Acute Kawasaki Disease Patients Research Articles

Involvement of IL-17 A/IL-17 Receptor A with Neutrophil Recruitment and the Severity of Coronary Arteritis in Kawasaki Disease

PurposeTo assess the role of the interleukin (IL)-17 A/IL-17 receptor A (IL-17RA) in Kawasaki disease (KD)-related coronary arteritis (CA).MethodsIn human study, the plasma levels of IL-17 A and coronary arteries were concurrently examined in acute KD patients. In vitro responses of human coronary endothelial cells to plasma stimulation were investigated with and without IL-17RA neutralization. A murine model of Lactobacillus casei cell-wall extract (LCWE)-induced CA using wild-type Balb/c and Il17ra-deficient mice were also inspected.ResultsThe plasma levels of IL-17 A were significantly higher in KD patients before intravenous immunoglobulin therapy, especially in those with coronary artery lesion. The pre-IVIG IL-17 A levels positively correlated with maximal z scores of coronary diameters and plasma-induced endothelial mRNA levels of chemokine (C-X-C motif) ligand-1, IL-8, and IL-17RA. IL-17RA blockade significantly reduced such endothelial upregulations of aforementioned three genes and inducible nitric oxide synthase, and neutrophil transmigration. IL-17RA expression was enhanced on peripheral blood mononuclear cells in pre-IVIG KD patients, and in the aortic rings and spleens of the LCWE-stimulated mice. LCWE-induced CA composed of dual-positive Ly6G- and IL-17 A-stained infiltrates. Il17ra-deficient mice showed reduced CA severity with the fewer number of neutrophils and lower early inducible nitric oxide synthase and chemokine (C-X-C motif) ligand-1 mRNA expressions than Il17ra+/+ littermates, and absent IL-17RA upregulation at aortic roots.ConclusionIL-17 A/IL-17RA axis may play a role in mediating aortic neutrophil chemoattraction, thus contributory to the severity of CA in both humans and mice. These findings may help to develop a new therapeutic strategy toward ameliorating KD-related CA.

Bioinformatics analysis and identification of hub genes of neutrophils in Kawasaki disease: a pivotal study.

Kawasaki disease (KD) is considered the main contributor to acquired heart diseases in developed countries. However, the precise pathogenesis of KD remains unclear. Neutrophils play roles in KD. This study aimed to select hub genes in neutrophils in acute KD. mRNA microarray of neutrophils from four acute KD patients and three healthy controls was performed to screen differentially expressed mRNAs (DE-mRNAs). DE-mRNAs were analyzed and predicted by Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways, and protein-protein interaction networks. Real time-PCR was finally conducted to confirm the reliability and validity of the expression level of DE-mRNAs from blood samples of healthy controls and KD patients in both acute and convalescent stage. A total of 1950 DE-mRNAs including 1287 upregulated and 663 downregulated mRNAs were identified. GO and KEGG analyses revealed the DE-mRNAs were mainly enriched in the regulation of transcription from RNA polymerase II promoter, apoptotic process, intracellular signal transduction, protein phosphorylation, protein transport, metabolic pathways, carbon metabolism, lysosome, apoptosis, pyrimidine metabolism, alzheimer disease, prion disease, sphingolipid metabolism, huntington disease, glucagon signaling pathway, non-alcoholic fatty liver disease, pyruvate metabolism, sphingolipid signaling pathway, and peroxisome. Twenty hub DE-mRNAs were selected including GAPDH, GNB2L1, PTPRC, GART, HIST2H2AC, ACTG1, H2AFX, CREB1, ATP5A1, ENO1, RAC2, PKM, BCL2L1, ATP5B, MRPL13, SDHA, TLR4, RUVBL2, TXNRD1, and ITGAM. The real-time PCR results showed that BCL2L1 and ITGAM mRNA were upregulated in acute KD and were normalized in the convalescent stage. These findings may improve our understanding of neutrophils in KD. Key Points • Neutrophilic BCL2L1 and ITGAM mRNA were first reported to be correlated with the pathogenic mechanism of KD.

The Therapeutic Effects of EFNB2-Fc in a Cell Model of Kawasaki Disease.

The EphrinB2/EphB4 signaling pathway involves the regulation of vascular morphogenesis and angiogenesis. However, little is known about EphrinB2/EphB4 in the pathogenesis of Kawasaki disease (KD) and coronary artery aneurysm formation. Hence, this study aimed to explore the role of EphrinB2/EphB4 and the potential therapeutic effect of EphrinB2-Fc in the coronary arterial endothelial injury of KD. The levels of EphB4 were compared between KD patients and healthy children. Human coronary artery endothelial cells (HCAECs) were stimulated with sera from acute KD patients to establish the KD cell model. The overexpression of EphB4 or treatment with EphrinB2-Fc was found to intervene in the cell model. The cell migration, angiogenesis, and proliferation ability were assessed, and the expression of inflammation-related factors was measured. Our study showed that EphB4 showed low expression in both KD patients and the cell model of KD. The EphB4 protein levels in the CECs of CAA+ KD patients were much lower than those in healthy children. EphrinB2-Fc treatment of KD sera-activated HCAECs suppressed cell proliferation, reduced the expression of inflammation-related factors (such as IL-6 and P-selectin), and elevated cell angiogenesis ability. The results reveal that EphrinB2-Fc has a protective function in endothelial cells and has promising clinical applications for protecting vascular endothelium in patients with KD.

Circulating Endothelial Cells: A New Possible Marker of Endothelial Damage in Kawasaki Disease, Multisystem Inflammatory Syndrome in Children and Acute SARS-CoV-2 Infection.

Background: Kawasaki Disease (KD) and Multisystem Inflammatory Syndrome in Children (MIS-C) are pediatric diseases characterized by systemic inflammation and vascular injury, potentially leading to coronary artery lesions (CALs). Data on vascular injury occurring during acute COVID-19 (AC19) in children are still lacking. The aim of our study was to investigate endothelial injury in KD-, MIS-C- and AC19-dosing circulating endothelial cells (CECs). Methods: We conducted a multicenter prospective study. CECs were enumerated by CellSearch technology through the immunomagnetic capture of CD146-positive cells from whole blood. Results: We enrolled 9 KD, 20 MIS-C and 10 AC19. During the acute stage, the AC19 and KD patients had higher CECs levels than the MIS-C patients. From the acute to subacute phase, a significant CEC increase was observed in the KD patients, while a mild decrease was detected in the MIS-C patients. Cellular clusters/syncytia were more common in the KD patients. No correlation between CECs and CALs were found in the MIS-C patients. The incidence of CALs in the KD group was too low to investigate this correlation. Conclusions: Our study suggests a possible role of CECs as biomarkers of systemic inflammation and endothelial dysfunction in KD and MIS-C and different mechanisms of vascular injury in these diseases. Further larger studies are needed.

Abstract 476: Kawasaki Disease, Metabolic Switch, And Endothelial Dysfunction

Kawasaki Disease (KD) induces endothelial dysfunction, in part, through down-regulation of miR-483. Metabolic reprogramming, including increased glycolysis and reduced oxidative phosphorylation, is associated with endothelial cell (EC) dysfunction. However, whether metabolic reprogramming is involved in KD-induced EC dysfunction is unknown. Using RNA-seq and pathway analyses, we determined that genes involved in glycolysis were upregulated by sera from acute KD patients compared with convalescent control sera. We validated by qPCR and found increased mRNA levels of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), hexokinase 1 (HK1), and hexokinase 3 (HK3), as well as the decrease in glucokinase regulator (GCKR) in cultured ECs incubated with acute KD sera. Moreover, PFKFB3 protein level was elevated in ECs cultured with acute KD sera and in the intima of coronary arteries of mice with LCWE-induced KD vasculitis. Seahorse assay revealed that KD sera increased extracellular acidification rate (ECAR) and decreased oxygen consumption rate (OCR) in ECs, indicating that serum factors in acute KD patients increased glycolysis and impaired mitochondrial function. Given the presence of a miR-483 targeting site in the 3’ untranslated region (3’UTR) of PFKFB3 mRNA, we cloned a PFKFB3-3’UTR luciferase reporter (PFKFB3-Luc) to test whether PFKFB3 is regulated by miR-483. As anticipated, miR-483 overexpression downregulated luciferase activity in ECs transfected with PFKFB3-Luc. Moreover, miR-483 overexpression decreased PFKFB3 and ECAR in ECs. In contrast, anti-miR-483 increased PFKFB3 and ECAR levels, which phenocopied acute KD-induced EC metabolic reprogramming. We conclude that glycolysis in ECs is augmented during the acute phase of KD, which promotes EC dysfunction. Mechanistically, the acute KD-associated metabolic switch in ECs is regulated, at least in part, by increased PFKFB3 as a consequence of reduced level of miR-483.

Co-culture of peripheral blood mononuclear cell (PBMC) and human coronary artery endothelial cell (HCAEC) reveals the important role of autophagy implicated in Kawasaki disease.

BackgroundKawasaki disease (KD) is a systemic vasculitis syndrome that commonly occurs in children. Autophagy has been increasingly shown to be involved in various cardiovascular diseases, including endothelial dysfunction and vascular endothelial injury. However, whether autophagy is implicated in the pathogenesis of KD remains poorly understood, and particularly, how the dysfunction of human coronary artery endothelial cells (HCAECs) is associated with autophagy in peripheral blood mononuclear cells (PBMCs) from KD patients awaits further investigation.MethodsPeripheral blood samples were collected from KD patients, common fever patients, and healthy controls. The PBMC samples were isolated from KD blood samples collected at three different phases: the acute phase before therapy (acute-KD), 1 week (subacute-KD), and 4 weeks (convalescent-KD) after drug administration.ResultsThe autophagy flux was significantly increased in the PBMCs of KD patients at acute phase. The PBMCs of acute KD patients could induce autophagy in HCAECs and promote the secretion of chemokines and pro-inflammatory factors after cocultured with HCAECs whereas 3-methyladenine (3-MA) drug could partly reverse this process.ConclusionsAutophagy is involved in the inflammatory injury of vascular endothelial cells associated with PBMCs in KD patients, and may play a crucial role in regulating inflammation. Hence, we identify a novel regulatory mechanism of vascular injury in this disease.

Abstract 13032: Endothelial Cell Response to Intravenous Immunoglobulin, Infliximab and Atorvastatin Treatment During the Acute Phase of Kawasaki Disease

Introduction: Damage to coronary arteries (CA) during the acute phase of Kawasaki disease (KD) is linked to inflammatory cell infiltration, myofibroblast generation (EndMT: endothelial-mesenchymal transition), and endothelial cell (EC) dysfunction. To promote EC health, we performed a Phase I/IIa clinical trial (6-week course) of atorvastatin in patients with acute KD and coronary artery Z score > 2.5. We demonstrated that atorvastatin was safe and well-tolerated. However, its effect on EC in KD has not been systematically studied. Hypothesis: Treatment with atorvastatin added to intravenous immunoglobulin (IVIG) and infliximab (IFX) promotes EC health in acute KD patients. Methods: We performed RNAseq on human umbilical vein ECs (HUVECs) incubated with pooled sera from KD patients (pre-treatment or subacute illness day 17-25) treated with IVIG and IFX with or without atorvastatin added to their initial treatment. Results: Differentially expressed genes (DEGs) from HUVECs incubated with sera pooled from 5 KD patients collected pre- and post-treatment (IVIG + IFX) that were suppressed by treatment included IL-8 , CCL2 and ICAM1 , molecules in the TNFα and IL1β pathways that have key roles in recruitment of inflammatory cells to EC. Subacute sera pooled from 5 patients treated with IVIG + IFX + atorvastatin induced expression of NOS3 , KLF2 and KLF4 , molecules that promote EC homeostasis and angiogenesis, and suppressed expression of TGFB2 and Dickkopf WNT signaling pathway inhibitor 1 ( DKK1 ), molecules in the TGFβ and WNT signaling pathways. These results were validated by RT-PCR on HUVECs incubated with independent individual sera from patients treated with/without atorvastatin. Conclusions: HUVECs incubated with pre- or post-treatment sera from KD patients showed upregulation of IL1 and TNFα pathway genes that were dramatically suppressed after treatment with IVIG and IFX. Incubation of HUVECs with post-treatment sera from patients who also received atorvastatin showed reduced expression of DKK1 and TGFB2 , suggesting reduced EndMT, and increased NOS3 and KLF4 , key mediators of EC homeostasis. These results suggest that atorvastatin improves EC health and may block KD-induced myofibroblast proliferation in the CA wall.

Single-cell RNA sequencing of peripheral blood mononuclear cells from acute Kawasaki disease patients

Kawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although functional and phenotypic changes of immune cells have been reported, a global understanding of immune responses underlying acute KD is unclear. Here, using single-cell RNA sequencing, we profile peripheral blood mononuclear cells from seven patients with acute KD before and after intravenous immunoglobulin therapy and from three age-matched healthy controls. The most differentially expressed genes are identified in monocytes, with high expression of pro-inflammatory mediators, immunoglobulin receptors and low expression of MHC class II genes in acute KD. Single-cell RNA sequencing and flow cytometry analyses, of cells from an additional 16 KD patients, show that although the percentage of total B cells is substantially decreased after therapy, the percentage of plasma cells among the B cells is significantly increased. The percentage of CD8+ T cells is decreased in acute KD, notably effector memory CD8+ T cells compared with healthy controls. Oligoclonal expansions of both B cell receptors and T cell receptors are observed after therapy. We identify biological processes potentially underlying the changes of each cell type. The single-cell landscape of both innate and adaptive immune responses provides insights into pathogenesis and therapy of KD.

Clinical implications of procalcitonin in Kawasaki disease: a useful candidate for differentiating from sepsis and evaluating IVIG responsiveness.

ObjectiveKawasaki disease (KD) is an acute systemic vasculitis and suspected to be triggered by several potential infections in which procalcitonin (PCT) experiences an increase to some extent. However, whether PCT can serve as a useful candidate for differentiating KD from sepsis, and even for predicting incomplete KD, intravenous immunoglobulin (IVIG) nonresponsiveness and coronary artery abnormalities (CAAs) remains unclear.MethodsA total of 254 Chinese KD children were enrolled and divided into 6 subgroups, including complete KD, incomplete KD, IVIG-responsive KD, IVIG-nonresponsive KD, KD with CAAs and KD without CAAs. Blood samples were collected from all subjects within 24-h pre- and 48-h post-IVIG infusion, respectively. PCT, C-reactive protein, erythrocyte sedimentation rate and blood cell counts were detected. In addition, both 261 children with sepsis and 251 healthy children sex- and age-matched with KD children were enrolled in the same period.Results(1) PCT experienced the highest increase in sepsis patients before antibiotic therapy, followed by acute KD patients and the healthy controls. (2) The proportion of KD patients with a PCT concentration below 0.25 ng/ml was 11 folds higher than that of sepsis patients. (3) PCT had a sensitivity of 91.7% and a specificity of 30.3% at a cutoff value of > 0.15 ng/ml to predict IVIG nonresponsiveness, and the proportion of IVIG-nonresponders with a PCT concentration of 0.25–0.50 ng/ml was 2 folds higher than that of IVIG-responders.ConclusionsThe PCT concentrations below 0.25 ng/ml may be useful for discriminating KD from sepsis, and moreover, the PCT concentrations of 0.25–0.50 ng/ml may be helpful in predicting IVIG nonresponsiveness.

In silico Identification of 10 Hub Genes and an miRNA-mRNA Regulatory Network in Acute Kawasaki Disease.

Kawasaki disease (KD) causes acute systemic vasculitis and has unknown etiology. Since the acute stage of KD is the most relevant, the aim of the present study was to identify hub genes in acute KD by bioinformatics analysis. We also aimed at constructing microRNA (miRNA)–messenger RNA (mRNA) regulatory networks associated with acute KD based on previously identified differentially expressed miRNAs (DE-miRNAs). DE-mRNAs in acute KD patients were screened using the mRNA expression profile data of GSE18606 from the Gene Expression Omnibus. The functional and pathway enrichment analysis of DE-mRNAs were performed with the DAVID database. Target genes of DE-miRNAs were predicted using the miRWalk database and their intersection with DE-mRNAs was obtained. From a protein–protein interaction (PPI) network established by the STRING database, Cytoscape software identified hub genes with the two topological analysis methods maximal clique centrality and Degree algorithm to construct a miRNA-hub gene network. A total of 1,063 DE-mRNAs were identified between acute KD and healthy individuals, 472 upregulated and 591 downregulated. The constructed PPI network with these DE-mRNAs identified 38 hub genes mostly enriched in pathways related to systemic lupus erythematosus, alcoholism, viral carcinogenesis, osteoclast differentiation, adipocytokine signaling pathway and tumor necrosis factor signaling pathway. Target genes were predicted for the up-regulated and down-regulated DE-miRNAs, 10,203, and 5,310, respectively. Subsequently, 355, and 130 overlapping target DE-mRNAs were obtained for upregulated and downregulated DE-miRNAs, respectively. PPI networks with these target DE-mRNAs produced 15 hub genes, six down-regulated and nine upregulated hub genes. Among these, ten genes (ATM, MDC1, CD59, CD177, TRPM2, FCAR, TSPAN14, LILRB2, SIRPA, and STAT3) were identified as hub genes in the PPI network of DE-mRNAs. Finally, we constructed the regulatory network of DE-miRNAs and hub genes, which suggested potential modulation of most hub genes by hsa-miR-4443 and hsa-miR-6510-5p. SP1 was predicted to potentially regulate most of DE-miRNAs. In conclusion, several hub genes are associated with acute KD. An miRNA–mRNA regulatory network potentially relevant for acute KD pathogenesis provides new insights into the underlying molecular mechanisms of acute KD. The latter may contribute to the diagnosis and treatment of acute KD.

Leukocyte proteomics coupled with serum metabolomics identifies novel biomarkers and abnormal amino acid metabolism in Kawasaki disease

Kawasaki disease (KD) is a systemic vasculitis that can lead to severe cardiovascular complications, whereas the development and clinical usage of specific biomarkers might help diagnose KD and avoid certain complications. To this end, the molecular profiles of acute KD patients with coronary artery lesions (CAL) were first investigated through leukocyte proteomics and serum metabolomics assays. A total of 269 differentially abundant proteins and 35 differentially abundant metabolites with the top fold-changed levels were identified in acute KD patients compared to those in the healthy controls. Among them, several highly promising candidate marker proteins and metabolites indicative of KD progression were further analysed, such as the increased proteins ALPL, NAMPT, and S100P, as well as the decreased proteins C1QB and apolipoprotein family members. Moreover, metabolites, including succinic acid, dGMP, hyaluronic acid, L-tryptophan, propionylcarnitine, inosine, and phosphorylcholine, were found to be highly accurate at distinguishing between KD patients and healthy controls. Interestingly, the abnormal expression levels of a distinct set of proteins and metabolites in acute KD patients can be restored to normal levels upon intravenous immunoglobulin (IVIG) treatment. Overall, this work has revealed novel biomarkers and abnormal amino-acid metabolism as a prominent feature involved in KD patients with CAL. SignificanceKD is frequently concomitant with the development of life-threatening coronary vasculitis. Here, the profiles of leukocyte proteomics and serum metabolomics in acute KD patients with CALs were first investigated, and several hub molecules identified here could be used as supplemental biomarkers for KD diagnosis. Moreover, the metabolomic abnormalities especially the amino acids are particularly prominent in KD patients. Interestingly, the abnormal expression levels of a distinct set of proteins and metabolites in acute KD patients can be restored to normal levels upon IVIG treatment. Therefore, these findings might help understand the IVIG activities and also the underlying mechanisms of IVIG-resistant patients, thereby providing a new perspective for the exploration of mechanisms related to KD.

The Expression and Role of microRNA-133a in Plasma of Patients with Kawasaki Disease

ABSTRACT Kawasaki disease (KD)), also known as mucocutaneous lymph node syndrome (MCLS), is an autoimmune and systemic vasculitis syndrome. Its etiology and pathogenesis are still unclear. microRNAs (miRNA), a novel class of small non-coding RNAs, regulate the expression of multiple protein-encoding genes at the post-transcriptional level. We intend to study the change of miRNA-133a in the plasma of patients with KD, explore the role of miRNA-133a on HUVEC and define the pathogenesis of vascular dysfunction in KD. miRNA-133a expression and the mRNA and protein expression of protein phosphatase 2 catalytic subunit alpha (PPP2CA) were assessed by RT-qPCR and Western blot, respectively. The PPP2CA mRNA 3ʹUTR was predicted to be the potential target of miRNA-133a by using the miRNA databases and verified by the luciferase assay. The plasmids of miRNA-133a mimics and inhibitors were transfected into HUVEC cells. The plasma soluble vascular endothelial cadherin (sVE-cadherin, the excised extracellular part of VE-cadherin) levels were investigated by ELISA. The results suggested that miRNA-133a was increased by 3.8 times in the acute KD group and by 2.7 times in the convalescent KD group compared with the control group (both P = .000). PPP2CA is the target gene of miRNA-133a and its expression was inhibited by miRNA-133a acting on PPP2CA mRNA 3ʹUTR (P = .013). The plasma sVE-cadherin levels in the acute KD groups were increased compared with the control group (P = .024). The ROC curve analysis showed that the expression of miRNA-133a segregate acute KD patients from convalescent KD patients and healthy children. Our results suggest that miRNA-133a might be a new biomarker for KD.

Biomarkers for the Discrimination of Acute Kawasaki Disease From Infections in Childhood.

Background: Kawasaki disease (KD) is a vasculitis of early childhood mimicking several infectious diseases. Differentiation between KD and infectious diseases is essential as KD's most important complication—the development of coronary artery aneurysms (CAA)—can be largely avoided by timely treatment with intravenous immunoglobulins (IVIG). Currently, KD diagnosis is only based on clinical criteria. The aim of this study was to evaluate whether routine C-reactive protein (CRP) and additional inflammatory parameters myeloid-related protein 8/14 (MRP8/14 or S100A8/9) and human neutrophil-derived elastase (HNE) could distinguish KD from infectious diseases.Methods and Results: The cross-sectional study included KD patients and children with proven infections as well as febrile controls. Patients were recruited between July 2006 and December 2018 in Europe and USA. MRP8/14, CRP, and HNE were assessed for their discriminatory ability by multiple logistic regression analysis with backward selection and receiver operator characteristic (ROC) curves. In the discovery cohort, the combination of MRP8/14+CRP discriminated KD patients (n = 48) from patients with infection (n = 105), with area under the ROC curve (AUC) of 0.88. The HNE values did not improve discrimination. The first validation cohort confirmed the predictive value of MRP8/14+CRP to discriminate acute KD patients (n = 26) from those with infections (n = 150), with an AUC of 0.78. The second validation cohort of acute KD patients (n = 25) and febrile controls (n = 50) showed an AUC of 0.72, which improved to 0.84 when HNE was included.Conclusion: When used in combination, the plasma markers MRP8/14, CRP, and HNE may assist in the discrimination of KD from both proven and suspected infection.

Increased concentrations of growth differentiation factor-15 in children with Kawasaki disease

BackgroundWe investigated the serum concentrations of growth differentiation factor-15 (GDF-15) in children with acute Kawasaki disease (KD) and evaluate its role in predicting coronary artery lesions (CALs) and no response KD. MethodsWe obtained blood sample from 30 healthy children and 131 children with KD before intravenous immunoglobulin therapy. Serum GDF-15 concentrations were measured using ELISA kits. Univariate and multivariate logistic regression analysis were conducted to evaluate the potential association between GDF-15 and the occurrence of CALs and treatment responses. ResultsSerum GDF-15 concentrations in KD were remarkably increased compared with healthy control. Serum GDF-15 concentrations in KD with CALs were also increased compared with no CALs, and in no response KD compared with response KD. Serum of GDF-15 concentrations was positively correlated with white blood cell count, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and negatively associated with albumin and pre-albumin in all patients with KD. GDF-15 was an independent predictor of CALs and no response KD. GDF-15 was superior to CRP and ESR, while it was not inferior to the combination of CRP and ESR for predicting CALs. ConclusionsSerum of GDF-15 concentrations was significantly increased in acute KD patients, especially in KD with CALs and no response KD. GDF-15 could sever as an independent predictor for CALs and no response KD.

Hemolysis From Intravenous Immunoglobulin in Obese Patients With Kawasaki Disease.

Objective: We assessed the risk of IVIG-associated hemolytic anemia in patients with acute Kawasaki disease (KD) and evaluated the risk of weight-based dosing in our obese patients.Methods: IVIG-associated hemolytic anemia was assessed in acute KD patients treated with IVIG at Rady Children's Hospital-San Diego. Patients in whom hemolytic anemia was suspected had a decrease in z-score of their hemoglobin (zHgb) at least two standard deviations below the cohort's mean change in zHgb from baseline to 2 weeks post-IVIG treatment. These patients were further evaluated for spherocytosis, blood type, need for transfusion, red cell distribution width, reticulocytosis, and direct Coombs test. Body mass index was calculated.Results: Of the 30 IVIG-resistant KD patients who received a second dose of IVIG, 2 (6.7%) developed hemolytic anemia after a total of 4 g/kg of IVIG dosed on actual body weight, or a mean of 4.6 g/kg of IVIG based on lean body mass. Compared to 496 non-obese KD patients who received a single dose of IVIG with no cases of hemolytic anemia, two (5.6%) of 36 obese KD patients developed hemolytic anemia after a single dose of IVIG (2 g/kg) dosed on actual body weight, or a mean of 2.7 g/kg IVIG based on lean body mass.Conclusions: In addition to following patients carefully for hemolytic anemia after a second dose of IVIG, physicians should consider IVIG dosing based on lean body mass for obese patients.

Increased levels of circulating fibroblast growth factor 21 in children with Kawasaki disease.

The purpose of this study was to examine the serum levels of fibroblast growth factor 21 (FGF21) in children with acute Kawasaki disease (KD) and to investigate its relationship with coronary artery lesions (CALs). Blood samples from 58 children with KD before intravenous immunoglobulin treatment and from 28 healthy children as control group were collected. Serum FGF21 levels in all participants were measured using enzyme-linked immunosorbent assay, and clinical parameters were tested in all KD patients. Serum FGF21 levels were significantly increased in acute KD patients as compared to the control group. Serum levels of FGF21 were substantially higher in the group of KD patients with CALs (KD-CALs) than in KD patients without CALs (KD-NCALs). Positive relationships between serum levels of FGF21 and percentage of leukomonocytes (L %), C-reactive protein, activated partial thromboplastin time and D-dimer were observed in KD patients. Furthermore, serum FGF21 levels were negatively correlated with red blood cell counts, hemoglobin (Hb), percentage of neutrophils (N %) and albumin. Serum level of FGF21 is associated with inflammation and coagulation. The paradoxical increase in serum FGF21 in acute KD patients may indicate a protective compensatory response.

MiR-222-3p in Platelets Serves as a Distinguishing Marker for Early Recognition of Kawasaki Disease.

Kawasaki disease (KD) is an acute vasculitis, which leads to 20% of sufferers developing coronary artery aneurysm in children if not appropriately treated. Therefore, the early diagnosis of KD is essential for alleviating the risk of developing heart disease. MicroRNAs (miRNAs) are a large class of small non-coding RNAs which post-transcriptionally regulate gene expression and have been shown to play critical roles in numerous biological processes and diseases. In this study, we used high-throughput miRNA sequencing and found dozens of miRNAs are highly expressed in platelets. By comparing the miRNA expression profile of platelets of acute KD patients and other febrile patients, miR-222-3p is validated to be significantly upregulated in platelets of acute KD patients. Furthermore, KEGG pathway analysis shows that targets of miR-222-3p are enriched in immune-related signaling pathways. Our study uncovers the potential of miR-222-3p in platelets as biomarker for early diagnosis of Kawasaki disease.

Changes and significance of histone acetylation associated with forkhead box P3 gene during the acute phase of Kawasaki disease

Objective To investigate the histone acetylation status of forkhead box P3(Foxp3) gene and its roles in immunological pathogenesis of Kawasaki disease(KD). Methods Forty-two children with KD and 32 age-matched healthy children consented to participate in this study as a control group.Co-immunoprecipitation and real-time PCR were performed to determine acetylation levels of histone H4 associated with Foxp3 gene and binding abilities of Kruppel like factor 10(KLF10) and p300/CBP-associated factor (PCAF) with Foxp3 gene in CD4+ T cells.The proportion of CD4+ CD25high Foxp3+ cells (Treg) and protein levels of Foxp3, KLF10, PCAF, phosphated SMAD family member 2/3(pSmad2/3) and itchy E3 ubiquitin protein ligase(Itch) were analyzed by flow cytometry.Quantitative real-time PCR was used to evaluate the levels of Foxp3, transforming growth factor-β1 receptor Ⅱ (TGF-βRⅡ), transforming growth factor-β1 receptor Ⅰ(TGF-βRⅠ), tyrosine kinase 2(Tyk2), SH2 domain-containing protein-tyrosine phosphatase-2(SHP2) and cytotoxic T-lymphocyte-associated protein 4(CTLA4) mRNA in Treg.Plasma concentrations of TGF-β and interleukin-6(IL-6) were measured by enzyme-linked immunosorbent assay. Results (1)Compared with the control group, the proportion of Treg, expression levels of Foxp3 and TGF-β, and histone acetylation levels of Foxp3 promoter decreased remarkably during acute KD, and the differences were all statistically significant(all P<0.05), and restored after IVIG therapy(all P<0.05). Meanwhile, the 5 indexes aforementioned in KD patients with coronary artery lesions (KD-CAL+ ) were lower than those without coronary artery lesions(KD-CAL-), and the differences were all statistically significant (all P<0.05). (2)Compared with the control group, protein levels of KLF10 and PCAF in Treg cells, and their binding abilities with Foxp3 gene were significantly down-regulated during acute KD, and the differences were all statistically significant(all P<0.05), and restored to some extent after IVIG treatment(all P<0.05). Positive correlations between protein levels of KLF10 and PCAF, and mRNA level of Foxp3 were detected in acute KD patients (r=0.47, 0.59, all P<0.05). Furthermore, protein levels of KLF10 and PCAF and their binding abilities with Foxp3 gene in KD-CAL+ group were lower than those in KD-CAL- group, and the differences were all statistically significant(all P<0.05). (3) Compared with the control group, the plasma concentration of TGF-β and expressions of TGF-βRⅡ/Ⅰ, pSmad2/3, Itch, CTLA4 and SHP2 in Treg were down-regulated during the acute phase of KD, and the differences were all statistically significant(all P<0.05), as well as plasma concentration of IL-6 and expression its downstream molecule Tyk2 increased remarkably in patients with acute KD(all P<0.05). All the indexes mentioned before restored significantly after IVIG treatment (all P<0.05). Simultaneously, the 7 former indexes in KD-CAL+ group were found to be lower than those in KD-CAL- group, while 2 indexes of the latter in KD-CAL+ group were higher than those in KD-CAL- group, and the differences were all statistically significant(all P<0.05). Conclusion Histone hypoacetylation of Foxp3 promoter might be one of the important factors contributing to insufficiency and dysfunction of regulatory T cells during acute Kawasaki disease. Key words: Kawasaki disease; Foxp3; Histone; Acetylation; Immune

Safety of Infliximab Following Live Virus Vaccination in Kawasaki Disease Patients.

No published data are available regarding infliximab use in Kawasaki disease patients with recent live virus vaccination. We investigated 38 acute Kawasaki disease patients who received infliximab within 90 days of receiving measles-mumps-rubella, varicella-zoster virus, or Rotavirus vaccines. There were no adverse events or serious infections, which suggests that infliximab use in acute Kawasaki disease patients with recent live viral vaccinations is safe.

MicroRNA-93 may control vascular endothelial growth factor A in circulating peripheral blood mononuclear cells in acute Kawasaki disease.

Kawasaki disease (KD) is the most common systemic vasculitis syndrome primarily affecting medium-sized arteries, particularly the coronary arteries. Though KD may be associated with immunological problems, the involvement of microRNAs (miRs) has not been fully described. We enrolled 23 KD patients and 12 controls. We performed miR and mRNA microarray analysis of peripheral blood mononuclear cells (PBMCs) isolated from acute KD patients and controls. Continuously, we measured specific miRs, mRNA and the expression of proteins by using reverse-transcriptase PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). We identified strikingly high levels of miR-182 and miR-296-5p during the acute febrile phase, and of miR-93, miR-145-5p, miR-145-3p, and miR-150-3p in the defervescence stage, especially in refractory KD patients. The expression of vascular endothelial growth factor A (VEGFA) mRNA, previously reported to be controlled by miR-93, was significantly elevated during the febrile phase and normalized upon treatment, negatively correlating with the expression of miR-93. Further, plasma levels of VEGF-A correlated with PBMC VEGFA mRNA expression. Several miRs are highly specific to the acute phase of KD, and may participate in regulating the expression of genes in pathways associated with KD. In particular, miR-93 may participate in regulating expression of VEGF-A and contribute to the pathogenesis of arteritis in acute KD.