Indoxyl Sulfate Activates NLRP3 Inflammasome to Induce Cardiac Contractile Dysfunction Accompanied by Myocardial Fibrosis and Hypertrophy.

In patients with chronic kidney diseases (CKD), high serum indoxyl sulfate (IS) levels correlate with cardiac fibrosis and hypertrophy and thus a critical risk factor for heart failure. The aim of this study was to determine the effects of IS on cardiac function and inflammasome pathway in a rat model of CKD. We assessed the physiological and pathological changes and measured biomarkers of fibrosis and hypertrophy in the hearts of Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH + IS). Low left ventricular (LV) ejection fraction, LV dilatation, and advanced myocardial fibrosis and hypertrophy were observed in DH + IS, which resemble changes found in uremic cardiomyopathy. These changes were independent of renal function and blood pressure. RT-PCR and western blotting analysis showed upregulation of fibrosis and hypertrophy-related biomarkers and adhesion molecules in the hearts of DH + IS rats. IS activated aryl hydrocarbon receptor (AHR) pathway, nuclear factor kappa B p65 (NF-κB p65), and inflammasome in the myocardium of DH + IS rat. Moreover, IS upregulated the expression of critical NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and increased production of IL-1ß and IL-18. Finally, IS upregulated various inflammatory cytokines, such as MCP-1, TNF-α, IL-6, and TGFß1, in the myocardium. Our results suggested that IS induced cardiac fibrosis and hypertrophy and impaired LV function through activation of cardiac NLRP3 inflammasome via the AHR/NF-κB pathway.

Notch1 haploinsufficiency in mice accelerates adipogenesis.

Notch signaling has been recognized recently as a key regulator of metabolism. Here, we determined the role of Notch1 in adipogenesis in wild-type (WT) and Notch1 hetero-mutant (N1+/-) mice provided with 12-week normal or high-fat diet. Haploinsufficiency of Notch1 was associated with adipose tissue accumulation despite similar food intake. White adipose tissue (WAT) of N1+/- showed accumulation of adipogenic cells (CD34+CD68+ cells), crown-like structures, and upregulation of cell proliferation markers (cyclin D1 and Ki67). Notch1 expression in WAT reached peak levels in 8-week-old WT mice in parallel with fat accumulation, especially under HF/HS-feeding, whereas such increment was blunted in N1+/- mice. Downstream of Notch1 haploinsufficiency, over-expression of adipogenic factors PPARγ and C/EBPα was noted following down-regulation of downstream transcriptional factors of Notch signaling (Hes-1, Pref-1, and Sox9). Both pharmacological Notch signal inhibition and Notch1 knockdown enhanced adipogenesis of 3T3-L1 preadipocytes. N1+/- mice showed impaired glucose and insulin tolerance with downregulation of IRS-1 and GLUT4 in WAT after high-fat diet. Taken together, our results suggest that haploinsufficiency of Notch1 promotes fat accumulation and adipogenesis and provides a mechanistic link between Notch signaling and development of metabolic syndrome.

Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells.

Introduction: The aim of this study was to determine the role of Notch in indoxyl sulfate (IS)-induced vascular calcification (VC). Materials and methods: VC and expression of Notch-related and osteogenic molecules were examined in Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH+IS). The effects of IS on expression of Notch receptors, apoptotic activity, and calcification were examined in cultured aortic smooth muscle cells (SMCs). Results: Medial calcification was noted only in aortas and coronary arteries of DH+IS rats. Notch1, Notch3, and Hes-1 were expressed in aortic SMCs of all rats, but only weakly in the central areas of the media and around the calcified lesions in DH+IS rats. RT-PCR and western blotting of DH+IS rat aortas showed downregulation of Notch ligands, Notch1 and Notch3, downstream transcriptional factors, and SM22, and conversely, overexpression of osteogenic markers. Expression of Notch1 and Notch3 in aortic SMCs was highest in incubation under 500 µM IS for 24hrs, and then decreased time- and dose-dependently. Coupled with this decrease, IS increased caspase 3/7 activity and TUNEL-positive aortic SMCs. In addition, pharmacological Notch signal inhibition with DAPT induced apoptosis in aortic SMCs. ZVAD, a caspase inhibitor abrogated IS-induced and DAPT-induced in vitro vascular calcification. Knockdown of Notch1 and Notch3 cooperatively increased expression of osteogenic transcriptional factors and decreased expression of SM22. Conclusion: Our results suggested that IS-induced VC is mediated through suppression of Notch activity in aortic SMCs, induction of osteogenic differentiation and apoptosis.

Reaction Monitoring of Gold Oxides Prepared by an Oxygen-dc Glow Discharge from Gold Films in Various Aqueous Solutions by a Surface Plasmon Resonance-based Optical Waveguide Sensing System and X-ray Photoelectron Spectroscopy.

The chemical properties of thin (∼0.6 nm) gold oxide layers prepared by an oxygen-dc glow discharge from gold films at room temperature in various solvents and solutions were studied using a surface plasmon resonance (SPR)-based optical waveguide sensing system and X-ray photoelectron spectroscopy (XPS). New insights into the stability and reactivity of gold oxides under these conditions were obtained. The O 1s XPS spectra show three oxygen species, comprising components I, II, and III in the gold oxides, and components I and II are present in this order from the top surface of the oxide (component III). The gold oxide is stable in various solvents (water, methanol, ethanol, acetone, acetonitrile, diethyl ether, chloroform, hydrocarbons) for 10 - 30 min at room temperature. The gold oxide decomposes in aqueous 10-2 M solutions of acetaldehyde, hydrochloric acid, and sodium hydroxide during these periods. Gold oxide decomposition in these solutions was monitored by SPR and the decomposition rates were obtained. Decomposition of the gold oxides was confirmed and their surfaces were characterized after decomposition by XPS.

A case of extensive encircling pulmonary vein isolation in a patient with severe scoliosis.

The patient was a 62-year-old man with atrial fibrillation and severe scoliosis. Scoliosis may impair cardiorespiratory function. Enhanced computed tomography (CT) was helpful for the Brockenbrough method. Three-dimensional (3D) mapping also demonstrated clockwise rotation of the heart. We successfully isolated extensive encircling pulmonary vein in this patient using enhanced CT and 3D mapping. The CT venous images revealed appropriate localization of the vein and heart. CT and 3D mapping may ensure a more stable and safer procedure.

Allergic airway hyperresponsiveness, inflammation, and remodeling do not develop in phosphoinositide 3-kinase gamma-deficient mice.

BACKGROUND: Bronchial asthma is characterized by chronic airway inflammation caused by inflammatory cells. Phosphoinositide 3-kinases (PI3Ks) are known to play a prominent role in fundamental cellular responses of various inflammatory cells, including proliferation, differentiation, and cell migration. PI3Ks therefore are expected to have therapeutic potential for asthma. Although some investigations of the involvement between the pathogenesis of asthma and PI3K have been performed, it is unknown whether PI3Kgamma, a PI3K isoform, is involved in the pathogenesis of asthma. OBJECTIVE: We investigated the role of PI3Kgamma in allergen-induced allergic airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling with PI3Kgamma-deficient mice. METHODS: After ovalbumin (OVA) sensitization, wild-type (WT) and PI3Kgamma-deficient mice were exposed to aerosolized OVA 3 days per week for 5 weeks. RESULTS: In OVA-sensitized and OVA-challenged (OVA/OVA) PI3Kgamma-deficient mice, levels of airway inflammation, AHR, and airway remodeling were significantly decreased compared with those in OVA/OVA WT mice. On the other hand, no significant differences were detected in serum OVA-specific IgE and IgG1 levels and CD4/CD8 balance in bronchoalveolar lavage fluid between OVA/OVA WT mice and OVA/OVA PI3Kgamma-deficient mice. To determine in which phase of allergic responses PI3Kgamma plays a role, we transferred splenocytes from OVA-sensitized WT or PI3Kgamma-deficient mice to naive mice of either genotype. Similar increased levels of eosinophils were induced in both WT recipient mice but not in both PI3Kgamma-deficient recipient mice. CONCLUSION: PI3Kgamma might be involved in allergic airway inflammation, AHR, and airway remodeling by regulating the challenge/effector phase of allergic responses.

Activation of eosinophils by lipopolysaccharide-induced monocyte-derived cytokines.

BACKGROUND: Interactions between eosinophils and monocytes after lipopolysaccharide inhalation are yet to be investigated. The mechanism of eosinophil activation induced by lipopolysaccharide in the presence of monocytes was investigated. METHODS: Expression of ICAM-1 and Mac-1 on eosinophils was evaluated after lipopolysaccharide stimulation in the presence of monocytes or monocyte culture supernatants. Cytokines in the supernatant of lipopolysaccharide-stimulated monocytes were measured using a cytokine array. RESULTS: Expression of ICAM-1 and Mac-1 on eosinophils was up-regulated after lipopolysaccharide stimulation in the presence of monocytes or monocyte culture supernatant. Lipopolysaccharide induced secretion of ENA-78, GMCSF, GRO, IL-1 beta, IL-6, IL-10, MCP-1, TNF-alpha and MIP-3 alpha from monocytes. The up-regulation of ICAM-1, but not Mac-1, on eosinophils was attenuated by anti-TNF-alpha neutralizing antibody. CONCLUSIONS: Monocyte-derived TNF-alpha plays an important role in the up-regulation of ICAM-I on eosinophils induced by lipopolysaccharides.

Hepatocyte growth factor suppresses production of reactive oxygen species and release of eosinophil-derived neurotoxin from human eosinophils.

BACKGROUND: Reactive oxygen species (ROS) and eosinophilic granule proteins such as eosinophil-derived neurotoxin (EDN) are known to damage bronchial tissue and cause airway hyperresponsiveness (AHR) in asthma. Hepatocyte growth factor (HGF) regulates various biological activities and is known to be a multifunctional factor. In our previous study, we found that HGF suppressed allergic airway inflammation and AHR in a murine model of asthma. However, there have been few reports regarding the detailed mechanism of the anti-allergic effect of HGF in asthma. In this study, we investigated the potential of recombinant HGF to regulate the production of ROS and the release of EDN from human eosinophils. METHODS: Eosinophils were isolated from subjects with mild eosinophilia by modified CD16-negative selection. We investigated the expression of CD69, an activation marker of eosinophils, on eosinophils, using flow cytometry. Further, ROS production from eosinophils was analyzed using luminol-dependent chemiluminescence, and EDN release was measured by ELISA. RESULTS: Treatment with HGF suppressed interleukin-5-induced upregulation of CD69 expression, ROS production and EDN release from human eosinophils. CONCLUSION: Taken together, these data suggest that in asthma, HGF attenuates allergic airway inflammation and AHR through at least the suppression of ROS production and EDN release from eosinophils.

Co-existence of Mycobacterium tuberculosis and Mycobacterium intracellulare in one sputum sample.

An 81-year-old man was admitted to the hospital with a severe sore throat and a low grade fever. A chest radiograph showed bilateral diffuse reticulonodular shadows. By fluorescent stain for mycobacteria, his sputum smear showed acid-fast bacteria. The initial polymerase chain reaction (PCR) of his sputum revealed Mycobacterium intracellulare (M. intracellulare), but not Mycobacterium tuberculosis (M. tuberculosis). However, a repeat PCR was performed because M. tuberculosis could not be ruled out due to his clinical symptoms and chest imaging. The second PCR detected both M. intracellulare and M. tuberculosis. From the standpoint of infection control, this case illustrates the possibility that M. tuberculosis could be a threat if a second PCR is not done. While PCR is a useful exam for diagnosing M. tuberculosis, it can produce false negative results. Therefore, for diagnosing tuberculosis, particularly in a case such as the present case, a second PCR, which is not normally necessary, should be done.

Anti-allergic inflammatory effects of hepatocyte growth factor.

Hepatocyte growth factor (HGF) is known to influence a number of cell types and regulate various biological activities including cytokine production, cell migration, proliferation and survival. Thus, HGF is now recognized to be a key factor in the prevention and attenuation of disease progression. We have reported that HGF reduces allergic airway inflammation, airway hyperresponsiveness, remodeling and development of Th2 cytokines as well as growth factors such as transforming growth factor-beta in vivo. In vitro, HGF directly attenuates chemotaxis of eosinophils in the absence of Th2 cytokines and modulates mitogen-activated protein kinases, which play an important role in eosinophil migration. In this review, we discuss the physiological role of HGF in allergic inflammation and its mechanism of anti-inflammatory effects, including the regulation of eosinophil functions.

[Eosinophil chemotaxis assay using novel device EZ-TAXIScan].

BACKGROUND: Eosinophils are major effector cells in the pathogenesis of allergic inflammation such as bronchial asthma, and eosinophil migration to sites of inflammation is an important step. To date, several approaches have been developed to study eosinophil chemotaxis. Among them, the Boyden chamber method has been widely used, although this system requires a relatively large number of cells, and it usually provides no longitudinal information. In this study, we investigated real-time eosinophil chemotaxis using EZ-TAXIScan, a novel horizontal microchannel device. METHODS: Eosinophils were isolated from subjects with mild eosinophilia by modified CD16-negative selection. Eosinophil chemotaxis and migration speed induced by various chemoattractants including eotaxin, RANTES, PAF, and prostaglandin (PGD2) were measured by EZ-TAXIScan. We also determined the time course of chemotaxis using Boyden chambers. RESULT: By using EZ-TAXIScan, rapid (a few minutes after stimulation) and fast (20-30 microm/min) eosinophil chemotactic responses were observed by stimulation with PAF or PGD2, although eosinophils stimulated with eotaxin or RANTES showed relatively late (60 minutes after stimulation) and slow (15 microm/min) responses. In contrast, using a Boyden Chamber, the chemotactic responses we tested showed a similar time course peaking at 20-60 min. CONCLUSION: The availability of EZ-TAXIScan for investigation of eosinophil chemotaxis was confirmed. However, it should be noted that EZ-TAXIScan showed a different response to certain chemoattractants compared with the conventional method.

Possible novel receptor for PGD2 on human bronchial epithelial cells.

Prostaglandin D(2) (PGD(2)), a major prostanoid produced by activated mast cells, has long been implicated in allergic diseases. Recent studies have shown that PGD(2) exerts its effects through two different G-protein-coupled receptors (GPCRs), the D-prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on T helper type-2 cells (CRTH2), expressed in various human tissues. The PGD(2)/CRTH2 system mediates the chemotaxis of eosinophils, basophils, and Th2 cells, which are involved in the induction of allergic inflammation. We have reported that normal human bronchial epithelial cells (NHBE) and epithelial cell lines (NCI-H(292)) expressed CRTH2, and PGD(2) induces production of IL-8 and GM-CSF. This review discusses the role of CRTH2/DP on epithelial cells and mentions a possible novel receptor for PGD(2).

[Clinical evaluation after an introduction of our manual for antibiotic use during perioperative period and a notification policy of use of the antibiotics for MRSA].

Abuse of antibiotics results in unfavorable consequences in healthcare associated infection control, such as emergence of multiple-resistant bacteria, and increased medical cost and nosocomial infection. In 2003, we had an outbreak of Methicillin-resistant Staphylococcus aureus (MRSA) in a surgical ward of Akita University Hospital. With the aim of preventing inappropriate use of antibiotics during perioperative period, a guideline for usage of antibiotics, which is made by surgeons themselves, was introduced, and a notification policy of the antibiotic use for MRSA was also applied in 2004. In this study, we evaluated the influence of the antibiotic restriction on changes in antibiotics consumption, prevalence of multiple-drug-resistant pathogens, and their susceptibility to antibiotics. The notification policy reduced the cost and amount of antibiotics and the prevalence of multiple-drug-resistant pathogens, such as MRSA and Pseudomonas aeruginosa. The susceptibility of bacteria, such as Staphylococcus aureus, MRSA, Serratia marcescens and Pseudomonas aeruginosa, to antibiotics showed a remarkable improvement. The introduction of notification policy for usage of antibiotics has a favorable influence on the infection control in hospitals and re education of doctors.

[Physiopathological evaluation of defecatory problems in elderly people using saline enema test and fecoflowmetry].

The population of the aged people is increasing rapidly in developed countries. Bowel care is recognized as an important factor to the wellbeing of the disabled elderly people. To evaluate the physiopathology of defecatory problems in the elderly, we applied saline enema test and fecoflowmetry; that is, pressure fluctuations of the rectum and anal canal were simultaneously recorded during saline infusion in the rectum, and then the saline evacuation curve was recorded in the elderly subjects. The patterns of the pressure fluctuations in the rectum and anal canal were analyzed in saline enema test. In fecoflowmetry, the shape of the evacuation curve and several parameters such as, flow rate and evacuation time were evaluated. The saline volume required to elicit rectal contractions and relaxations of the anal canal were significantly decreased in the patients with the lesions narrowing the spinal canal. The shape of evacuation curve represented the state of defecation in each subject. Subjects without defecatory problems had high flow rates and short evacuation time, while subjects with incontinence and/or constipation had low flow rates and long evacuation time. Furthermore, big and slow periodic pressure fluctuations of the anal canal, so called ultra slow waves, were seen in some patients with severe chronic constipation accompanying megacolon. These findings suggested that the physiopathology of defecatory problems in the elderly is variable and complicated, and that the appropriate treatment for these patients is achieved through appropriate evaluation.

RANTES production from mononuclear cells in response to the specific allergen in asthma patients.

BACKGROUND: Eosinophils are considered to be the major inflammatory cells in asthma. Since regulated on activation, normal T expressed and secreted (RANTES) is a potent chemoattractant for various important inflammatory cells such as eosinophils as well as memory T cells potentially recruiting these cells to an inflamed focus, RANTES has been considered to play a key role in various allergic disorders such as asthma. METHODS: To extend our understanding of the participation of eosinophils and T cells in relation to the production of RANTES in response to the specific allergen in asthma, we examined the production of RANTES from peripheral blood mononuclear cells cultured with specific allergen in atopic asthma patients by a sandwich enzyme-linked immunosorbent assay. RESULTS: It was revealed that mononuclear cells produced RANTES but not eotaxin in response to the specific allergen in asthma. RANTES production from mononuclear cells of asthma patients with eosinophilia was greater than that of asthma patients without eosinophilia. Moreover, in this study, no differences in RANTES production between CD4 negative cells and CD8 negative cells were observed. CONCLUSIONS: Taken together, these findings may suggest that mononuclear cells play a crucial role in the pathogenesis, particular in eosinophil and T lymphocyte recruitment into the inflamed focus of asthma through RANTES production in response to the specific allergen.

The synthetic PPARgamma agonist troglitazone inhibits IL-5-induced CD69 upregulation and eosinophil-derived neurotoxin release from eosinophils.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates lipid metabolism. Recently, PPARgamma was reported to be a negative regulator in the immune system. Eosinophils also express PPARgamma, however, the role of PPARgamma in eosinophil functions is not well understood. Surface expression of CD69 and eosinophil-derived neurotoxin (EDN) release are well-known activation markers of eosinophils. We investigated the effect of a PPARgamma agonist on human eosinophil functions such as IL-5-induced CD69 surface expression and EDN release. IL-5 significantly induced eosinophil CD69 surface expression analyzed using flow cytometry and EDN release measured by ELISA. IL-5-induced eosinophil CD69 surface expression and EDN release were significantly inhibited by the synthetic PPARgamma agonist troglitazone, and these effects were reversed by a PPARgamma antagonist. The PPARgamma agonist troglitazone has a potent inhibitory effect on activation and degranulation of eosinophils, and it may be a therapeutic modality for the treatment of allergic diseases.

Activation of eosinophils by rice-husk dust exposure: a possible mechanism for the aggravation of asthma during rice harvest.

Grain dust and other irritants affect the airway of allergic patients in rice-growing area during the harvest. The aim of this study was to elucidate the mechanism of airway hypersensitivity in rice-growing areas during the harvest. Firstly, the effect of rice-husk dust on eosinophil activation was studied. Secondary, the concentration of lipopolysaccharides (LPS), a potent activator of inflammatory cells, in rice-husk dust was measured. Since it is possible for LPS, a component of gram-negative bacterial cell wall, to adhere to the particle of smoke generated from rice-husk dust, LPS contained in the smoke was also measured. Furthermore, chemical irritants contained in the smoke generated from the rice-husk dust were analyzed. Microscopically, the dust contained fine thorns dropped off from the outer sheath of the rice, and irritated the skin, throat and eyes. The grain dust extract increased the expressions of eosinophil activation markers. These up-regulatory effects were largely dependent on LPS. The smoke contained LPS and several chemical irritants such as formaldehyde and acetaldehyde. Rice-husk dust and its smoke, hazardous air pollutants, probably play a major role in the aggravation of airway diseases in agricultural areas.

Effect of ketotifen on the production of reactive oxygen species from human eosinophils primed by eotaxin.

Ketotifen is an antiallergic drug and may have direct inhibitory effects on eosinophils. To investigate the anti-eosinophilic effect of ketotifen, we examined the effect of ketotifen on the production of reactive oxygen species (ROS) from eotaxin-primed human eosinophils. Ketotifen at 10(-10)-10(-6) mol/l significantly reduced the production of ROS evoked by A23187 from eosinophils primed by eotaxin. In contrast, ketotifen at 10(-5) mol/l significantly augmented the production in the absence of eotaxin. We demonstrated that appropriate concentrations of ketotifen may have direct inhibitory effects on eosinophil oxidative metabolism primed by eotaxin. Ketotifen may contribute to the treatment of allergic disease through its anti-eosinophilic effects.