CD25+CD4+ regulatory T cells in patients with Kawasaki disease

OBJECTIVE: To investigate whether the CD25 + CD4 + regulatory T-cell population, which plays important roles not only in maintaining immunologic self-tolerance but also in controlling the magnitude and character of antimicrobial immune responses, is related to the pathophysiology of Kawasaki disease (KD). STUDY DESIGN: The patient group consisted of 54 patients (median age, 30 months; 27 female and 27 male patients) fulfilling the criteria for KD. Age-matched control subjects included 17 patients with active infections and 24 healthy children. We analyzed CD25 + CD4 + cells and the mRNA expression of Foxp3, cytotoxic T lymphocyte-associated antigen 4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), and transforming growth factor beta in peripheral blood mononuclear cells and purified CD4 + T cells. RESULTS: The proportions of CD25 + CD4 + cells in patients with acute-phase KD (median, 2.35% of total lymphocytes) were significantly lower than those in healthy control subjects (median, 3.14%) and control subjects with disease (median, 3.15%). The proportions returned to the normal level after intravenous gammaglobulin treatment (median, 3.86%). The mRNA expression of Foxp3, CTLA4, and GITR showed similar tendencies. CONCLUSIONS: The decrease of CD25 + CD4 + regulatory T cells in the acute phase might have a role in the development of KD.     

Heat Shock Protein as a Predisposing and Immunopotentiating Factor in Kawasaki Disease

Local inflammatory reactivation of a previous BCG inoculation site is a specific and early manifestations of Kawasaki disease (KD). It is postulated that the erythematous changes at the BCG site are part of a generalized activation of the immune system, and that molecules cross-reacting between the infectious agent involved and mycobacterial BCG antigens contribute to the inflammatory process. To detect the cross-reacting molecules, the antibody responses to mycobacterial antigens were examined in 21 patients by IgG-immuno-blotting using acute and convalescent phase sera. Markedly increased levels of antibody against mycobacterial 65kDa protein were detected in all convalescent but in none of the acute sera. Since the antibodies detected are also directed to recombinant 65kDa heat shock protein (HSP65), HSP65 may be related to the initial phase of KD. Moreover, peripheral lymphocytes from convalescent KD patients proliferated in culture in the presence of HSP65, suggesting the sensitization of lymphocytes to HSP65, and γδTCR(+) T cells in the peripheral blood were increased in half the KD patients. Thus, HSP65 is a potent factor involved in activation of the immune system, and consequent endothelial damage in the initial phase of KD.     

Macrophage infiltration of pancreatic acini and islets in acute Kawasaki disease

A report of 2 patients who developed diabetes after Kawasaki disease (KD) led us to determine whether macrophages and/or T cells infiltrate acute KD pancreas. Three of 10 acute fatal KD cases had diffuse macrophage infiltration of the pancreas; T cells were not prominent. Affected islets were seen in close proximity to normal islets. These findings may explain the rarity of diabetes after KD.     

Immunological Abnormalities in Kawasaki Disease with Coronary Artery Lesions

A review of our previous immunological studies on Kawasaki disease (KD) was undertaken. The results showed that peripheral blood macrophages/monocytes, T-cells and B-cells become activated during acute KD in terms of numerical changes in immunocompetent cells, expression of activated antigens on the cell surfaces and cytokine production. Also, during acute KD with coronary artery lesions (CALs) the numbers of macrophages/monocytes are increased. In addition, both the increased levels of tumor necrosis factor-α and shed intercellular adhesion molecule-1 in serum are more evident in KD patients with CALs than in those without. Our results further suggest that the main characteristics of the pathogenesis of KD are increased numbers of peripheral blood macrophages/monocytes with the secretion of monokines by these activated cells, and the expression of adhesion molecules on immunocompetent cells. These immune responses develop more vigorously in KD patients with CALs.     

The Potential Role of Cytokine-Mediated Vascular Endothelial Activation in the Pathogenesis of Kawasaki Disease

Kawasaki disease (KD) is an acute febrile illness of infancy and early childhood characterized by diffuse vasculitis. Although the disease is generally self-limited, up to 30% of untreated patients with KD may develop coronary artery (CA) abnormalities. The acute phase of KD is characterized by marked activation of the immune system leading to increased cytokine production by immune effector cells, the induction of activation antigens on their vascular endothelium and the generation of lytic antibodies directed against vascular endothelial cells (EC) stimulated with cytokines. Treatment with intravenous gammaglobulin (IVGG) usually rapidly reduces acute clinical symptoms and prevents CA abnormalities. Immunologically, successful IVGG treatment is associated with decreased lymphocyte activation, reduced cytokine secretion and the loss of cytokine induced expression of leukocyte adhesion molecules on vascular endothelium. The association between improvement of clinical symptoms with the reduction of cytokine secretion, and reversal of EC activation supports a role for immune mediated injury to cytokine induced EC antigens in the pathogenesis of this disorder.     

辅助性T淋巴细胞17/调节性T淋巴细胞偏倚在川崎病发病机制中的作用

目的 探讨辅助性T淋巴细胞17(Th17)和Foxp3+CD4+调节性T淋巴细胞在儿童川崎病(KD)免疫发病机制中的作用.方法 急性期KD患儿40例,同龄健康对照儿童30例.KD患儿在静脉丙种球蛋白(IVIG)治疗前直接取血备检.流式细胞术检测其外周血Th17细胞及Foxp3+CD4+T淋巴细胞比例;荧光定量PCR检测其CD4+T淋巴细胞转录因子ROR-γt、Foxp3 mRNA表达;ELISA法检测其血浆中IL-17、IL-6、转化生长因子-β(TGF-β)、IL-23水平.结果 急性期KD患儿外周血Th17细胞明显升高(P<0.01),而Foxp3+CD4+T淋巴细胞明显降低(P<0.01);急性期KD患儿Th17细胞转录因子ROR-γt转录水平明显高于健康对照组(P<0.01),Foxp3+CD4+T淋巴细胞转录因子Foxp3表达明显降低(P<0.01);血清IL-6、IL-23和IL-17水平明显升高(Pa<0.01),而TGF-β水平无明显变化(P>0.05).结论 Th17促炎性T淋巴细胞高表达和Foxp3+CD4+调节性T淋巴细胞数量减少导致免疫抑制效应不足是导致儿童KD免疫失衡的重要原因,体内相关细胞因子的变化是引起上述改变的原因.     

川崎病白细胞介素-4基因组蛋白乙酰化修饰改变及意义

目的探讨白细胞介素-4(IL-4)基因组蛋白乙酰化修饰水平改变及其在川崎病(KD)发病机制中的作用。方法选取2016年10月至2018年12月在深圳市儿童医院就诊的KD患儿36例为研究对象,同年龄健康儿童28例作为对照组。KD患儿分别于急性期及静脉用丙种球蛋白(IVIG)治疗有效后4~5 d取血备检。采用染色质免疫共沉淀-荧光定量PCR检测外周血CD4+T淋巴细胞IL-4基因启动子、增强子Va组蛋白H4乙酰化和p300、CREB结合蛋白(CBP)水平;流式细胞术检测外周血Ⅱ型辅助性T淋巴细胞(Th2)(CD4+IL-4+)比例及CD4+T淋巴细胞中磷酸化信号转导及转录活化因子6(pSTAT6)、GATA结合蛋白3(GATA3)、活化T细胞核因子1(NFAT1)、Ⅱ型转化生长因子β受体(TGF-βRⅡ)、磷酸化L型氨基酸转运蛋白1(pLAT1)蛋白表达水平;荧光定量PCR检测CD4+T淋巴细胞IL-4、IL-5、IL-13、IL-4受体α(IL-4Rα)、Ⅰ型转化生长因子β受体(TGF-βRⅠ)、性别决定区Y框蛋白4(SOX4)mRNA表达水平;酶联免疫吸附试验测定血浆IL-4、转化生长因子β(TGF-β)水平。结果1.KD患儿Th2细胞比例、功能相关分子(IL-4、IL-5和IL-13)表达及IL-4基因启动子、增强子Va组蛋白乙酰化水平均明显高于对照组,差异均有统计学意义(均P<0.05),其中冠状动脉损伤组(CAL)前述指标均高于无冠状动脉损伤组(NCAL),差异均有统计学意义(均P<0.05),经IVIG治疗后显著降低,差异均有统计学意义(均P<0.05)。2.与对照组比较KD患儿外周血CD4+T淋巴细胞p300、CBP与IL-4基因启动子、增强子Va结合水平明显上调,差异均有统计学意义(均P<0.05),且p300与IL-4基因启动子、增强子Va结合水平与后者表达均呈正相关(r=0.72、0.43,均P<0.05),经IVIG治疗后呈不同程度下降,差异均有统计学意义(均P<0.05)。其中CAL组p300、CBP与IL-4基因启动子、增强子Va结合水平明显高于NCAL组,差异均有统计学意义(均P<0.05)。3.与对照组比较,KD患儿血浆IL-4水平及CD4+T淋巴细胞IL-4Rα/STAT6/GATA-3、pLAT1/NFATc1表达显著增高,差异均有统计学意义(均P<0.05),血浆TGF-β水平及下游信号分子TGF-βRⅡ/TGF-βRⅠ/SOX4表达明显下调,差异均有统计学意义(均P<0.05),其中CAL组血浆IL-4水平及CD4+T淋巴细胞IL-4Rα/STAT6/GATA-3、pLAT1/NFATc1表达均高于NCAL组,血浆TGF-β水平及下游信号分子TGF-βRⅡ/TGF-βRⅠ/SOX4表达低于NCAL组,差异均有统计学意义(均P<0.05),经IVIG治疗后呈不同程度的回调,差异均有统计学意义(均P<0.05)。结论IL-4基因组蛋白H4过度乙酰化可能是导致KD患儿免疫功能异常的重要原因之一。     

Advances in Kawasaki disease

Recent studies have increased our understanding of the etiopathogenesis of Kawasaki disease (KD). The inflammatory infiltrate in KD coronary artery aneurysms has been shown to consist of CD8 T lymphocytes, macrophages, and IgA plasma cells, consistent with an immune response to an intracellular pathogen with a mucosal portal of entry. The identification of an oligoclonal IgA response in the vascular wall and the detection of a KD-associated antigen in inflamed KD tissues using a synthetic antibody derived from KD oligoclonal IgA antibodies have provided new approaches to identification of the etiologic agent. Highly effective therapy has evolved for KD, even in the absence of identification of the etiologic agent. The existence of incomplete KD cases remains a significant diagnostic dilemma for the clinician. Conclusion: the development of a diagnostic test, more specific therapy, and ultimate prevention of this potentially fatal illness of childhood are dependent upon continued advances in determining the etiopathogenesis of this fascinating disorder.Abbreviations KD Kawasaki disease - IVIG intravenous immunoglobulin     

川崎病患儿CD4+T淋巴细胞表面CD40L表达及其与冠状动脉损伤的关系

目的通过检测川崎病(KD)患儿静脉输注入血丙种球蛋白(丙球)治疗前后外周血T细胞表面CD40L(CD154)表达,探讨KD冠状动脉损伤的发病机制。方法采用流式细胞仪检测26例KD患儿静脉输注丙球治疗前后、16例其他发热性疾病患儿、15例正常儿童外局血T细胞表面的CD40L表达。采用酶联免疫吸附试验检测相应血清中E-选择素。结果KD患儿CD4 T细胞表面CD40L表达及血清E-选择素显著高于其他发热性疾病组及正常对照组(P<0.01),KD患儿静脉输注丙球治疗后明显下降(P<0.01)。CD4 T细胞表面CD40L表达及E-选择素与KD冠状动脉损伤有关,而CD8 T细胞表面CD40L的表达与冠状动脉损伤无关。KD患儿CD4 T细胞表面CD40L表达与E-选择素水平正相关(r=0.626P<0.05)。结论CD40L异常表达及血清E-选择素在KD发病机制中起重要作用。静脉输注丙球能下调CD40L表达及血清E-选择索,有利于血管炎治疗。     

川崎病冠状动脉病变相关细胞因子基因多态性

川崎病是好发于5岁以下儿童的急性全身性中小血管炎性综合征,以全身中小动脉急性炎症反应为主要病理改变,可导致冠状动脉扩张、心肌梗死及猝死.细胞因子是主要由免疫细胞分泌的、能调节细胞功能的小分子多肽.在免疫应答过程中,细胞因子对于炎症细胞间相互作用、细胞的生长和分化有重要调节作用.川崎病急性期超抗原激活自身免疫反应,大量细胞因子及炎症介质释放入血,参与川崎病的发生发展及血管病变.许多炎症反应细胞因子与川崎病并发冠状动脉损害有关.该文就多种细胞因子在川崎病合并冠状动脉病变中的作用机制进行综述.     

Surface and cytoplasmic immunoglobulin expression in circulating B-lymphocytes in acute Kawasaki disease

Kawasaki disease (KD) is an acute vasculitis of young childhood predominantly affecting the coronary arteries. IgA plasma cells have been found to infiltrate vascular and nonvascular tissues in fatal acute KD. To determine whether IgA B-lymphocytes were increased in the peripheral blood of patients with KD, we performed three-color flow cytometry to detect surface and cytoplasmic immunoglobulin expression (IgA, IgM, IgD, and IgG) of peripheral B-lymphocytes in KD patients during the acute, subacute, and convalescent stages of illness and in age-matched febrile and afebrile pediatric controls. Surprisingly, absolute numbers of B-lymphocytes expressing IgA were found to be significantly lower in peripheral blood of acute KD patients compared with febrile and afebrile pediatric controls. These findings indicate that IgA plasma cells are not present in KD tissue as a result of excess numbers of these IgA B-lymphocytes in peripheral blood. We speculate that IgA B-lymphocytes are selectively withdrawn from the peripheral circulation into KD target tissues as part of a specific IgA immune response.     

Peripheral blood eosinophilia and eosinophil accumulation in coronary microvessels in acute Kawasaki disease

BACKGROUND: Early stage Kawasaki disease (KD) histopathology includes perivasculitis and vasculitis of the microvessels. The lesions then extend to larger vessels. Therefore the analysis of microvessel lesions is important to better understand the initial pathogenesis of KD vasculitis. METHODS AND RESULTS: We studied epicardial microvessel lesions (<50 microm) and aneurysm lesions of paraffin-embedded cardiac tissues from 4 Japanese patients who died 7 to 22 days after KD onset. The cellular composition in the microvessel lesions was different from that in coronary aneurysm lesions; eosinophils were preferentially accumulated in the microvessel lesions. The average population of eosinophils was 16% of total infiltrated cells in the microvessel lesions, whereas it was 3% in the intima of aneurysm walls. We examined peripheral blood eosinophil cell counts in 95 KD patients and 95 febrile age-matched controls. Baseline eosinophil cell counts in KD patients were higher than those in febrile control patients (361 +/- 441 65 +/- 133; < 0.0001). Eosinophilia (>350 cells/microl) before therapy was documented in 36% of KD patients, but in only 4% of febrile controls ( < 0.0001). Sixty-six KD patients (69%) developed eosinophilia within 2 weeks of illness. CONCLUSIONS: Because the numbers of circulating eosinophils in the body are tightly regulated, eosinophil accumulation in blood or tissues may reflect the host's immune response against KD related antigen(s).     

BTNL2 gene polymorphisms may be associated with susceptibility to Kawasaki disease and formation of coronary artery lesions in Taiwanese children

The butyrophilin-like 2 (BTNL2) gene is a member of the B7 receptor family that probably functions as a T cell costimulatory molecule. Because altered T cell functions are implicated in dysregulation of the immune response seen in Kawasaki disease (KD), it is reasonable to speculate that BTNL2 gene is involved in the pathophysiology of KD. The purpose of this study was to investigate whether polymorphisms of the BTNL2 gene are associated with KD and the development of coronary artery lesions (CALs) in Taiwanese children. Nine-three patients with KD and 669 ethnically matched healthy controls were genotyped for BTNL2 gene rs1555115 C/G and rs2395158 A/G polymorphisms. The frequency of GG genotype of rs 1555115 was significantly higher in KD patients compared with controls (2.2% vs 0.2%, P = 0.012). The odds ratio for developing KD in individuals with rs 1555115 GG genotype was 14.7 (95% confidence interval, 2.04–105.5, P = 0.003) compared with individuals with rs 1555115 CG and CC genotypes. No significant difference was observed in the genotype and allelic frequencies of rs 2395158 polymorphism between KD patients and controls. However, the frequency of the G allele of rs 2395158 was significantly higher in KD patients with CALs than in those without CALs (P = 0.001). No significant difference was observed in the genotype and allelic frequencies of rs 1555115 polymorphism between KD patients with and without CALs. In conclusion, our results suggest that BTNL2 gene polymorphisms might be genetic markers of KD susceptibility and risk of coronary artery complication in Taiwanese children.     

Pyuria in patients with Kawasaki disease

Kawasaki disease(KD) is an acute, febrile vasculitis that predominantly develops in children ≤ 5 years of age and can lead to multiple organ injuries including the kidneys. Of these injuries, pyuria is a common feature of patients with KD, occurring in 30%-80% of patients. Sterile pyuria is most common in KD patients ≤ 1 year of age. KD patients with sterile pyuria exhibit more severe inflammatory reactions and may have subclinical renal injuries. Sterile pyuria in KD is associated with mononuclear cells(not neutrophils) in the urine. Although sterile pyuria in KD was at one time thought to be due to urethritis caused by a non-specific vasculitis of the urethra, recent studies suggest that sterile pyuria in KD originates from the urethra, the kidney as a resultof mild and sub-clinical renal injuries, and/or the bladder due to cystitis. Pyuria is not always sterile in KD, but can result from a urinary tract infection(UTI). As causative pathogens, Escherichia coli and Klebsiella oxytoca have been reported. The clinical phenotypes do not differ between those with or without UTI. Because some KD patients with UTIs have urinary tract abnormalities such as vesicoureteral reflux, a complete UTI workup including renal ultrasound, voiding cystourethrogram and/or dimercaptosuccinic acid renal scan recommended in KD patients with UTIs.     

急性期川崎病IL-4基因组蛋白甲基化的改变及意义

目的 探讨急性期川崎病(KD)患儿IL-4基因组蛋白甲基化改变及其在KD Th2细胞异常机制中的作用。方法 KD患儿急性期及IVIG治疗4～5 d后取血备检,同年龄健康儿童为对照组。采用流式细胞术检测外周血Th2比例及CD4⁺ T细胞IL-4、pSTAT6和GATA3蛋白水平;染色质免疫共沉淀分析外周血CD4⁺ T细胞IL-4基因CNS1、HSⅡ、HSVa组蛋白甲基化H3K4me3及GATA3、MLL1结合水平;荧光定量PCR分析CD4⁺ T细胞IL-4、IL-5、IL-13、IL-4Rα、IL-2Rγ、SOCS5 mRNA水平;双抗体夹心ELISA检测血浆细胞因子IL-4、IL-5和IL-13蛋白浓度。结果 KD组42例,对照组36例。① KD患儿急性期Th2细胞比例,功能相关分子(IL-4、IL-5和IL-13)mRNA表达,血浆蛋白水平,IL-4基因CNS1、HSⅡ、HSVa位点组蛋白甲基化H3K4me3修饰水平均明显增加(P<0.05),且冠状动脉损伤组(CAL)前述指标均高于无冠状动脉损伤组(NCAL)(P<0.05),经IVIG治疗后明显降低(P<0.05); ②KD患儿急性期外周血CD4⁺ T细胞转录因子GATA-3、组蛋白甲基化酶MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平显著增高(P<0.05),且MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平与IL-4 mRNA表达显著正相关(r分别为0.42、0.33和0.39, P均<0.05),经IVIG治疗后均表现不同水平的恢复(P<0.05)。其中CAL组MLL1、GATA-3与IL-4基因CNS1、HSⅡ、HSVa结合水平明显高于NCAL组 (P<0.05); ③KD患儿急性期血浆IL-4浓度和CD4⁺ T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA-3、SOCS5表达显著增高(P<0.05),其中CAL组血浆IL-4浓度和CD4+T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA3表达均高于NCAL组、SOCS5表达低于NCAL组(P<0.05),经IVIG治疗后均表现不同水平的下调(P<0.05)。结论 IL-4基因组蛋白甲基化修饰异常可能是导致KD患儿急性期Th2细胞异常的始动因素之一。     

Increased production of serum IgA-class antibody to lipid A in Kawasaki disease

BACKGROUND: The etiology of Kawasaki disease (KD) remains unknown. To investigate whether a conventional bacterial antigen is involved in the pathogenesis of KD, we studied the serum response to lipopolysaccharide (LPS). METHODS: We measured the serum levels of IgG-, IgM- and IgA-class antibodies (Ab) to lipid A, a toxic site of LPS, using enzyme-linked immunosorbent assay in 20 patients with KD, 11 patients with Gram-negative bacterial infection (GNBI), 27 healthy children and 12 healthy adults. RESULTS: The serum levels of anti-lipid A IgG, IgM and IgA tended to increase with advancing age in healthy children older than 6 months of age. The mean level of anti-lipid A IgM in the acute phase of GNBI and the mean levels of anti-lipid A IgM and IgA in the acute phase of KD were found to increase significantly, in comparison to the age-matched controls. Furthermore, the mean level of anti-lipid A IgA also showed a significant increase from the acute to the subacute phases of KD. Regarding the IgA-subclass response, higher titers of anti-lipid A specific Ab were seen in the IgA2 subclass than in the IgA1 subclass. CONCLUSION: These findings indicate that KD patients demonstrate an intense response to lipid A in the IgA, especially IgA2-subclass, thus suggesting that an unusual activation of the mucosal immune response to a ubiquitous antigen derived from Gram-negative bacteria may be involved in the pathogenesis of KD.     

急性期川崎病IL-4基因组蛋白甲基化的改变及意义

目的 探讨急性期川崎病(KD)患儿IL-4基因组蛋白甲基化改变及其在KD Th2细胞异常机制中的作用。方法 KD患儿急性期及IVIG治疗4～5 d后取血备检,同年龄健康儿童为对照组。采用流式细胞术检测外周血Th2比例及CD4⁺ T细胞IL-4、pSTAT6和GATA3蛋白水平;染色质免疫共沉淀分析外周血CD4⁺ T细胞IL-4基因CNS1、HSⅡ、HSVa组蛋白甲基化H3K4me3及GATA3、MLL1结合水平;荧光定量PCR分析CD4⁺ T细胞IL-4、IL-5、IL-13、IL-4Rα、IL-2Rγ、SOCS5 mRNA水平;双抗体夹心ELISA检测血浆细胞因子IL-4、IL-5和IL-13蛋白浓度。结果 KD组42例,对照组36例。① KD患儿急性期Th2细胞比例,功能相关分子(IL-4、IL-5和IL-13)mRNA表达,血浆蛋白水平,IL-4基因CNS1、HSⅡ、HSVa位点组蛋白甲基化H3K4me3修饰水平均明显增加(P<0.05),且冠状动脉损伤组(CAL)前述指标均高于无冠状动脉损伤组(NCAL)(P<0.05),经IVIG治疗后明显降低(P<0.05); ②KD患儿急性期外周血CD4⁺ T细胞转录因子GATA-3、组蛋白甲基化酶MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平显著增高(P<0.05),且MLL1与IL-4基因CNS1、HSⅡ、HSVa结合水平与IL-4 mRNA表达显著正相关(r分别为0.42、0.33和0.39, P均<0.05),经IVIG治疗后均表现不同水平的恢复(P<0.05)。其中CAL组MLL1、GATA-3与IL-4基因CNS1、HSⅡ、HSVa结合水平明显高于NCAL组 (P<0.05); ③KD患儿急性期血浆IL-4浓度和CD4⁺ T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA-3、SOCS5表达显著增高(P<0.05),其中CAL组血浆IL-4浓度和CD4+T细胞IL-4Rα、IL-2Rγ、pSTAT6、GATA3表达均高于NCAL组、SOCS5表达低于NCAL组(P<0.05),经IVIG治疗后均表现不同水平的下调(P<0.05)。结论 IL-4基因组蛋白甲基化修饰异常可能是导致KD患儿急性期Th2细胞异常的始动因素之一。     

Analysis of tumor necrosis factor-α production and polymorphisms of the tumor necrosis factor-α gene in individuals with a history of Kawasaki disease

BACKGROUND: Tumor necrosis factor (TNF)-alpha plays a central role in the pathogenesis of vasculitis in Kawasaki disease (KD). To address the genetic background of KD, we investigated the level of TNF-alpha production and genetic polymorphisms in the 5' flanking region of the TNF-alpha gene in healthy children with a history of KD. METHODS: For TNF-alpha production, peripheral blood mononuclear cells (PBMC) of children with a history of KD (n = 61) and of non-KD children (n = 35) were stimulated with phorbol 12-myristate 13-acetate, toxic shock syndrome toxin-1 (TSST-1) and the culture supernatant of Staphylococcus aureus derived from a KD patient (S-6), which had several superantigenic activities. The genetic background of KD was addressed by studying polymorphisms in the 5' flanking region of the TNF-alpha gene at positions -1031 (thymine (T) to cytosine (C) change, termed -1031C), -863 (C to adenine (A), -863A), -857 (C to T, -857T), -308 (guanine (G) to A, -308A) and -238 (G to A, -238A) in KD, using dot-blot hybridization with sequence-specific oligonucleotide probes. RESULTS: The PBMC of KD patients with coronary artery lesions produced slightly higher levels of TNF-alpha in response to the bacterial products (such as TSST-1 and S-6). None of the polymorphisms in the 5' flanking region of the TNF-alpha gene were related to KD. CONCLUSIONS: These results suggest that a genetic disposition towards overproduction of TNF-alpha in response to bacterial products may be involved in the pathogenesis of KD.