Seiichiro Takeshita

Inhibitory effect of serine protease inhibitors on neutrophil‐mediated endothelial cell injury

To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil‐mediated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbilical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose‐dependently by urinary trypsin inhibitor (ulinastatin, UTI) and ONO‐5046, which have the ability to inactivate neutrophil elastase, but not by gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban, which have no ability to inactivate neutrophil elastase. In addition, when UTI and ONO‐5046 were added to the tumor necrosis factor α‐primed neutrophils alone, they showed a dose‐dependent inhibition of the intracellular elastase activity, but the other SPI did not, for either flow cytometry or confocal microscopy. Therefore, UTI and ONO‐5046 may protect EC against the neutrophil‐mediated injury not only by inactivating the extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils and suppressing the production and secretion of activated elastase from them.

Circulating endothelial cells in Kawasaki disease

Recent reports have demonstrated that circulating endothelial cells (CECs) are observed in several diseases with vascular injury. Because Kawasaki disease (KD) is one type of systemic vasculitis, we hypothesized that an increased number of CECs may be associated with the appearance of complicated coronary artery lesions (CAL). In the present study we investigated the enumeration and origin of CECs in 20 patients with KD, using an immunohistochemical method with monoclonal antibodies: clone P1H12 against ECs and clone AC133 against endothelial progenitor cells (EPCs), which were derived from the bone marrow. The mean number of CECs increased significantly (P < 0·05) from the acute through the subacute phases of KD compared with both the convalescent phase of KD and healthy children. The mean number of CECs was significantly (P < 0·05) higher in six KD patients with CAL than in 14 KD patients without CAL. The population of EPCs in the total CECs in KD was 4·4 ± 1·2% (range 0–18%). The number of EPCs during the subacute phase was also significantly higher (P < 0·05) in KD patients with CAL than in those without CAL. Our findings indicate that the number of CECs increase in KD vasculitis and suggest that the increased numbers of CECs and EPCs may reflect the EC damage of this disease.

Elevated serum levels of matrix metalloproteinase-9 (MMP-9) in Kawasaki disease

Matrix metalloproteinases (MMPs) play an important role in the progression of tumour cells and the invasion of inflammatory cells by degrading the extracellular matrix. In the MMP family, MMP‐9 gelatinase is thought to contribute to the pathogenesis of inflammatory arteritis by disrupting the elastic lamina. The aim of the present study is to investigate the potential role of MMP‐9 in Kawasaki disease (KD), an acute type of systemic vasculitis in children. We studied the total levels of MMP‐9 (free proMMP‐9 and free MMP‐9) in the sera using a new assay system and the expression of MMP‐9 mRNA in the leucocytes using reverse transcription‐polymerase chain reaction in 18 patients with KD, 10 patients with sepsis and 10 healthy children (HC). The serum MMP‐9 levels were significantly higher (P < 0·01) in the acute phase of KD than in the acute phase of sepsis and HC. In the time course of KD, the serum MMP‐9 levels decreased significantly (P < 0·01) from the subacute through the convalescent phases. In the acute phase of KD, the serum MMP‐9 levels showed a significantly positive correlation (P < 0·05) with the circulating leucocyte counts, especially the neutrophil counts. Furthermore, the expression of MMP‐9 mRNA in the circulating leucocytes increased in the acute phase of KD and decreased from the subacute through the convalescent phases. These findings indicate that an excessive amount of MMP‐9 is present in the plasma during the acute phase of KD, thus suggesting that circulating leucocytes may be a source of the MMP‐9 secreted into the circulation.

Increased levels of circulating soluble CD14 in Kawasaki disease

The CD14 molecule, which is known to be a receptor for endotoxin, is expressed on monocytes and neutrophils. It is found as a soluble CD14 (sCD14) in circulation, and the plasma level has been shown to be increased in some infectious diseases, including sepsis. To investigate the potential significance of circulating sCD14 in Kawasaki disease (KD), the plasma level of sCD14 was measured using ELISA in patients with KD, patients with a Gram‐negative bacterial infection (GNBI) including sepsis, patients with viral infection (VI), and healthy controls. We also analysed CD14 receptor expression in monocytes and neutrophils using flow cytometry and a semiquantitative reverse transcription‐polymerase chain reaction. Although KD patients had significantly lower counts of peripheral neutrophils and monocytes than GNBI patients, KD patients had significantly higher levels of sCD14 than GNBI. No significant correlations were observed between sCD14 levels and clinical laboratory values or the cytokine (interferon‐gamma, tumour necrosis factor‐alpha) levels in the acute phase. The mean intensity of CD14 receptor expression on neutrophils markedly increased in the acute phases of KD and GNBI compared with that in their convalescent phases, while that on monocytes decreased. The expression of CD14 mRNA in neutrophils increased in the acute phases of KD and GNBI, while that in monocytes did not decrease but instead remained quite abundant. The present findings suggest that the elevated level of circulating sCD14 appears to be an important parameter for KD and that sCD14 shedding is accompanied by different kinetics regarding the expression of CD14 antigen and CD14 gene between monocytes and neutrophils.

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.

The Role of Bacterial Lipopolysaccharide-Bound Neutrophils in the Pathogenesis of Kawasaki Disease

To investigate the possible role of lipopolysaccharide (LPS, endotoxin) in the pathogenesis of Kawasaki disease, neutrophils from 15 patients with the disease and 7 with sepsis (4 infected with gram-negative bacteria and 3 with gram-positive bacteria) were analyzed by flow cytometry using anti-LPS and anti-CD14 monoclonal antibodies. The number of LPS- and CD14-positive neutrophils was dramatically higher early after the onset of Kawasaki disease and gram-negative sepsis but not with gram-positive sepsis. An immunoprecipitation analysis revealed LPS was bound to CD14 in vivo on neutrophils from Kawasaki disease patients. The mean plasma level of neutrophil elastase was significantly higher in the acute phase of Kawasaki disease than in the acute phase of sepsis. These findings suggest that exposure to LPS occurs at the onset of Kawasaki disease when LPS-bound neutrophils secrete excess protease (implicated in neutrophil-mediated endothelial injury) into the circulation.

Delayed apoptosis of circulating neutrophils in Kawasaki disease

Circulating polymorphonuclear neutrophils (PMNs) are known to increase in number and are functionally activated in the acute phase of Kawasaki disease (KD). In the present study, we investigated whether the apoptosis of PMNs is deregulated in KD. When the isolated PMNs were cultured in vitro, the proportions of spontaneous apoptotic PMNs (annexin V+ cells and cells with fragmented DNA) were found to be significantly lower (P < 0·01) in the patients with KD (n = 25) than in the patients with a bacterial infection (n = 20) or a viral infection (n = 20), or in healthy children (n = 20). The proportion of circulating Fas‐positive PMNs was also significantly lower (P < 0·01) in the acute KD patients than in the other groups. In the acute phase of KD, the proportion of spontaneous apoptotic PMNs showed a significant positive correlation (P < 0·01) with the proportions of circulating Fas‐positive PMNs. Furthermore, the agonistic anti‐Fas MoAb (CH‐11) induced a significant increase in the proportion of apoptotic PMNs in the patients with a viral infection and healthy children, but not in either the patients with KD or the patients with a bacterial infection. In the intracellular expression of anti‐ and pro‐apoptotic proteins, the A1/Bax ratio was significantly higher in acute KD than in the other groups. These findings indicate that PMN apoptosis is inhibited during the acute phase of KD and also suggest that both the resistance against the Fas‐mediated death signal and the down‐regulation of the mitochondrial apoptotic signalling pathway due to an altered balance of Bcl‐2 protein expression are responsible for the delayed PMN apoptosis.

Circulating soluble selectins in Kawasaki disease

To investigate the significance of circulating adhesion molecules associated with leucocyte–endothelial cell interaction in Kawasaki disease (KD), serum levels of soluble E‐, P‐, L‐selectin (sE‐, sP‐, sL‐selectin), and vascular cell adhesion molecule‐1 (VCAM‐1) were measured in 16 patients with KD, eight with other febrile diseases, six with Henoch–Schönlein purpura (HSP), and 10 healthy children using an ELISA. Serum sE‐selectin levels from patients in the acute phase of KD were significantly higher than from those in other groups (P<0.01). The levels of sP‐selectin in the subacute phase of KD were significantly higher than in other groups (P<0.01). Serum sL‐selectin levels tended to rise in the convalescent phase of KD. There were also significant correlations between sE‐selectin levels and C‐reactive protein (r=0.80, P<0.0001), and between sP‐selectin levels and platelet counts (r=0.57, P<0.0001) in KD patients. These data indicate that circulating soluble forms of three selectins may have different kinetics during the clinical course of KD, suggesting that they may reflect its inflammatory process.

Isolated congenital left ventricular diverticulum with perinatal dysrhythmia: a case report and review of the literature.

Abstract. We report a case of isolated congenital left ventricular diverticulum (LVD) with perinatal dysrhythmia, which disappeared spontaneously 1 week after birth. The LVD arose from the lateral wall of the LV, and the contraction of the LVD was synchronous with the kinetics of the main LV chamber. The LVD changed very little in size during the first 30 months after birth, and its relative size to the growing LV main chamber decreased. The patient had neither any symptoms nor complications during this time. The available literature on prenatal and neonatal cases with isolated LVD or LV aneurysm is also reviewed.

Intravenous immunoglobulin (IVIG) preparations induce apoptosis in TNF-α-stimulated endothelial cells via a mitochondria-dependent pathway

Endothelial cells (ECs) are a target in inflammation, and the death of EC is regulated by various factors. Although intravenous immunoglobulin (IVIG) preparations are known to be beneficial therapeutic agents for the treatment of autoimmune diseases and systemic inflammatory disorders, their mechanism of action have not yet been completely elucidated. The aim of the present study is to investigate the possible role of IVIG in EC apoptosis. We demonstrate herein that IVIG induced the apoptosis of human umbilical vein ECs (HUVECs) prestimulated by TNF‐αin vitro, but not in unstimulated HUVECs, in a dose‐ and time‐dependent manner, using a proportion of cells with hypodiploid DNA, DNA ladder formation and morphological changes. Anti‐Fas MoAbs had no effect on the IVIG‐induced apoptosis in the TNF‐α‐stimulated HUVECs. IVIG decreased the intracellular expression of anti‐apoptotic proteins of the Bcl‐2 family (A1 and Bcl‐XL) while IVIG increased the intracellular expression of pro‐apoptotic proteins (Bax and Bcl‐XS) in the TNF‐α‐stimulated HUVECs. Furthermore, IVIG increased the intracellular production of reactive oxygen species and decreased the mitochondrial membrane potential (Δψm). Caspase‐inhibitors inhibited the IVIG‐induced apoptosis of the TNF‐α‐stimulated HUVECs. The present results show a novel action in which IVIG can induce the apoptosis of TNF‐α‐stimulated HUVECs through a mitochondrial apoptotic signalling pathway. These observations suggest that the clinical use of IVIG preparations may thereby regulate the cell death of activated ECs in inflammation.