Kawasaki disease (KD) is the most common childhood vasculitis. Approximately 25% of KD patients are refractory to standard intravenous immunoglobulin (IVIG) therapy and frequently develop coronary artery lesions (CAL) that result in long-term complications. Transcriptome studies utilizing blood cells from KD patients and reported animal model studies had identified interleukin (IL)-1β as a crucial component of an essential immune pathway in the formation of CAL. We previously reported that high-dose immunoglobulin G (IgG) treatment completely inhibited tumor necrosis factor (TNF)-α-stimulated inflammatory responses in an in vitro human coronary artery endothelial cells (HCAECs) model. Here, we show that IL-1β, but not TNF-α, stimulation markedly induced nuclear protein expression of NF-kappa-B inhibitor zeta (IκBζ) in HCAECs. It is of particular significance that IL-1β-induced IκBζ expression is entirely refractory to high-dose IgG treatment. Therefore, IκBζ may be a critical factor in the IVIG-resistant vascular inflammatory responses in severe KD. Itaconate is a Krebs cycle-derived metabolite with several immunomodulatory effects. Dimethyl itaconate (DI), a membrane-permeable derivative of itaconate, can significantly suppress IL-1β-induced IκBζ expression in HCAECs. DI is an analog of dimethyl fumarate (DMF), which is already in clinical use for some diseases. Like DI, DMF suppressed IL-1β-induced IκBζ expression and subsequent production of inflammatory cytokines, including IL-6 and G-CSF. This study identified IκBζ as an essential inflammatory factor in IVIG-resistant inflammatory responses in HCAECs. Immunomodulatory substances, such as DI and/or DMF, may be therapeutically exploited as a novel drug to alleviate inflammation in severe IVIG-resistant KD patients.

BACKGROUND:We aimed to identify distinct disease patterns and improve the understanding of treatment responses in Kawasaki disease (KD). Patients were classified into subgroups using pre-intravenous immunoglobulin (IVIG) blood tests and cytokine profiles. METHODS:We retrospectively analyzed 212 KD patients admitted between November 2008 and April 2015. Principal component analysis and hierarchical clustering used 20 variables including age, blood tests, and nine cytokines. RESULTS:Five subgroups were identified: Cluster 1 (Toddler Favorable Outcome), Cluster 2 (Severe Vasculitis), Cluster 3 (Moderate Vasculitis), Cluster 4 (Infant Favorable Outcome), and Cluster 5 (Infant Cytokine Storm). Cluster 4 demonstrated the highest IVIG response rate (87%), whereas Clusters 2 and 5 showed significantly lower IVIG response rates (20% and 21%, respectively). The acute-phase peak CAL occurrence was highest in Cluster 5 (53%; odds ratio 10.00, 95% CI: 3.14-31.88), compared to 45% in Cluster 2, 10% in Cluster 1, and 8.9% in Cluster 4. CONCLUSION:Two of the five subgroups showed increased risk of IVIG resistance and CAL development. A logistic regression model incorporating age and monocyte count achieved an AUC of 0.897 for identifying the highest-risk subgroup, suggesting its potential utility as a practical screening tool in routine clinical practice. IMPACT:Five distinct Kawasaki disease subgroups were identified using pre-intravenous immunoglobulin (IVIG) blood tests and cytokine profiles. Two high-risk subgroups were identified, both associated with IVIG resistance and coronary artery lesion (CAL) development: toddlers with highly elevated inflammatory markers and infants with cytokine storm despite modest elevation of standard inflammatory markers. In the infant cytokine storm subgroup, the acute-phase CAL occurrence was 53% (odds ratio 10.00), the highest among all subgroups. Conventional risk stratification tools, including the Kobayashi score, may fail to identify these high-risk infants. A prediction model incorporating age and monocyte count achieved AUC of 0.897, supporting its utility.

Infection with Yersinia pseudotuberculosis, a known causal pathogen of human bacterial gastroenteritis, causes various symptoms and complications. A previously healthy 7-year-old girl was admitted because of fever and gastrointestinal symptoms. She was initially diagnosed with intussusception by abdominal ultrasonography. Although the patient was successfully treated by air enema, the fever persisted. The patient was then diagnosed with incomplete Kawasaki disease based on the presence of four principal clinical features. Intravenous immunoglobulin and oral aspirin were initiated. The patient defervesced and the other symptoms subsided after the treatment. Cardiac ultrasound results showed normal coronary arteries. Because of the gastrointestinal symptoms, stool samples were cultured repeatedly, only to yield normal flora. However, serum levels of anti-Y. pseudotuberculosis-derived mitogen antibody were elevated between the 7th and 18th days of the disease, thereby confirming Y. pseudotuberculosis infection. Because Y. pseudotuberculosis infection results in various clinical manifestations, we must be aware of each symptom and address them systematically.

Diagnostic guidelines for Kawasaki Disease was revised to meet the present situation in 2002. This issue intends to explain new guidelines and their backgrounds. Major alterations are interpretation of cases with 4 or fewer febrile days shortened by early intravenous immunoglobulin treatment, and the clinical importance of atypical (incomplete, or suspected) cases.

To the Editor, Kawasaki disease (KD) is a pediatric systemic vasculitis of unknown etiology. Intravenous immunoglobulin (IVIG) has been the standard treatment for KD and is highly effective in almost 80% of patients. However, the remaining patients are resistant to initial IVIG treatment and are at high risk for coronary artery lesions (CALs) due to severe coronary inflammation. That resistance is the most important issue needing to be clinically resolved for KD. Therefore, there is an urgent need for the development of novel therapies for IVIGresistant KD based on an understanding of the mechanism of that resistance. We show here that activation of two transcription factors, CCAAT/enhancer-binding protein beta and delta (C/EBPβ and C/EBPδ), whose target genes include IL-6 and G-CSF, plays a crucial role in IVIG-resistant inflammation of human coronary artery endothelial cells (HCAECs). Accumulating evidence suggests that both IL-1β and IL-17A are crucially involved in IVIG resistance in KD patients.1-6 We previously demonstrated that IVIG could specifically and completely suppress TNF-α-induced, but not IL-1β-induced, protein production of such KD-related cytokines as IL-6 and G-CSF by HCAECs.7 Furthermore, the dramatic and specific anti-inflammatory effects of IVIG were associated with complete inhibition of C/EBP activity. In this study, we aimed to elucidate the effects of IL-1β and IL-17A on the anti-inflammatory effects of IVIG and the involvement of C/EBPβ and C/EBPδ in those effects. The detailed methods are provided in Appendix S1. Consistent with our previous study, IVIG completely inhibited TNF-α-induced production of IL-6 and G-CSF by HCAECs. In contrast, we newly found that the striking anti-inflammatory effects of IVIG were almost completely abolished by the addition of IL-1β to TNF-α even at a ratio of only 1:100 (Figure 1A, upper panels). Similar to IL-1β, adding IL-17A to TNF-α attenuated IVIG's anti-inflammatory effects, but the effect was weaker than that of IL-1β (Figure 1A, lower panels). Indeed, IL-17A must be present in at least a 1:1 ratio to TNF-α. The mRNA expression patterns for both IL-6 and G-CSF (Figure 1B) were in line with their protein production patterns (Figure 1A). Since the levels of IL-6 and G-CSF production induced by combined TNF-α and IL-1β stimulation (Figure 1A, upper panels) and those induced by IL-1β stimulation alone (Figure S1A) were almost the same, it was clear that TNF-α had no additive effect on IL-1β stimulation of HCAECs. Therefore, the Western blot (WB) and electrophoretic mobility shift assay (EMSA) experiments shown in Figure 2 were conducted using only IL-1β stimulation. IVIG treatment completely suppressed TNF-α–induced nuclear expression of C/EBPβ and C/EBPδ (Figure 2A; WB) and their DNA-binding activity (Figure 2B; EMSA). Conversely, those effects were abolished when the cells were stimulated with IL-1β alone or a combination of TNF-α and IL-17A (Figure 2). These results strongly suggest that C/EBPβ and C/EBPδ activities elicited by IL-1β or IL-17A could be crucially involved in attenuation of IVIG's anti-inflammatory effect on HCAECs. To confirm the involvement of C/EBPs in cytokine-induced expression of IL-6 and G-CSF by HCAECs, HCAECs were transfected with siRNAs targeting C/EBPβ and C/EBPδ. Transfection of siRNAs for C/EBPβ and C/EBPδ into HCAECs successfully suppressed their respective mRNAs (Figure S2A). siRNAs for each of C/EBPβ and C/EBPδ independently suppressed IL-6 and G-CSF production by HCAECs. Moreover, co-transfection with siRNAs for both C/EBPβ and C/EBPδ showed a tendency for synergistic inhibition, especially upon IL-1β stimulation (Figure S2B). These results suggest that both C/EBPβ and C/EBPδ are required for cytokine-induced IL-6 and G-CSF production by HCAECs. 4. Dewald G. A missense mutation in the plasminogen gene, within the plasminogen kringle 3 domain, in hereditary angioedema with normal C1 inhibitor. Biochem Biophys Res Commun. 2018;498:193-198. 5. Ghannam A, Sellier P, Defendi F, et al. C1 inhibitor function using contact-phase proteases as target: evaluation of an innovative assay. Allergy. 2015;70:1103-1111. 6. Defendi F, Charignon D, Ghannam A, et al. Enzymatic assays for the diagnosis of bradykinin-dependent angioedema. PLoS ONE. 2013;8:e70140. 7. Baroso R, Sellier P, Defendi F, et al. Kininogen cleavage assay: diagnostic assistance for kinin-mediated angioedema conditions. PLoS ONE. 2016;11:e0163958. 8. Takada A, Takada Y. The activation of two isozymes of glu-plasminogen (I and II) by urokinase and streptokinase. Thromb Res. 1983;30:633-642. 9. Gonzalez-Gronow M, Gawdi G, Pizzo SV. Tissue factor is the receptor for plasminogen type 1 on 1-LN human prostate cancer cells. Blood. 2002;99:4562-4567.

Objective To conduct a prospective study to evaluate the immunogenicity and safety of live attenuated vaccines in patients with nephrotic syndrome receiving immunosuppressive agents. Study design Patients with nephrotic syndrome receiving immunosuppressive agents with negative or borderline antibody titers (virus-specific IgG levels <4.0) against measles, rubella, varicella, and/or mumps fulfilling the criteria of cellular and humoral immunity were enrolled. Virus-specific IgG levels were measured using an enzyme immunoassay. The primary endpoint was the seroconversion rate (ie, achievement of virus-specific IgG levels >= 4.0) at 2 months after vaccination. Virus-specific IgG levels at 1 year, breakthrough infections (wild-type infections), and adverse events were also evaluated. Results A total of 116 vaccinations were administered to 60 patients. Seroconversion rates were 95.7% for measles, 100% for rubella, 61.9% for varicella, and 40.0% for mumps. More patients with a borderline antibody titer before vaccination achieved seroconversion than those with negative antibody titer. with statistical significance after varicella and mumps vaccination. The rate of patients who maintained seropositivity at 1 year after vaccination was 83.3% for measles, 94.1% for rubella, 76.7% for varicella, and 20.0% for mumps. No patient experienced breakthrough infection. No serious adverse events, including vaccine-associated infection, were observed. Conclusion Immunization with live attenuated vaccines may be immunogenic and is apparently safe in our cohort of patients with nephrotic syndrome receiving immunosuppressive agents if their cellular and humoral immunologic measures are within clinically acceptable levels.

The etiology of Kawasaki disease (KD) remains unknown. However, many studies have suggested that specific genetic factors and/or some infectious agents underlie the onset of KD. Previous studies have suggested that human adenovirus (HAdV) is one of the triggering pathogens of KD. Here, we report monozygotic twin boys who sequentially developed KD in conjunction with acute HAdV type 3 (HAdV-3) infection.

Kawasaki disease (KD), an acute childhood panvasculitis, presents a variety of ocular complications as well as conjunctival injection among the principal symptoms. However, most pediatricians are unfamiliar with the ophthalmological complications of KD. A 2-year-old girl was referred to us from the ophthalmology department due to injected bulbar conjunctivae and optic disc swelling. She had familial exudative vitreoretinopathy as an underlying disease and the ocular findings had been made by chance while the patient was receiving an eye examination. Although she was afebrile at the time of her first medical interview, KD was diagnosed based on the presence of four of the principal symptoms including fever and dilatation of the coronary arteries. Intravenous immunoglobulin (IVIG) therapy was administered on Day 15 from the onset of fever. After IVIG administration, her laboratory test results showed rapid improvement but her optic disc swelling continued for six months. Eye complications in KD generally occur in the anterior segment, and recovery occurs within two months. Past reports have shown that in three of seven KD cases with optic disc involvement, optic disc swelling lasted over two months. This is the second case in which the condition lasted six months.

The standard treatment for Kawasaki disease (KD) is high-dose intravenous immunoglobulin (IVIG). Some patients experienced recurrent fever after IVIG following defervescence. However, little is known about the frequency of such episodes and the clinical outcome for such patients. We classified 195 KD patients into 4 groups based on their fever patterns after initial IVIG treatment: group NR (no response), group EF (early recurrent fever within 72 hours after defervescence), group LF (late recurrent fever >72 hours after defervescence), and group GR (good response). We compared the clinical characteristics and laboratory data among these groups retrospectively. Nearly a third of patients had recurrent fever (group EF, n = 45; group LF, n = 11). In two-thirds of these patients, the fever had subsided spontaneously without retreatment; 2 patients in Group LF with smoldering KD symptoms had developed coronary artery lesions (CALs) even after additional IVIG. Recurrent fever and smoldering symptoms might be risk factors for CAL.

Kawasaki disease (KD) is a systemic vasculitis in infants. In KD, encephalopathy is rarely (0.1%) associated, however, clinically mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) has previously been reported in some pediatric patients. Here, we report on a 2-year-old girl who had KD complicated with MERS. The patient experienced generalized clonic convulsion and prolonged consciousness disturbance with fever for 2 days. Her head MRI showed a high signal intensity lesion in the splenium of the corpus callosum in diffusion-weighted images, and low apparent diffusion coefficient (ADC) values on day 3. An electroencephalogram showed high voltage slow waves on the occipital and parietal head. On the same day, it was confirmed that the patient showed all the main symptoms of KD. Based on these findings, we diagnosed her with MERS-complicated KD. Even though she was treated with immunoglobulin (total 4 g/kg) and pulsed-dose methylprednisolone, her fever and consciousness disturbance continued, and blood tests showed that inflammation markers remained high. We then treated the patient with infliximab on day 9, and within a few hours of the treatment her fever dropped and all symptoms of KD and consciousness disturbance disappeared. No recurrence of KD or other complications of KD occurred, and she was discharged on day 23. We propose that infliximab is an effective optional treatment for immunoglobulin/glucocorticoid-resistant KD with MERS. To clarify this possibility, further case accumulation is warranted.

Background: Infliximab (IFX) therapy is increasingly used in the treatment of intravenous immunoglobulin (IVIG)-resistant patients with Kawasaki disease (KD).Purpose: To analyze the effects and safety of IFX therapy in IVIG-resistant patients with KD.Methods: Fifty-five patients who were administered IFX after initial IVIG between December 2008 and September 2016 were included in this study.Results: Of the 55 patients treated with IFX for KD, 34 (62％) were male, with a median age of 31 (4-131) months.The median number of days of illness at the initiation of IFX therapy was 10 (7-24) days.In 40 (73％) patients, this treatment was provided as part of second-or third-line therapy.Following IFX administration, 38 (69％) patients became afebrile within 48 h (responders).Ten patients (18％) were given additional treatments after IFX administration.No significant differences in sex, age, or days of IFX administration were observed between the responders (n＝38) and non-responders (n＝17).After IFX administration, the levels of WBC and 2016

We report the case of an 11-year-old girl with ulcerative colitis refractory to conventional therapy, who was subsequently treated successfully with repeated fecal microbiota transplantation (FMT). The patient was steroid dependent despite several infliximab treatments, and colectomy was proposed to improve quality of life. After repeated FMT, she was able to maintain remission with on minimal dose of steroid. Although her fecal microbiota was dysbiotic before FMT, it was restored to a similar pattern as the donor after repeated FMT.

Background: Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is heavily upregulated at sites of inflammation. We conducted a retrospective study to assess the utility of TN-C as a novel biomarker to predict the risk of developing coronary artery lesions (CAL) and resistance to intravenous immunoglobulin (IVIG) in patients with Kawasaki disease (KD).Methods and Results: We collected blood samples of 111 KD patients (IVIG-responder: 89, IVIG-resistant: 22; CAL: 8) and 23 healthy controls, and measured the serum levels of TN-C. TN-C levels on admission were significantly higher in patients than in healthy controls and in patients during convalescence after IVIG administration (69.6 vs. 20.4 vs. 39.7 ng/ml, respectively; P<0.001), and correlated positively with C-reactive protein (P<0.001), neutrophil (percentage; P=0.005), and ALT (P<0.001), and negatively with platelet count (P=0.023) and sodium level (P=0.025). On admission, TN-C levels in patients who later developed CAL were significantly higher than in those without CAL (P=0.010), and significantly higher in IVIG-resistant subjects than in IVIG-responders (P=0.003). The accuracy of TN-C testing for the prediction of IVIG resistance was comparable to that of the Kobayashi score.Conclusions: Serum TN-C could be a biomarker for predicting the risk of developing CAL and IVIG resistance during the acute phase of KD.

Background: Kawasaki disease (KD) patients who do not respond to initial intravenous immunoglobulin (IVIG) therapy have a higher risk of developing coronary artery lesions (CALs). Infliximab (IFX) has been reported to be effective in alleviating fever in these patients and has been increasingly used. However, little is known about how IFX suppresses inflammation in KD. We analyzed immunological parameters in KD patients who responded to IFX, as well as those who did not, after the initial IVIG therapy. Methods: Twenty-six KD patients hospitalized and treated with IFX after initial IVIG and aspirin between August 2009 and July 2014 were enrolled in this study. We analyzed electronic medical records of baseline characteristics and laboratory data (WBC, neutrophil, lymphocyte and platelet counts, levels of GOT, GPT, Alb, Na, CRP and D-dimer), and measured plasma levels of cytokines (G-CSF, IL-6, IL-8, IL-10, IL-12p70, IL-17, TNF-α, IFN-γ, sIL-2R, sTNFR1 and sTNFR2) before and after IFX. We defined responders as patients who became afebrile within two days after IFX treatment. Results: The median age of 17 responders (11 males and 6 females) and 9 non-responders (5 males and 4 females) was 31 mo (9-84 mo) and 39 mo (16-64 mo), respectively (p=0.65). The initiation of IFX treatment after the onset of fever was compatible in both the group (day 9 vs day 9 in medians) (p=0.85). The incidence of CALs one month after onset was 0% (0/17) and 22% (2/9), respectively (p=0.11). Observed only in responders, WBC and neutrophil counts, and the levels of CRP, D-dimer, G-CSF, IL-6, sIL-2Rα, TNF-α, sTNFR-1 and sTNFR-2 decreased remarkably after IFX (p<0.01). In non-responders, serum level of Na was significantly lower (p=0.04) before IFX, while WBC (p=0.04) and neutrophil (p=0.01) counts, and the levels of CRP (p=0.02), IL-6 (p=0.01), IL-10 (p=0.03) were higher. After IFX, the level of Alb (p=0.02) was lower. Conclusions: After IFX, the level of TNF-α, together with many pro- and anti-inflammatory mediators, decreased in responders but not in non-responders. In particular, suppression of IL-6 and IL-10 was insufficient in non-responders, suggesting that inflammation mechanisms other than the TNF-α axis would be important in KD patients who are unresponsive to IFX.

ABSTRACT Bacterial superantigens (SAgs) are immunostimulatory toxins that induce acute diseases mainly through the massive release of inflammatory cytokines. Yersinia pseudotuberculosis is the only Gram-negative bacterium known to produce a SAg ( Y. pseudotuberculosis -derived mitogen [YPM]). This SAg binds major histocompatibility complex class II molecules on antigen-presenting cells and T cell receptors (TcR) bearing the variable region Vβ3, Vβ9, Vβ13.1, or Vβ13.2 (in humans) and Vβ7 or Vβ8 (in mice). We have previously shown that YPM exacerbates the virulence of Y. pseudotuberculosis in mice. With a view to understanding the mechanism of YPM's toxicity, we compared the immune response in BALB/c mice infected with a YPM-producing Y. pseudotuberculosis or the corresponding isogenic, SAg-deficient mutant. Five days after infection, we observed strong CD4 + Vβ7 + T cell expansion and marked interleukin-4 (IL-4) production in mice inoculated with SAg-producing Y. pseudotuberculosis . These phenomena were correlated with the activation of ypm gene transcription in liver and spleen. A transcriptomic analysis revealed that the presence of YPM also increased expression of granzyme and perforin genes in the host's liver and spleen. This expression was attributed to a CD4 + T cell subset, rather than to natural killer T (NKT) cells that display a TcR with a Vβ region that is potentially recognized by YPM. Increased production of cytotoxic molecules was correlated with hepatotoxicity, as demonstrated by an increase in plasma alanine aminotransferase activity. Our results demonstrate that YPM activates a potentially hepatotoxic CD4 + T cell population.

Background The etiology of Kawasaki disease (KD) is unknown. Reportedly, there is an association between KD and Yersinia pseudotuberculosis (YPT). Steroid therapy for KD patients with high risk of cardiac sequelae (CS) has been reported; however, the number of reports is limited. Methods We conducted a prospective study of 108 patients with newly diagnosed KD in one year to determine how many KD patients have positive anti-YPT antibody titers and/or positive anti-YPT-derived mitogen (YPM) antibody titers. In addition, we tried to identify clinical differences between KD patients in whom YPT infection was or not a contributing factor. We also compared clinical characteristics of patients treated with the protocol of the Randomized controlled trial to Assess Immunoglobulin plus Steroid Efficacy for Kawasaki disease (RAISE) study (RAISE group) and with the conventional Intravenous immunoglobulin (IVIG) protocol (conventional group). Results Eleven patients (10 %) were positive for anti-YPT and/or anti-YPM antibodies (positive group) and 97 (90 %) were negative (negative group). Cardiac sequelae (CS) occurred significantly more frequently in the positive than the negative group (two patients, 18 % vs one patient, 1 %, p = 0.027). Forty patients were in the RAISE group. Two of 40 (5 %) in the RAISE group and one of 68 (1.47 %) in the conventional group had CS ( p = 0.55). Conclusions KD patients with YPT infection had CS significantly more frequently and treatment with RAISE protocol did not decrease the frequency of CS in our cohort, nor did YPT infection affect risk scores of no response to IVIG. However, our sample size was overly small to draw such conclusions. Further investigation in a larger cohort is necessary to confirm our findings. Additionally, further research is needed to determine whether early diagnosis of YPT can prevent KD from developing and reduce the incidence of CS.

Background: We have reported that cyclosporin A (CsA) therapy may be a promising and safe option for patients with Kawasaki disease (KD) resistant to initial and additional intravenous immunoglobulin (IVIG). Up to now, it has been considered that CsA exerts effects on the intracellular phosphatase calcineurin, and subsequently inhibits activation of nuclear factor of activated T cells (NFAT). However, the functional mechanism of CsA therapy in KD patients has remained unclear. Methods: The KD patients enrolled in this study were treated with CsA between April 2012 and December 2013. In accordance with our treatment protocol, KD patients are initially treated with IVIG and aspirin. If there is no response, a further course of IVIG is given, and if there is no response to this additional IVIG, the patients are treated with CsA. Peripheral blood samples were obtained just before and after the initial course of IVIG, additional IVIG, and CsA therapy. To evaluate the NFAT pathway and activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, we examined the gene expression of cytokines and intracellular signal transducers using real-time RT-PCR and phospho-STAT3 (pSTAT3) and STAT5 (pSTAT5) using flow cytometry. Results: Thirty-six KD patients were divided into three groups: responsive to initial IVIG (n=19), responsive to additional IVIG (n=7), and treated with CsA group (n=10). In the CsA group, expression of mRNAs for interleukin (IL)-2, NFATc1 and NFATc2 was significantly increased, whereas the mean fluorescence intensity (MFI) of pSTAT3 (CD3+ T cell) and pSTAT3 (CD16b+ granulocyte) was decreased after CsA treatment. In the group responsive to initial IVIG, the expression of mRNAs for IL-2, IL-6, NFATc1 and NFATc2 was significantly increased, whereas that for STAT3, 5A, 5B was decreased after IVIG treatment; on the other hand, the MFI of pSTAT3 (CD3+ T cell) and pSTAT3 (CD16b+ granulocyte) was decreased, and that of pSTAT5 (CD16b+ granulocyte) was increased, after IVIG. Conclusion: In patients with refractory KD, CsA exerts effects on the activity of the NFAT and JAK-STAT pathways. However, our results suggest that in patients with immunoglobulin-resistant KD, CsA may act through a mechanism other than the NFAT pathway.

Objective: We studied microbiome of throat swab, rectal swab, and venous blood obtained from patients with Kawasaki disease (KD) to determine whether bacterial nuclei are detectable in circulating blood of KD patients and whether the microbial composition resembles to that of oral cavity or gut. Methods: We initially studied 7 patients (4 males and 3 females, 20-59 months) and obtained swabs and blood samples before IVIG treatment (day 4-5) to prepare DNA and cDNA library. Next, we studied 14 patients (8 males and 6 females, 2-47 months) and prepared blood cDNA library before IVIG treatment (day 2-8) and before discharge (day 10-15). Samples were sequenced by Illumina HiSeq2000 and the results were analyzed using MePIC for human genome subtraction and megablast search, followed by taxonomic analysis with MEGAN viewer. Results: The number of sequences homologous to bacterial genome per swab sample obtained from DNA or cDNA libraries varied 20,000~4,700,000 or 500,000~2,400,000, respectively. In most of the throat samples, the sequences were classified in descending order of phyla, Firmicutes > Proteobacteria > Bacteroidetes > Actinobacteria. In rectal samples, Proteobacteria and Bacteroidetes were more abundantly and Firmicutes was less abundantly represented than throat samples. The total number of sequences per blood sample was 30,000,000~70,000,000 and 99.0~99.9% of them were homologous to human genome. The number of sequences homologous to bacterial genome per DNA or cDNA library was 221-286 or 125-593, respectively. In blood samples, the frequencies of phyla were arranged in descending order, Proteobacteria = Actinobacteria > Firmicutes >> Bacteroides. The sequences homologous to Burkholderia, Streptococcus and Neisseria, the indigenous bacteria in the oral cavity, were present in most of the patients. Among them, the abundance of Streptococcus was significantly (p=0.03) reduced in the cDNA libraries during convalescence. Conclusion: A variety of sequences homologous to bacterial genome were detected from the venous blood obtained from KD patients during the acute as well as the convalescent phase. Further study is necessary to investigate the specificity and the characteristics of these sequences in patients with KD.