Kunio Shirato

Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis.

BACKGROUND Epidemiological and clinical evidence suggests that an increased intake of long-chain n-3 fatty acids protects against mortality from coronary artery disease. We aimed to test the hypothesis that long-term use of eicosapentaenoic acid (EPA) is effective for prevention of major coronary events in hypercholesterolaemic patients in Japan who consume a large amount of fish. METHODS 18 645 patients with a total cholesterol of 6.5 mmol/L or greater were recruited from local physicians throughout Japan between 1996 and 1999. Patients were randomly assigned to receive either 1800 mg of EPA daily with statin (EPA group; n=9326) or statin only (controls; n=9319) with a 5-year follow-up. The primary endpoint was any major coronary event, including sudden cardiac death, fatal and non-fatal myocardial infarction, and other non-fatal events including unstable angina pectoris, angioplasty, stenting, or coronary artery bypass grafting. Analysis was by intention-to-treat. The study was registered at ClinicalTrials.gov, number NCT00231738. FINDINGS At mean follow-up of 4.6 years, we detected the primary endpoint in 262 (2.8%) patients in the EPA group and 324 (3.5%) in controls-a 19% relative reduction in major coronary events (p=0.011). Post-treatment LDL cholesterol concentrations decreased 25%, from 4.7 mmol/L in both groups. Serum LDL cholesterol was not a significant factor in a reduction of risk for major coronary events. Unstable angina and non-fatal coronary events were also significantly reduced in the EPA group. Sudden cardiac death and coronary death did not differ between groups. In patients with a history of coronary artery disease who were given EPA treatment, major coronary events were reduced by 19% (secondary prevention subgroup: 158 [8.7%] in the EPA group vs 197 [10.7%] in the control group; p=0.048). In patients with no history of coronary artery disease, EPA treatment reduced major coronary events by 18%, but this finding was not significant (104 [1.4%] in the EPA group vs 127 [1.7%] in the control group; p=0.132). INTERPRETATION EPA is a promising treatment for prevention of major coronary events, and especially non-fatal coronary events, in Japanese hypercholesterolaemic patients.

Chemosensitivity and Perception of Dyspnea in Patients with a History of Near-Fatal Asthma

BACKGROUND Many deaths from attacks of asthma may be preventable. However, the difficulty in preventing fatal attacks is that not all the pathophysiologic risk factors have been identified. METHODS To examine whether dyspnea and chemosensitivity to hypoxia and hypercapnia are factors in fatal asthma attacks, we studied 11 patients with asthma who had had near-fatal attacks, 11 patients with asthma who had not had near-fatal attacks, and 16 normal subjects. Their respiratory responses to hypoxia and hypercapnia, determined by the standard rebreathing technique while the patients were in remission, were assessed in terms of the slopes of ventilation and airway occlusion pressure as a function of the percentage of arterial oxygen saturation and end-tidal carbon dioxide tension, respectively. The perception of dyspnea was scored on the Borg scale during breathing through inspiratory resistances ranging from 0 to 30.9 cm of water per liter per second. RESULTS The mean (+/- SD) hypoxic ventilatory response (0.14 +/- 0.12 liter per minute per percent of arterial oxygen saturation) and airway occlusion pressure (0.05 +/- 0.05 cm of water per percent of arterial oxygen saturation) were significantly lower in the patients with near-fatal asthma than in the normal subjects (0.60 +/- 0.35, P < 0.001, and 0.16 +/- 0.08, P < 0.001, respectively) and the patients with asthma who had not had near-fatal attacks (0.46 +/- 0.29, P = 0.003, and 0.15 +/- 0.09, P = 0.004). The Borg score was also significantly lower in the patients with near-fatal asthma than in the normal subjects, and their lower hypoxic response was coupled with a blunted perception of dyspnea. CONCLUSIONS Reduced chemosensitivity to hypoxia and blunted perception of dyspnea may predispose patients to fatal asthma attacks.

Effects of EPA on coronary artery disease in hypercholesterolemic patients with multiple risk factors: sub-analysis of primary prevention cases from the Japan EPA Lipid Intervention Study (JELIS).

BACKGROUND Japan EPA Lipid Intervention Study (JELIS) was a large-scale clinical trial examining the effects of eicosapentaenoic acid (EPA) on coronary artery disease (CAD) in hypercholesterolemic patients. Herein, we focused on risk factors other than low-density lipoprotein cholesterol (LDL-C) to investigate the effects of EPA on CAD among JELIS primary prevention cases. METHODS Hypercholesterolemic patients on statin therapy but without evidence of CAD (n=14,981) were randomly assigned to an EPA group (n=7503) or a control group (n=7478). The relationships between incident CAD, the number of CAD risk factors (hypercholesterolemia; obesity; high triglyceride (TG) or low high-density lipoprotein cholesterol (HDL-C); diabetes; and hypertension) and EPA treatment were investigated. RESULTS For the control and EPA groups combined, a higher number of risk factors was directly associated with an increased incidence of CAD. Incidence was lower for the EPA group than for the control group regardless of the numbers of risk factors. Compared to patients with normal serum TG and HDL-C levels, those with abnormal levels (TG >or=150 mg/dL; HDL-C <40 mg/dL) had significantly higher CAD hazard ratio (HR: 1.71; 95% CI: 1.11-2.64; P=0.014). In this higher risk group, EPA treatment suppressed the risk of CAD by 53% (HR: 0.47; 95% CI: 0.23-0.98; P=0.043). CONCLUSIONS Multiple risk factors besides cholesterol are associated with markedly increased incidence of CAD. High TG with low HDL-C represents a particularly potent risk factor. EPA was effective in reducing the incidence of CAD events for patients with this dyslipidemic pattern, suggesting that EPA may be especially beneficial in patients who with abnormal TG and HDL-C levels.

In vivo release of glutamate in nucleus tractus solitarii of the rat during hypoxia.

1. An attempt has been made to test the hypothesis that, in the caudal part of nucleus tractus solitarii (NTS) where carotid sinus nerve (CSN) afferents project, L‐glutamate (Glut) modulates the hypoxic ventilatory response. 2. Unanaesthetized, peripherally chemodenervated (carotid body denervated; CBD) and sham‐operated, freely moving rats were used. During peripheral chemoreceptor stimulation by hypoxia (10% O2 for 30 min) or doxapram (Dox) infusion (2 mg kg‐1 (30 min)‐1), ventilation was recorded and successively, under the same conditions, the extracellular Glut concentration ([Glut]o) in the caudal NTS was measured by in vivo microdialysis. [Glut]o was also measured during hyperoxic hypercapnia (10% CO2‐30% O2 for 30 min). 3. Furthermore, the effects on ventilation of exogenous Glut, the NMDA (N‐methyl‐D‐aspartate) receptor antagonist MK‐801 or the ionotropic receptor antagonist kynurenate microinjected into the caudal NTS were investigated in sham‐operated rats. 4. In sham‐operated rats, both ventilation and [Glut]o in NTS were increased during peripheral chemoreceptor stimulation. On the other hand, no increases in either ventilation or Glut release were observed in CBD rats. In spite of ventilatory augmentation during hypercapnia, no response of [Glut]o to hypercapnia was observed in either group. 5. Local Glut application into NTS increased ventilation. Pretreatment with MK‐801 or kynurenate reduced the hypoxic ventilatory response. This reduction in ventilation was mainly due to the decrease in tidal volume. 6. These results suggest that hypoxia induced the release of Glut in NTS and that this effect was mediated by arterial chemosensory input.

Regulation of Murine Airway Eosinophilia and Th2 Cells by Antigen-Conjugated CpG Oligodeoxynucleotides as a Novel Antigen-Specific Immunomodulator

The characteristic features of bronchial asthma reflect the orchestrated activity of Th2 cells. Oligodeoxynucleotides containing CpG motifs (CpG) have recently been highlighted as an immunomodulator that biases toward a Th1-dominant phenotype. We have previously reported that intratracheal coadministration of CpG and allergen inhibited airway eosinophilia and hyperresponsiveness in a synergistic manner. To substantiate the synergism between CpG and Ag, we introduced a covalently linked conjugate between CpG and Ag and examined the efficacy on airway eosinophilia and Th2 cytokine production. We found that the conjugated form of CpG plus Ag was 100-fold more efficient in regulating airway eosinophilia than the unconjugated mixture. The inhibitory effects lasted for at least 2 mo. The inhibition of airway eosinophilia by the conjugate was Ag specific and associated with an improvement of the airway hyperresponsiveness and the unresponsiveness of the Ag-specific Th2 cells in the regional lymph nodes. The CpG-Ag conjugate was 100-fold more effective than the unconjugated mixture for inducing in vitro Th1 differentiation in an IL-12-dependent manner. Our data show that CpG conjugated to Ag can work as a novel Ag-specific immunomodulator and imply that inhalation of allergen-CpG conjugate could be a desensitization therapy for patients with bronchial asthma.

Reduction in the Recurrence of Stroke by Eicosapentaenoic Acid for Hypercholesterolemic Patients: Subanalysis of the JELIS Trial

Background and Purpose— The JELIS trial examined the preventive effect of eicosapentaenoic acid (EPA) against coronary artery diseases. Hypercholesterolemic patients received statin only (no EPA group: n=9319) or statin with EPA (EPA group: n=9326) for around 5 years. EPA significantly suppressed the incidence of coronary events in previous analysis. Herein, we investigated the effects of EPA on the primary and secondary prevention of stroke. Methods— We conducted a subanalysis of JELIS with respect to stroke incidence in the primary and secondary prevention subgroups defined as those without and with a prior history of stroke using Cox proportional hazard ratios, adjusted for baseline risk factor levels. Results— As for primary prevention of stroke, this occurred in 114 (1.3%) of 8862 no EPA group and in 133 (1.5%) of 8841 EPA group. No statistically significant difference in total stroke incidence (Hazard Ratio, 1.08; 95% confidence interval, 0.95 to 1.22) was observed between the no EPA and the EPA groups. In the secondary prevention subgroup, stroke occurred in 48 (10.5%) of 457 no EPA group and in 33 (6.8%) of 485 EPA group, showing a 20% relative reduction in recurrent stroke in the EPA group (Hazard Ratio, 0.80; 95% confidence interval, 0.64 to 0.997). Conclusions— Administration of highly purified EPA appeared to reduce the risk of recurrent stroke in a Japanese population of hypercholesterolemic patients receiving low-dose statin therapy. Further research is needed to determine whether similar benefits are found in other populations with lower levels of fish intake. The trial is registered at ClinicalTrials.gov (number NCT00231738).

Continuity of airway goblet cells and intraluminal mucus in the airways of patients with bronchial asthma

The aim of this study was to elucidate the mechanism of the formation of the widespread mucous-plugging observed in autopsied lungs from patients with bronchial asthma. We performed morphometric analysis of airways of autopsied lungs from eight patients with bronchial asthma (Group BA), and compared it with those of six chronic bronchitics (Group CB) and four control patients (Control). The following parameters were measured in paraffin sections: volume proportion of bronchial glands to bronchial wall (Gland%); goblet cell granules to total epithelial layer (Goblet %); intraluminal mucus expressed as the mucus occupying ratio (MOR); volume ratio of intraluminal mucus continuous with goblet cells to total intraluminal mucus (Vc/Vtol %); and surface ratio of the contact surface of intraluminal mucus continuous with goblet cells to the total luminal surface (Sc/Stot %). Gland%, Goblet %, and MOR or inflammatory cell numbers in the airway walls both from Group BA and CB were larger than those from the Control group. However, no significant differences were observed between Group BA and CB in Gland%, Goblet %, MOR or inflammatory cell numbers, except for the eosinophil number: i.e. 23 +/- 3, 22 +/- 3 and 6 +/- 2% in Gland%; 22 +/- 9, 5 +/- 4 and 2 +/- 2% in Goblet%; 10 +/- 3, 18 +/- 3 and 0.3 +/- 0.5% in MOR; 199 +/- 68, 10 +/- 3 and 2 +/- 2 cells. mm-2 in eosinophil number of the peripheral airways from Groups BA, CB and Control, respectively. In contrast, marked and significant increases were observed both in Vc/Vtot% and Sc/Stot% in Group BA compared to Groups CB and Control both in central and peripheral airways: i.e. Vc/Vtot% in the peripheral airways was 53 +/- 5, 4 +/- 3 and 0.8 +/- 0.8% from Groups BA, CB and Control, respectively (BA vs CB or BA vs Control, p < 0.01 each). These findings suggest that the continuity of goblet cells and intraluminal mucus or lack of full release of mucus, from goblet cells, is peculiar to asthmatic airways, and may contribute to the formation of mucous-plugs.

Nitric oxide as a retrograde messenger in the nucleus tractus solitarii of rats during hypoxia.

1. We examined the role of nitric oxide (NO) in respiratory regulation in the nucleus tractus solitarii (NTS), where L‐glutamate release associated with peripheral chemoreceptor activation modulates the hypoxic ventilatory response. 2. Experiments were performed in unanaesthetized freely moving rats. First, the effects on the hypoxic ventilatory response of sodium nitroprusside (SNP, a NO donor) or NG‐monomethyl‐L‐arginine (L‐NMMA, a NO synthase inhibitor), microinjected into the NTS, were investigated. Second, using in vivo microdialysis, changes in extracellular L‐glutamate during hypoxia were examined in the presence of L‐NMMA. Third, the effect of L‐NMMA on ventilatory augmentation by exogenous L‐glutamate was examined. Furthermore, we measured extracellular L‐citrulline concentration changes during hypoxia in the NTS to assess NO formation indirectly and also examined the effect of MK‐801 (an NMDA receptor antagonist) on L‐citrulline levels during hypoxia. 3. SNP increased ventilation during both normoxia and hypoxia. L‐NMMA did not alter ventilation or L‐glutamate levels during normoxia but significantly attenuated the hypoxic ventilatory response and the increase in L‐glutamate during hypoxia. The inhibition by L‐NMMA was blocked by L‐arginine. The ventilatory augmentation by exogenous L‐glutamate was attenuated by L‐NMMA. L‐Citrulline increased during hypoxia, and this increase was inhibited by MK‐801. 4. We provide the first in vivo evidence that, in the NTS, NO works as a retrograde messenger in an L‐glutamate‐releasing positive feedback system contributing to the augmentation of ventilation during hypoxia.

Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG-tagged antigen by dendritic cells

Oligodeoxynucleotides containing CpG motifs have been highlighted as potent Th1 activators. We previously reported that Ag and CpG, when conjugated together, synergistically promoted the Ag-specific Th1 development and inhibited the Th2-mediated airway eosinophilia. In this study, we examined the mechanisms underlying the synergism of the covalent conjugation. The CpG-OVA conjugate enhanced the Th1 activation and development. These characteristic features of the conjugate could not be ascribed to the polymerization of OVA, but mirrored the augmented binding of the CpG-tagged Ag to dendritic cells (DCs) in a CpG-guided manner, because phycobiliprotein, R-PE, conjugated to CpG stained a higher proportion of DCs with higher intensity than the mixture. R-PE fluorescence was emitted from cytoplasmic portions of the DCs, which simultaneously expressed costimulatory molecules and IL-12. The CpG-conjugated R-PE trafficking described above actually served as a potent Ag. These results indicate that CpG conjugated to Ag exhibit novel joint properties as promoters of Ag uptake and DC activators, thereby potentiating the ability of DCs to generate Th1 cells. The DNA-mediated promotion of Ag uptake would be advantageous for evoking host immune responses against invading microorganisms.

Increased von Willebrand Factor in the Endocardium as a Local Predisposing Factor for Thrombogenesis in Overloaded Human Atrial Appendage

OBJECTIVES We investigated immunoreactive von Willebrand factor (vWF), a platelet adhesion molecule, in the endocardial endothelium and its relationship to thrombogenesis in the human atrial appendage. BACKGROUND Intra-atrial thrombogenesis is generally thought to be induced by blood stasis in the atrial appendage involved with atrial fibrillation (AF). Little attention has been paid to alterations of the endocardial endothelium on which the thrombus develops. METHODS Atrial appendage tissue was obtained at heart surgery or at autopsy from AF and non-AF cardiac patients and from noncardiac patients. Immunohistochemistry for endothelial cell markers including vWF, CD31, CD34 and endothelial nitric oxide synthase (eNOS) and platelet glycoprotein Ib/IX or IIb/IIIa was performed and semiquantitatively graded. RESULTS In contrast to the apparent immunostaining for CD31, CD34 and eNOS, only focal or little immunoreactive vWF was seen in the endocardium of noncardiac patients. Immunoreactive vWF in the endocardial endothelium was increased in most cardiac patients, particularly in the left, but not in the right, atrial appendage of patients with mitral valvular disease, irrespective of whether AF was present. Platelet adhesion/thrombus formation in the endocardium was found in limited sites in which the overlying endothelium was deficient in eNOS and CD34. When warfarin-treated cases were excluded, there was a significant correlation between the immunohistochemical grade for vWF and the degree of platelet adhesion/thrombus formation in the endocardium. CONCLUSIONS Immunoreactive vWF in the endocardial endothelium was increased in overloaded human atrial appendage, which may be a local predisposing factor for intraatrial thrombogenesis.