado de aneurismas em pacientes com AACE na forma IgG (OR=2,58; p=0,77). Nenhuma outra característica clínica mostrou-se relevante com o anticorpo estudado. CONCLUSÃO: Nossos dados sugerem que o AACE na forma IgG pode ser uma marcador de lesão mais grave em pacientes com doença de Behçet baseado no fato de encontrarmos uma maior freqüência de história pregressa de manifestação de sistema nervoso central em pacientes com AACE circulante.

Role of Anti-Endothelial Cell Antibody in the Development of Coronary Arterial Lesions in Kawasaki Disease

BACKGROUND AND OBJECTIVES : Anti-endothelial cell antibodies (AECA) are found in the sera of many patients with Kawasaki disease (KD). In this study, the pathogenic role of AECA in the development of coronary arterial lesions of KD was investigated. SUBJECTS AND METHODS : Serum IgM-AECA concentrations were measured in 22 KD patients. Cultured human coronary artery endothelial cells (HCAEC) were incubated with either acute or convalescent phase sera, and their expressions of intercellular adhesion molecule-1 (ICAM-1) assessed. IgM fractions of the sera were purified, and their ability to induce ICAM-1 mRNA and protein expressions evaluated. To address the signal transduction pathways involved in IgM-AECA-induced ICAM-1 expression, the blocking effect of four protein kinase inhibitors, PD98059, SB203580, dimethylaminopurine (DMAP) and parthenolide were measured. RESULTS : IgM-AECA was present in 14 out of 22 (64%) acute KD sera. ICAM-1 expression of HCAEC incubated with acute KD sera (117.1+/-46.7) and AECA-positive acute KD sera (143.3+/-37.5) were significantly higher than those of the convalescent KD sera (88.9+/-14.4, p<0.05) or AECA-negative acute KD sera (71.2+/-11.8, p<0.05), respectively. IgM-AECA from KD patients significantly induced ICAM-1 protein and mRNA expression. The upregulation of ICAM-1 expression was significantly inhibited by SB203580, DMAP and parthenolide, but not by PD98059. CONCLUSION : IgM-AECA was detected in the sera of about 2/3 of acute KD patients, which activated endothelial cells by upregulation of ICAM-1 expression, possibly via p38, JNK MAPK and NF-kappaB signal transduction pathways. Thus, IgM-AECA may play a pathogenic role in the development of coronary arterial lesions in KD patients.