Enhanced formation of neutrophil extracellular traps in Kawasaki disease.

BACKGROUND: Neutrophils contribute to the clearance of pathogens through the formation of neutrophil extracellular traps (NETs) in a process known as NETosis, but the excessive release of NETs has been reported to be involved in the pathogenesis of various diseases, including vasculitis, by inducing tissue injury. The aim of the present study was to investigate whether or not NETosis is enhanced in the acute phase of Kawasaki disease (KD). METHODS: After neutrophils isolated from the peripheral blood of patients with KD and healthy control (HC) were cultured in vitro, the degree of spontaneous NETosis was evaluated by measuring the number of NETs formed and the titers of cell-free DNA (cfDNA) and neutrophil elastase (NE)-DNA complex. RESULTS: Spontaneous NET formation in vitro was observed in neutrophils isolated from KD patients, and the number of NET formations was significantly higher in acute KD than in convalescent KD and HC. The increased levels of cfDNA and NE-DNA complexes in the acute phase of KD tended to decrease in the convalescent phase. CONCLUSIONS: Spontaneous NET formation was enhanced in neutrophils from patients with acute KD, suggesting that circulating neutrophils may be primed to undergo NETosis in KD vasculitis.

The combination of the neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios as a novel predictor of intravenous immunoglobulin resistance in patients with Kawasaki disease: a multicenter study.

INTRODUCTION: The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been reported to be a predictor for intravenous immunoglobulin (IVIG) resistance in patients with Kawasaki disease (KD) recently. The objective of the present study was to elucidate the predictive validity of this new marker in a multicenter study. MATERIALS AND METHODS: We retrospectively reviewed the clinical records of 520 consecutive KD patients (development data set) and 332 subsequent patients (validation data set) at 7 hospitals in Japan. RESULTS: Both NLR and PLR were significantly higher in the IVIG-resistant group than in the IVIG-responsive group. When we set the cut-off point as NLR ≥ 4.11 and PLR ≥ 119, multiple logistic regression analyses showed that a high NLR and PLR before initial IVIG were independent predictors of IVIG resistance, and their combination was a stronger predictor than either alone. The sensitivity and specificity of the combination of NLR ≥ 4.11 and PLR ≥ 119 were 0.58 and 0.73 in the development data set. Validated using an independent data set, they were 0.54 and 0.72 in the validation data set. On comparing the AUC of this predictor with those of the Gunma and Kurume scores, the AUC was highest for this predictor, followed by the Gunma score and Kurume score (0.70, 0.68, and 0.64, respectively). DISCUSSION: The predictive validity of the combination of a high NLR and PLR, which is a simple and convenient indicator, was equal to or better than that of the existing scoring systems. The new predictive marker may be a suitable indicator for predicting IVIG resistance in KD patients.

Prognostic predictive value of gene mutations in Japanese patients with hypertrophic cardiomyopathy.

Although some studies have attempted to find useful prognostic factors in hypertrophic cardiomyopathy (HCM), those results are not fully helpful for use in actual clinical practice. Furthermore, several genetic abnormalities associated with HCM have been identified. However, the genotype-phenotype correlation in HCM remains to be elucidated. Here, we attempted to assess patients with different types of gene mutations causing HCM and investigate the prognosis. A total of 140 patients with HCM underwent a screening test for myofilament gene mutations by direct sequencing of eight sarcomeric genes. Patients with a single mutation in cardiac troponin T, cardiac troponin I, α-tropomyosin, and regulatory and essential light chains were excluded from the study because the number of cases was too small. The clinical presentations and outcomes of the remaining 127 patients with HCM, 31 ß-myosin heavy chain (MYH7) mutation carriers, 19 cardiac myosin-binding protein C (MYBPC3) mutation carriers, and 77 mutation non-carriers were analyzed retrospectively. MYBPC3 mutation carriers had a high frequency of ventricular arrhythmia and syncope. Kaplan-Meier curves revealed no significant difference in prognosis among the three groups, but a lack of family history of sudden death (SD) and a past history of syncope were significantly related to poor prognosis. An absence of family history of SD and past history of syncope are useful prognostic factors in patients with HCM. MYH7 and MYBPC3 mutations did not significantly influence prognosis compared to non-carriers. However, patients with the MYBPC3 mutation should be closely followed for the possibility of SD.

Low risk of treatment resistance in Down syndrome with Kawasaki disease.

BACKGROUND: A Japanese nationwide survey has reported that Down syndrome (DS) is a less-frequently occurring comorbidity in Kawasaki disease (KD). Although altered immune responses are frequently observed in DS, no studies have focused on the treatment response and risk for coronary artery abnormalities (CAA) in DS patients with KD. The aim of this study was therefore to evaluate the clinical manifestations, treatment response and prevalence of CAA in DS with KD. METHODS: We retrospectively reviewed the medical records of DS patients with KD from 2005 through 2012. The survey questionnaires were sent to facilities nationwide, and clinical data regarding KD in DS were collected. A control group consisted of non-DS patients with KD who were managed at Toho University. RESULTS: Of the 94 233 children diagnosed with acute KD from 2005 to 2012, 16 children with acute KD also had DS (0.017%). The DS-KD patients were significantly older than the non-DS patients (median, 8 years vs 1 year, P < 0.05, respectively). Half of the DS patients had incomplete KD. Although 50% of the DS children were at high risk of immunoglobulin resistance, all children responded to initial treatment and none had CAA. CONCLUSIONS: All DS-KD patients responded to initial i.v. immunoglobulin (IVIG) or aspirin despite having a high risk of IVIG resistance, and none of the DS patients had CAA. This suggests that the risk of treatment resistance and development of CAA may be not higher in DS patients with acute KD.

The Combined Usefulness of the Neutrophil-to-Lymphocyte and Platelet-to-Lymphocyte Ratios in Predicting Intravenous Immunoglobulin Resistance with Kawasaki Disease.

The laboratory records of 405 patients with Kawasaki disease before and after intravenous immunoglobulin (IVIG) therapy were compared between the IVIG-responsive (n = 320) and IVIG-resistant (n = 85) groups. A high neutrophil-to-lymphocyte ratio and a high platelet-to-lymphocyte ratio before IVIG, especially when combined, were useful predictors for IVIG resistance in Kawasaki disease.

Transarterial embolization for pediatric hepatocellular carcinoma with cardiac cirrhosis.

We describe the case of a 15-year-old boy with a history of Fontan operation and multiple intrahepatic tumors. Computed tomography showed multiple hepatic nodules with arterial enhancement. Because hepatocellular carcinoma (HCC) was not detected on biopsies and tumor markers were normal, progress was monitored on imaging. One hepatic tumor increased greatly in size during follow up. At 15 years of age, tumor markers rose rapidly, and he had upper abdominal swelling. Therefore, transarterial embolization (TAE) was performed for the largest tumor, suspected to be a HCC due to cardiac cirrhosis. This tumor had not increased at follow up 4 months later. The patient died from hepatic failure at the age of 17 years, and HCC was diagnosed at autopsy. Although pediatric HCC is rare, its incidence is likely to increase. TAE, with or without anticancer agents, is a therapeutic option for unresectable pediatric HCC, as it is for adult HCC.

Soluble ST2 and N-terminal pro-brain natriuretic peptide combination. Useful biomarker for predicting outcome of childhoodpulmonary arterial hypertension.

BACKGROUND: Some potential biomarkers have been reported recently in patients with pulmonary arterial hypertension (PAH), but the most clinically useful among these potential biomarkers, especially in childhood PAH, has not been identified. Therefore, this study investigated which biomarker is useful in assessing severity of and patient prognosis in childhood idiopathic PAH (IPAH)/heritable PAH (HPAH). METHODS AND RESULTS: Fifty-nine patients who were younger than 16 years at onset of IPAH/HPAH were selected. The following 10 biomarker candidates were quantified: high-sensitivity troponin T, human heart fatty acid-binding protein, N-terminal pro-brain natriuretic peptide (NT-proBNP), pentraxin-3, soluble ST2 (sST2), angiopoietin-2 (Ang-2), matrix metalloproteinase 2, tenascin C, endostatin (ES), and thymidine kinase. Functional characteristics and clinical outcomes were analyzed retrospectively. NT-proBNP, sST2, Ang-2, and ES correlated well with New York Heart Association class. On area under the receiver operating characteristic curve analysis, sST2 had a significantly good relationship with prognosis. On Kaplan-Meier curve and univariate Cox regression analyses, elevated sST2 and NT-proBNP level predicted poor outcome of the present patients with childhood IPAH/HPAH. Furthermore, patients with elevated sST2 had significantly worse prognosis among those with high NT-proBNP. CONCLUSIONS: The sST2 and NT-proBNP combination is a useful biomarker to predict clinical condition and outcome in patients with childhood IPAH/HPAH.

[Use of the urinary trypsin inhibitor ulinastatin for acute Kawasaki disease].

Intravenous immunoglobulin (IVIG) therapy is widely recognized as standard treatment for Kawasaki disease(KD). However, about 20 % of KD patients are resistant to IVIG and are considered to be a high risk group for coronary artery lesions (CAL). Ulinastatin(UTI) is one of the neutrophil elastase inhibitors used for patients with pancreatitis or circulatory shock, and several studies have shown its efficacy for KD. Recently, we demonstrated that initial UTI treatment combined with IVIG decreased the number of patients requiring addi- tional rescue treatment and the occurrence of CAL. In this study, no severe adverse events occurred. Further research and a prospective trial are needed to prove the clinical efficacy and demonstrate the limits of UTI in patients with KD.

Ulinastatin, a urinary trypsin inhibitor, for the initial treatment of patients with Kawasaki disease: a retrospective study.

BACKGROUND: Markedly activated neutrophils or higher plasma levels of neutrophil elastase are involved in the poor response to intravenous immunoglobulin (IVIG) and the formation of coronary artery lesions (CAL) in patients with acute Kawasaki disease. We hypothesized that ulinastatin (UTI), by both direct and indirect suppression of neutrophils, would reduce the occurrence of CAL. METHODS AND RESULTS: We retrospectively analyzed the clinical records of patients with Kawasaki disease between 1998 and 2009. Three hundred sixty-nine patients were treated with a combination of UTI, aspirin, and IVIG as an initial treatment (UTI group), and 1178 were treated with a conventional initial treatment, and IVIG with aspirin (control group). The baseline characteristics did not demonstrate notable differences between the two groups. The occurrence of CAL was significantly lower in the UTI group than in the control group (3% versus 7%; crude odds ratio [OR], 0.46; 95% confidence interval [CI], 0.25-0.86; P=0.01). The OR adjusted for sex, Gunma score (the predictive score for IVIG unresponsiveness), and dosage of initial IVIG (1 or 2 g/kg) was 0.32 (95% CI, 0.17-0.60; P<0.001). In addition, most CAL occurred in patients requiring additional rescue treatment and the proportion of those patients was significantly lower in the UTI group than in the control group (13% versus 22%; crude OR, 0.52; 95% CI, 0.38-0.73; P<0.001). The adjusted OR was 0.30 (95% CI, 0.20-0.44; P<0.001). CONCLUSIONS: UTI was associated with fewer patients requiring additional rescue treatment and reduction of CAL in this retrospective study.

Contribution of sarcoplasmic reticulum Ca²+ release and Ca²+ transporters on sarcolemmal channels to Ca²+ transient in fetal mouse heart.

Sarcoplasmic reticulum (SR) Ca release has been shown not to be the predominant mechanism responsible for excitation-contraction (E-C) coupling in fetal myocytes. However, most of the studies have been conducted either on primary cultures or acutely isolated cells, in which an apparent reduction of ryanodine receptor density have been reported. We aimed to elucidate the contribution of SR Ca release and Ca transporters on sarcolemmal channels to Ca transients in fetal mouse whole hearts. On embryonic day 13.5, ryanodine significantly reduced the amplitude of the Ca transient to 27.2 ± 4.4% of the control, and both nickel and SEA0400 significantly prolonged the time to peak from 84 ± 2 ms to 140 ± 5 ms and 129 ± 6 ms, respectively, whereas nifedipine did not alter it. Therefore, at early fetal stages, SR Ca release should be an important component of E-C coupling, and T-type Ca channel and reverse mode sodium-calcium exchanger (NCX)-mediated SR Ca release could be the predominant contributors. Using embryonic mouse cultured cardiomyocytes, we showed that both nifedipine and nickel inhibited the ability of NCX to extrude Ca from the cytosol. From these results, we propose a novel idea concerning E-C coupling in immature heart.

Fetal and neonatal development of Ca2+ transients and functional sarcoplasmic reticulum in beating mouse hearts.

BACKGROUND: It is generally accepted that Ca(2+)-induced Ca(2+) release is not the predominant mechanism during embryonic stages. Most studies have been conducted either on primary cultures or acutely isolated cells, in which an apparent reduction of ryanodine receptor density and alterations in the cell shape have been reported. The aim of the present study was to investigate developmental changes in Ca(2+) transients using whole hearts of mouse embryos and neonates. METHODS AND RESULTS: Fluo-3 fluorescence signals from stimulated whole hearts were detected using a photomultiplier and stored as Ca(2+) transients. The upstroke and decay of Ca(2+) transients became more rapid from the late embryonic stages to the neonatal stage. After thapsigargin application (an inhibitor of the sarcoplasmic Ca(2+)-ATPase [SERCA]), time to 50% relaxation (T(50)) of Ca(2+) transients was significantly prolonged. There were no significant changes in T(50) after Ru360 application (an inhibitor of mitochondrial Ca(2+) uniporter). The rate of increase in the amplitude of Ca(2+) transients after caffeine application became larger during developmental stages. CONCLUSIONS: Ca(2+) homeostasis developmentally changes from a slow rise and decay of Ca(2+) transients to rapid kinetics after the mid-embryonic stage. SERCA began to contribute significantly to Ca(2+) homeostasis at early embryonic stages and sarcoplasmic reticulum Ca(2+) contents increased from embryonic to neonatal stages, whereas mitochondrial Ca(2+) uptake did not contribute to Ca(2+) transients on a beat-to-beat basis.

Association of Severity of Coronary Artery Aneurysms in Patients With Kawasaki Disease and Risk of Later Coronary Events.

Importance: Few studies with sufficient statistical power have shown the association of the z score of the coronary arterial internal diameter with coronary events (CE) in patients with Kawasaki disease (KD) with coronary artery aneurysms (CAA). Objective: To clarify the association of the z score with time-dependent CE occurrence in patients with KD with CAA. Design, Setting, and Participants: This multicenter, collaborative retrospective cohort study of 44 participating institutions included 1006 patients with KD younger than 19 years who received a coronary angiography between 1992 and 2011. Main Outcomes and Measures: The time-dependent occurrence of CE, including thrombosis, stenosis, obstruction, acute ischemic events, and coronary interventions, was analyzed for small (z score, <5), medium (z score, ≥5 to <10; actual internal diameter, <8 mm), and large (z score, ≥10 or ≥8 mm) CAA by the Kaplan-Meier method. The Cox proportional hazard regression model was used to identify risk factors for CE after adjusting for age, sex, size, morphology, number of CAA, resistance to initial intravenous immunoglobulin (IVIG) therapy, and antithrombotic medications. Results: Of 1006 patients, 714 (71%) were male, 341 (34%) received a diagnosis before age 1 year, 501 (50%) received a diagnosis between age 1 and 5 years, and 157 (16%) received a diagnosis at age 5 years or older. The 10-year event-free survival rate for CE was 100%, 94%, and 52% in men (P < .001) and 100%, 100%, and 75% in women (P < .001) for small, medium, and large CAA, respectively. The CE-free rate was 100%, 96%, and 79% in patients who were not resistant to IVIG therapy (P < .001) and 100%, 96%, and 51% in patients who were resistant to IVIG therapy (P < .001), respectively. Cox regression analysis revealed that large CAA (hazard ratio, 8.9; 95% CI, 5.1-15.4), male sex (hazard ratio, 2.8; 95% CI, 1.7-4.8), and resistance to IVIG therapy (hazard ratio, 2.2; 95% CI, 1.4-3.6) were significantly associated with CE. Conclusions and Relevance: Classification using the internal diameter z score is useful for assessing the severity of CAA in relation to the time-dependent occurrence of CE and associated factors in patients with KD. Careful management of CE is necessary for all patients with KD with CAA, especially men and IVIG-resistant patients with a large CAA.

A comparison of the predictive validity of the combination of the neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio and other risk scoring systems for intravenous immunoglobulin (ivig)-resistance in Kawasaki disease.

BACKGROUND: We recently reported that the combination of the neutrophil-to-lymphocyte ratio (NLR) and the platelet-to-lymphocyte ratio (PLR) is a novel and useful predictor of intravenous immunoglobulin (IVIG)-resistance in Kawasaki disease (KD). In the present study, to evaluate the effectiveness of the new risk score, we compared its predictive validity to that of previously reported risk scores. MATERIALS AND METHODS: The laboratory records of 437 patients with KD before IVIG therapy were retrospectively analyzed, and the IVIG-responsive (n = 344) and IVIG-resistant (n = 93) patients were compared. The validity of the new score (the combination of NLR≥3.83 and PLR≥150) for predicting IVIG resistance in KD was compared to that of the Kobayashi, Egami and Sano risk scores. RESULTS: The new score and the Kobayashi score displayed high sensitivity (0.72 and 0.70 respectively) and specificity (0.67 and 0.68 respectively), while the Egami and Sano scores showed high specificity (0.71 and 0.81 respectively) but relatively low sensitivity (0.56 and 0.45 respectively). The odds ratios (ORs) for the new score, the Kobayashi score, the Egami score and the Sano score were 5.34 (95% confidence interval [CI] 3.22-8.85), 4.87 (95% CI 2.96-8.01), 3.14 (95% CI 1.96-5.03) and 3.53 (95% CI 2.17-5.77) respectively. CONCLUSIONS: The predictive validity of the combination of NLR≥3.83 and PLR≥150, which is a simple and convenient indicator, was equal to or higher than that of the other risk scores. This suggests that the new score could be a widely available marker for predicting IVIG resistance in KD.

Urinary Lactate Dehydrogenase Activity and Its Isozyme Patterns in Kawasaki Disease.

Abnormal urinary findings, such as sterile pyuria, proteinuria, and microscopic hematuria, are often seen in the acute phase of Kawasaki disease (KD). We investigated the potential significance of urinary lactate dehydrogenase (U-LDH) activity and its isozyme patterns in KD. Total U-LDH activity and its isozymes (U-LDH1-5) levels were compared among 120 patients with KD, 18 patients with viral infection (VI), and 43 patients with upper urinary tract infection (UTI) and additionally compared between intravenous immunoglobulin (IVIG) responders (n = 89) and nonresponders (n = 31) with KD. Total U-LDH activity was higher in KD (35.4 ± 4.8 IU/L, P < 0.05) and UTI patients (66.0 ± 8.0 IU/L, P < 0.01) than in VI patients (17.0 ± 6.2 IU/L). In the isozyme pattern analysis, KD patients had high levels of U-LDH1 and U-LDH2, while UTI patients had high levels of U-LDH3, U-LDH4, and U-LDH5. Furthermore, IVIG nonresponders of KD had significantly higher levels of total U-LDH activity (45.1 ± 4.7 IU/L, P < 0.05), especially U-LDH1 and U-LDH2 (P < 0.05), than IVIG responders (32.0 ± 2.8 IU/L). KD patients have increased levels of total U-LDH activity, especially U-LDH-1 and U-LDH2, indicating a unique pattern of U-LDH isozymes different from that in UTI patients.

[Development of a brief communication ability scale for Japanese demented elderly].

We developed a brief scale to evaluate communication ability of the demented elderly. This scale assesses not only abilities related to overall communication such as verbal function, judgment and emotional function, but also non-verbal communication such as eye-contact, nodding and smiling. The scale places little burden on the demented elderly subject and takes only a few minutes to perform, even if the dementia is severe. We evaluated 106 demented elderly residents of nursing homes using this brief communication ability scale, and the following results were obtained. The validity of this scale was confirmed by the high correlation coefficient between this scale and the formal caregiver questionnaire scores concerning communication ability, and the high-correlation coefficient between this scale and intellectual functions (r = -0.904), emotional functions (r = -0.841) and motor functions (r = -0.679) of dementia syndromes rating scale (Gottfries, Bråne, Steen scale; GBS scale), Hasegawa's Dementia Scale-Revised (HDS-R) (r = 0.625) and the Mini-Mental State Examination (MMS) (r = 0.733). The reliability of this scale was confirmed by the high interrater reliability coefficient of 0.828, test-retest reliability coefficient of 0.940 and Cronbach alpha coefficient of 0.938. These results indicate that the new scale is useful in the assessment of communication ability among the demented elderly.

Mutations of NOTCH3 in childhood pulmonary arterial hypertension.

Mutations of BMPR2 and other TGF-ß superfamily genes have been reported in pulmonary arterial hypertension (PAH). However, 60-90% of idiopathic PAH cases have no mutations in these genes. Recently, the expression of NOTCH3 was shown to be increased in the pulmonary artery smooth muscle cells of PAH patients. We sought to investigate NOTCH3 and its target genes in PAH patients and clarify the role of NOTCH3 signaling. We screened for mutations in NOTCH3, HES1, and HES5 in 41 PAH patients who had no mutations in BMPR2, ALK1, endoglin, SMAD1/4/8, BMPR1B, or Caveolin-1. Two novel missense mutations (c.2519 G>A p.G840E, c.2698 A>C p.T900P) in NOTCH3 were identified in two PAH patients. We performed functional analysis using stable cell lines expressing either wild-type or mutant NOTCH3. The protein-folding chaperone GRP78/BiP was colocalized with wild-type NOTCH3 in the endoplasmic reticulum, whereas the majority of GRP78/BiP was translocated into the nuclei of cells expressing mutant NOTCH3. Cell proliferation and viability were higher for cells expressing mutant NOTCH3 than for those expressing wild-type NOTCH3. We identified novel NOTCH3 mutations in PAH patients and revealed that these mutations were involved in cell proliferation and viability. NOTCH3 mutants induced an impairment in NOTCH3-HES5 signaling. The results may contribute to the elucidation of PAH pathogenesis.

A checklist to assess the quality of reports on spa therapy and balneotherapy trials was developed using the Delphi consensus method: the SPAC checklist.

OBJECTIVE: The purpose of this study was to develop a checklist of items that describes and measures the quality of reports of interventional trials assessing spa therapy. METHODS: The Delphi consensus method was used to select the number of items in the checklist. A total of eight individuals participated, including an epidemiologist, a clinical research methodologist, clinical researchers, a medical journalist, and a health fitness programmer. Participants ranked on a 9-point Likert scale whether an item should be included in the checklist. RESULTS: Three rounds of the Delphi method were conducted to achieve consensus. The final checklist contained 19 items, with items related to title, place of implementation (specificity of spa), care provider influence, and additional measures to minimize the potential bias from withdrawals, loss to follow-up, and low treatment adherence. CONCLUSION: This checklist is simple and quick to complete, and should help clinicians and researchers critically appraise the medical and healthcare literature, reviewers assess the quality of reports included in systematic reviews, and researchers plan interventional trials of spa therapy.