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Objective Kawasaki disease (KD) is characterized by systemic vasculitis, and it is the most common acquired heart disease in children. However, the etiology and immunopathogenesis of KD are still unclear. A genome-wide association study (GWAS) identified polymorphisms in CD40, BLK, and FCGR2A as the susceptibility genes for KD. No epigenetic array studies of KD have previously been published. This study was undertaken to investigate differences in DNA methylation in patients with KD as compared to controls. Methods The HumanMethylation27 BeadChip (Illumina) was used to survey the differences in DNA methylation between KD patients and controls. DNA methylation array validation was performed in a separate cohort by pyrosequencing assay and reporter gene assays. Messenger RNA (mRNA) expression was determined, and the association of methylation with response to intravenous immunoglobulin (IVIG) treatment was analyzed. Results HumanMethylation27 BeadChip assay showed a 15% difference in methylation of 10 genes between KD patients and controls. The FCGR2A cg24422489 group, which was recently reported to be associated with KD susceptibility in a GWAS, had significant hypomethylation of 15.54% less in the KD group than in the control group. Validation of FCGR2A methylation in another cohort also showed significant hypomethylation in the KD group (5 of 5 CpG sites [P < 0.01]; n = 43 in the KD group and n = 55 in the control group). KD patients with IVIG resistance showed hypomethylation of 5 CpG sites (P < 0.05). FCGR2A mRNA expression was significantly increased in patients in the acute stage of KD compared to controls. Reporter gene assays indicated that the CpG sites of the FCGR2A promoter region were sufficient to modulate gene expression. Conclusion This is the first study to examine the DNA methylation array in KD and identify a role of hypomethylation of FCGR2A in susceptibility to KD and IVIG resistance.

Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. Thymus and activation-regulated chemokine/chemokine ligand 17 (TARC/CCL17) is one of the Th2 chemokines and has been suggested as a candidate gene for conferring susceptibility to Th2 associated with allergy diseases. This study examined the correlation between gene polymorphisms and plasma levels of TARC/CCL17 in patients with KD and the outcomes of KD. A total of 381 KD patients and 564 controls were subjected to determination of five tagging single-nucleotide polymorphisms of TARC/CCL17. In addition, plasma TARC/CCL17 levels were measured by enzyme-linked immunosorbent assay. Polymorphisms of TARC/CCL17 were significantly different between normal children and patients with KD. A allele of rs4784805 has better intravenous immunoglobulin (IVIG) treatment response to KD. Furthermore, plasma TARC/CCL17 levels were higher in KD patients than that in controls before IVIG treatment. After IVIG treatment, plasma TARC/CCL17 levels decreased significantly. This study provides the first evidence supporting the association between TARC/CCL17 polymorphisms, susceptibility of KD, and IVIG responses in KD patients.

Background A form of systemic vasculitis, Kawasaki disease (KD) occurs most frequently in children under the age of five years old. Previous studies have found that Prostaglandin E2 (PGE2) correlates with KD, although the related mechanisms are still unknown. CD40L may also be a marker of vasculitis in KD, so this study focuses on PGE2 and CD40L expression in KD. Materials and Methods This study consisted of a total of 144 KD patients, whose intravenous immunoglobulin (IVIG)/coronary arterial lesion (CAL) formation resistance was evaluated. PGE2 levels were evaluated in vitro to study the effect of CD40L on CD4+ T lymphocytes. Results PGE2 levels significantly increased after IVIG treatment (p

Background. Kawasaki disease (KD) is characterized by systemic vasculitis of unknown etiology. Our previous studies showed expression ofCD40ligand on CD4+ T cells correlated to the coronary artery lesion (CAL) and disease progress in KD. Other studies from Japan suggested the role ofCD40Lin the pathogenesis of CAL, and this might help explain the excessive number of males affected with KD but cannot be reproduced by Taiwanese population. This study was conducted to investigate theCD40polymorphism in KD and CAL formation.Methods. A total of 950 subjects (381 KD patients and 569 controls) were investigated to identify 2 tagging single-nucleotide polymorphisms (tSNPs) ofCD40(rs4810485 and rs1535045) by using the TaqMan allelic discrimination assay.Results. A significant association was noted with regards toCD40tSNPs (rs1535045) between controls and KD patients (P=0.0405, dominant model). In KD patients, polymorphisms ofCD40(rs4810485) showed significant association with CAL formation (P=0.0436, recessive model). Haplotype analysis did not yield more significant results between polymorphisms ofCD40and susceptibility/disease activity of KD.Conclusions. This study showed for the first time that polymorphisms ofCD40are associated with susceptibility to KD and CAL formation, in the Taiwanese population.

Background: Exposure to cow’s milk protein in early infancy could lead to increased rates of allergic diseases later in life. We investigated whether feeding a protein-hydrolyzed formula (HF) in the first 6 months of life decreased allergic diseases up to 36 months later. Methods: Newborns who had at least 1 first-degree family member with a history of atopy and could not breast-feed were enrolled. They were fed with HF or cow’s milk infant formula (CM) for at least 6 months via an open-label protocol and were monitored prospectively at 6, 18 and 36 months of age to assess allergy sensitization and allergic diseases. Results: A total of 1,002 infants were enrolled and 679 infants were consistently fed the same formula for the first 6 months of life (345 HF and 334 CM). The percentage of food sensitization (especially to milk protein) was significantly lower in the HF group than in the CM group at 36 months (12.7 vs. 23.4%, p = 0.048). There was no significant difference in the prevalence of aeroallergen sensitization between the groups. Occurrence of allergic diseases during the first 3 years of life was significantly correlated with aeroallergen sensitization, but not to food allergen sensitization, parental atopy or feeding types. Conclusions: Infants fed with HF during the first 6 months of life had a significantly lower percentage of sensitization to milk protein allergens, but not allergic diseases during the first 3 years of life. Avoidance of cow’s milk protein alone in infancy is not enough to decrease rates of allergic diseases.

Objectives: Kawasaki disease (KD) is a systemic vasculitis primarily affecting children who are

Kawasaki disease (KD) is the leading cause of acquired heart disease during childhood in the developed countries. The mechanism and biomarkers of KD remain to be determined. In this study, we sought to elucidate potential plasma proteomic markers in KD patients in comparison to that in febrile controls. Plasma samples from KD patients and febrile controls were subjected to two-dimensional polyacrylamide gel electrophoresis analysis. Differential protein displays between KD patients and febrile controls were determined. Fibrinogen beta and gamma chains, alpha-1-antitrypsin (A1AT), CD5 antigen-like precursor (CD5L), and clusterin were increased in KD patients, whereas immunoglobulin free light chains were decreased, as compared with controls. The differential protein displays were validated with enzyme-linked immunosorbent assay tests. We found higher fibrinogen-related proteins (fibrinogen, A1AT, clusterin, and CD5L), along with a lower level of the immunoglobulin free light chains that involve fibrin degradation in KD. Results from this study showing a unique proteomic profiling with abnormal fibrinogen cascade may afford a good biomarker of KD and a better strategy to prevent cardiovascular complications of KD by correcting abnormal fibrin deposition or degradation.

To investigate the role of eosinophils in Kawasaki disease (KD) and the relationship to initial intravenous immunoglobulin (IVIG) treatment failure. A retrospective analysis of all children who were admitted and met the criteria of KD between 1999 and 2005. The patients were divided into IVIG-responsive and IVIG-resistant groups. A total of 185 patients were enrolled during the study period. A series of blood eosinophils and biochemistry studies were correlated to the effectiveness of IVIG. The neutrophils percentage before IVIG treatment (pre-IVIG), leukocyte counts within 3 days after IVIG treatment (post-IVIG), liver enzyme, albumin levels, and post-IVIG eosinophils percentage were all significantly different between the two groups in univariate analysis. Under multivariate analysis with logistic regression, post-IVIG eosinophilia [peripheral blood (PB) eosinophils >or=4%] had an inverse correlation to KD patients with IVIG-resistance (p = 0.003). Also, pre-IVIG hypoalbuminemia (albumin

Kawasaki disease is an acute multisystem vasculitic syndrome of unknown etiology occurring mostly in infants and children younger than 5 years of age. In developed countries, Kawasaki disease is currently the leading cause of acquired heart diseases in children. However, it is still a mysterious disease. In this article, we reviewed and summarized from the aspects based on infection agents, host immune dysregulation and genetic background intended to establish a feasible infection-immunogenetic pathogenesis for this mysterious disease and also provided the rational strategy to explore optimal treatment of this disease.

Granulocytes play an important role in inflammatory diseases. Neonates tend to develop granulocytopenia under sepsis and stress. It remains unclear whether apoptosis of neonatal granulocytes is different from that of adult granulocytes. In this study, we analyzed the discrepancy of granulocyte apoptosis between cord blood (CB) and adult blood (AB). We found that spontaneous and tumor necrosis factor-alpha (TNFalpha)-induced granulocyte apoptosis as determined by phosphatidylserine expression and DNA fragmentation were more prominent in CB granulocytes than AB granulocytes. CD95 ligand and TNFalpha levels were significantly higher in CB than in AB (p = 0.001 and p0.001, respectively). TNF receptor-2 and CD95 expression on CB granulocytes were not different from those on AB granulocytes. However, the TNF receptor-1 (TNFR1) expression was lower on CB granulocytes than that on AB granulocytes (69.98 +/- 7.32 versus 89.04 +/- 3.73%, p = 0.029). This decrease of TNFR1 expression on neonatal granulocytes might be related to a higher plasma TNFalpha level associated with an intrinsic defect on the dynamic change of membrane TNFR1 expression in neonatal granulocytes. The expression of anti-apoptotic molecule Bcl-2 in neonatal granulocytes was also lower than that in adult granulocytes (4.64 +/- 0.51 versus 7.24 +/- 1.17 unit/mg protein, p = 0.032). Moreover, another anti-apoptotic signal, nuclear factor-kappaB nuclear translocation, was also lower in CB than AB granulocytes. Results from this study suggest that higher plasma death ligands associated with lower anti-apoptotic molecules in granulocytes may act an important role in triggering neonatal granulocyte apoptosis.

Background: Many studies have shown that certain cytokines in amniotic fluids are correlated to premature labor and neonatal brain insults. Aims: We investigated whether different fetal phagocyte and vascular mediators including IL-8, myeloperoxidase (MPO), PGE 2 and soluble vascular cell adhesion molecule-1 (sVCAM-1) levels were correlated to prematurity and cerebral palsy (CP) of premature infants. Subjects: Umbilical cord blood samples from 96 preterm babies from 2250 cord blood collections were studied. Method: The enzyme-linked immunoassay (ELISA) technique was used to determine concentrations of cord blood IL-8, MPO, PGE 2 and sVCAM-1. Results: Preterm babies with gestational age (GA) ≦32 weeks had significant higher cord blood IL-8, MPO and PGE 2 levels than full-term babies. These mediators, however, were not correlated to gestational age, suggesting that increases of these mediators are more related to preterm delivery but not fetal maturation. Further analysis showed that IL-8, a mediator mainly from monocytes, but not MPO, another mediator mostly from granulocytes, was correlated to cerebral palsy. Conclusions: Taken together, these results suggest that both premature monocytes and granulocytes activation are involved in preterm delivery, but maybe only monocytes activation is correlated to premature infants' cerebral palsy. Selective manipulation of monocytes activation may be useful to prevent premature-related cerebral palsy.

The atopic history of parents has long been used to predict infant atopy. However, bias from questionnaires of allergic history are also frequently suspected, because a large number of vasomotor rhinitis, intrinsic asthma, and seborrheic dermatitis cases are probably misinterpreted to be atopic diseases.We attempted to identify a risk factor other than parental atopic history to predict elevated infant IgE levels and infant atopy.A total of 655 core families were prenatally recruited, and finally 545 families completed the study for the prospective analysis of infant atopy at 6 months of age. Atopic history and blood samples of parents were collected in the third trimester during pregnancy. Cord blood (CB) was collected immediately after birth. Infant blood samples and history of infant eczema were collected in the 6-month physical checkup clinic. Blood total IgE and specific IgE levels were determined by use of the Pharmacia CAP system.In univariate analysis, maternal, but not paternal, atopic history correlated with elevated CB IgE levels and the occurrence of infant eczema. Elevated maternal, but not paternal, total IgE levels (150 KU/L) significantly correlated with increases of CB IgE levels (median, 0.54 vs 0.17 KU/L, P.001), infant IgE levels (log-transformed mean values, 1.32 +/- 0.51 vs 1.13 +/- 0.51 KU/L, P.001), and infant eczema (P =.008). Multivariate logistical regression analysis, however, showed that only maternal total IgE levels correlated with CB and infant IgE levels and the development of infant eczema.The maternal, but not paternal, total IgE level correlates with elevated infant IgE levels and infant atopy. This provides a high specificity (83%) and a sensitivity of 34% for prediction of infant atopy. This suggests that maternal factors, placental factors, or both have an impact on perinatal allergic sensitization.

Objective: To assess nitric oxide (NO) production and different displays of NO synthase (NOS) isoforms in patients with Kawasaki disease (KD) before and after intravenous immunoglobulin (IVIG) treatment. Study design: Blood and urine samples were collected from patients with KD before IVIG treatment and 3 days afterward. Age-matched febrile patients with various diseases served as control patients. Plasma NOx (NO3- + NO2-) levels were measured as NO production. Real-time reverse transcriptase–polymerase chain reaction and immunohistochemical staining were used to detect differential NOS isoform expression in mononuclear leukocytes (MNCs). Results: Patients with KD (n = 40) had higher levels of NOx than the non-KD febrile control patients. The elevated NOx levels in patients with KD were significantly associated with the occurrence of coronary artery dilation (>3 mm). These elevated NOx levels significantly decreased after IVIG treatment (157.8 ± 15.9 μmol/L vs 57.0 ± 4.27 μmol/L, P

BackgroundKawasaki disease (KD) of unknown immunopathogenesis is an acute febrile systemic vasculitis and the leading cause of acquired heart diseases in childhood. To search for a better strategy for the prevention and treatment of KD, this study compared and validated human KD immunopathogenesis in a mouse model of Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis.MethodsRecruited subjects fulfilled the criteria of KD and were admitted for intravenous gamma globulin (IVIG) treatment at the Kaohsiung Chang Gung Memorial Hospital from 2001 to 2009. Blood samples from KD patients were collected before and after IVIG treatment, and cardiovascular abnormalities were examined by transthoracic echocardiography. Wild-type male BALB/c mice (4-week-old) were intraperitoneally injected with LCWE (1 mg/mL) to induce coronary arteritis. The induced immune response in mice was examined on days 1, 3, 7, and 14 post injections, and histopathology studies were performed on days 7 and 14.ResultsBoth human KD patients and LCWE-treated mice developed coronary arteritis, myocarditis, valvulitis, and pericarditis, as well as elevated plasma levels of interleukin (IL)-2, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF)-α in acute phase. Most of these proinflammatory cytokines declined to normal levels in mice, whereas normal levels were achieved in patients only after IVIG treatment, with a few exceptions. Toll-like receptor (TLR)-2, but not TLR4 surface enhancement on circulating CD14+ monocytes, was augmented in KD patients before IVIG treatment and in LCWE-treated mice, which declined in patients after IVIG treatment.ConclusionThis result suggests that that not only TLR2 augmentation on CD14+ monocytes might be an inflammatory marker for both human KD patients and LCWE-induced CAL mouse model but also this model is feasible for studying therapeutic strategies of coronary arteritis in human KD by modulating TLR2-mediated immune activation on CD14+ monocytes.

Trichomonas vaginalis virus is a double-stranded (ds) RNA virus found in the parasitic protozoan T. vaginalis. To examine the possible existence of related viruses in various T. vaginalis strains and isolates, we screened 20 such isolates and found the characteristic 5.5-kb viral ds RNA in 16 of them. An additional 0.5-kb ds RNA band was also identified in 10 of these virus-infected T. vaginalis isolates. In a local isolate T1, this 0.5-kb ds RNA cross-hybridized with the 5.5-kb ds RNA. Preliminary evidence from CsCl buoyant density gradient centrifugations suggests that the two ds RNA species are encapsidated in separate viral particles by the same capsid protein. The 5.5-kb ds RNA in isolate T1 exhibited moderate hybridization with the prototype 5.5-kb ds RNA in T. vaginalis stratin NIH-Cl. Little cross-hybridization was observed among the 5.5-kb ds RNAs from T. vaginalis isolates JH32A No. 4, T1 and T5, although the viral proteins in these isolates are of similar molecular weight and share common epitopes as viewed by Western blotting. Hybridization characteristics of the 5.5-kb ds RNAs in individual isolates were further analyzed, and the results suggest great ds RNA divergence among the T. vaginalis viruses. Interestingly, the 0.5-kb ds RNA in different isolates of T. vaginalis also exhibited divergence in patterns closely associated with that of the 5.5-kb ds RNA.

Kawasaki disease (KD) is a systemic vasculitis of unknown etiology and primarily affects children less than 5 years of age. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has been suggested as a candidate gene for conferring susceptibility to autoimmunity. This study examined the correlation of CTLA-4 gene polymorphisms in KD with and without coronary artery lesions (CAL). A total of 233 KD patients and 644 controls were subjected to determination of CTLA-4 polymorphisms at (−318) C/T and (+49) A/G positions by restriction fragment length polymorphism. Susceptibility, CAL, and intravenous immunoglobulin treatment response of KD were then analyzed with genetic variants. Polymorphisms of CTLA-4 (+49 A/G) and (−318 C/T) were not significantly different between normal children and patients with KD. The CTLA-4 (+49) A allele (AA+AG genotype), however, was significantly associated with CAL formation, especially in female patients. This study provides the first evidence supporting the association of CTLA-4 (+49) A/G polymorphism with the CAL formation of KD particularly in female patients.

Scrub typhus called tsutsugamushi disease, is a mite-born acute febrile disease caused by Rickettsia tsutsugamushi. To our knowledge, neonatal scrub typhus has not been reported in the literature. We report on a 26-day-old newborn with aseptic meningitis. The newborn showed an increased specific 1gM antibody to R.tsutsugamushi conjointly with his mother's 1gM titer to the organism. This is the first case in the literature documenting neonatal scrub typhus and may be related to infection in the mother. CASE REPORT A 26-day-old male newborn was well until 4 days prior to this admission. He suffered from intermittent high fever, abdominal distention, and skin pallor.

Kawasaki disease (KD) is a systemic febrile vasculitis particular coronary artery involvement. Eosinophilia has been found in our and other studies in KD. This study further investigates whether eosinophil-related T helper 2 (Th2) cytokines or the activation marker (eosinophil cationic protein - ECP) is involved in KD with coronary artery lesions (CAL). A total of 95 KD patients were enrolled for this study. Plasma samples were subjected to the measurement of interleukin (IL)-4, IL-5, and eotaxin by Luminex-Bedalyte multiplex beadmates system and to the measurement of ECP by fluoroimmunoassay. Patients with KD had higher eosinophils than controls. Eosinophil-related mediators: IL-4, IL-5, eotaxin, and ECP levels were also higher in KD patients than controls before intravenous immunoglobulin (IVIG) treatment. After IVIG treatment, ECP decreased but IL-4, IL-5, and eotaxin increased significantly. The higher the IL-5 and eosinophil levels after IVIG treatment, the lower rate of CAL was found. Changes of eosinophils after IVIG treatment were positively correlated to changes of IL-5 levels but not ECP levels. An increase of eosinophils and IL-5, but not ECP levels after IVIG treatment, was inversely correlated with CAL formation in KD.

Although maternal amniotic and vaginocervical cytokines are known to play a role in triggering preterm delivery, the effects of activating fetal phagocytes and platelets are not clear. In an attempt to clarify this issue, we measured levels of myeloperoxidase (MPO), a phagocyte activation marker, and soluble p-selectin (sCD62p), a platelet activation marker, in umbilical cord blood samples from 2200 consecutive cord blood collections, 106 of which were from preterm infants. MPO and sCD62p levels were correlated to gestational age and preterm delivery. It was found that MPO levels were significantly higher in preterm infants and were not significantly correlated to gestational age. In contrast, sCD62p levels were lower in preterm infants and were negatively correlated to gestational age. In summary, we showed that fetal phagocyte activation as demonstrated by higher cord blood MPO levels is associated with preterm delivery, but platelet activation as shown by lower sCD62p levels is not. This suggests that fetal phagocyte activation may be implicated in preterm delivery, and subsequently in pre-maturity-related inflammatory insults.

Enterovirus 71 (EV 71) infections have high neurovirulence and fatality. Immune responses were assessed in 78 patients with EV 71 infection. EV 71 meningoencephalitis occurred more frequently in younger children and in boys. C-reactive protein levels were not elevated, although total leukocyte counts were increased in these patients. The CD40-ligand expression on T cells significantly decreased in children with meningoencephalitis (P=.041). Polymorphism of the cytotoxic T lymphocyte antigen-4 (CTLA-4) at position 49 of exon 1 showed a higher frequency of G/G genotype in patients with EV 71 meningoencephalitis than in those without meningoencephalitis (18/31 vs. 14/47; P=.045) and in control subjects (18/31 vs. 25/93l; P=.007). Specific EV 71 neutralizing antibody titers were detectable but did not differ in children with and without meningoencephalitis in the acute and convalescent stages. Results from this study suggest that younger children with a certain CTLA-4 polymorphism and altered cellular but not humoral response may be linked to EV 71 meningoencephalitis.

To determine whether neural crest-derived neuroblastoma cells may release cytokines which regulate the function of leukocytes, we found that neuroblastoma (HTB-11) cells did not constitutionally express IL-1 beta, TNF alpha, or IL-8 mRNA. However, TNF alpha, which induced HTB-11 cells to differentiate to perineurium-like cells, induced expression of IL-8 mRNA in a dose- and time-dependent fashion. In contrast, pentoxifylline (1 mM), which promoted HTB-11 cells to differentiate to polygonal neuron-like cells, did not induce IL-8 mRNA expression. As determined by enzyme-linked immunoassay, high levels of IL-8 were detectable in the culture supernatants from TNF alpha-treated neuroblastoma cells, but not pentoxifylline-treated neuroblastoma cells (19.60 +/- 2.34 vs 0.10 +/- 0.06 ng/ml). Culture supernatants obtained from TNF alpha-treated neuroblastoma cells induced chemotaxis of neutrophils and lymphocytes that was significantly blocked by anti-IL-8 neutralizing antibodies. Detection of a leukocyte chemotactic factor was not observed in the culture supernatants from pentoxifylline-treated cells. These results suggest that neural crest-derived perineurium-like cells, but not neuron-like cells, may release a leukocyte chemotactic factor or factors such as IL-8 which could be involved in leukocyte recruitment seen in inflammatory diseases affecting peripheral nerves.