Vocal Fold Vibration of the Whistle Register Observed by High-Speed Digital Imaging.

INTRODUCTION: Singers use a whistle register to sing at a fundamental frequency above 1000 Hz. In previous studies, vocal fold vibrations with or without complete closure and partial vocal fold vibrations were observed depending on the subject. However, the production mechanism of the whistle register is not yet clearly understood because of the limitations of the imaging device for the glottis and subjects. OBJECTIVES: This study aims to examine vocal fold vibrations in a whistle register. METHODS: The dynamic behavior of the glottis was recorded for six singers (four females and two males) using a high-speed digital imaging device with a frame rate above 10,000 fps. Audio signals were recorded simultaneously. The data were analyzed in the form of topography, glottal area waveforms, spectrograms, and phonovibrography to examine spatiotemporal patterns of glottal motion. RESULTS: The vibratory motion of the vocal folds was classified into six patterns. The first pattern was the entire vocal fold vibration with complete closure during the closed phase. The second to fifth was the entire vocal fold vibration without complete closure, where a gap was observed for the full length of the vocal folds for the second, at the posterior part of the glottis for the third, at the anterior for the fourth, and at both ends for the fifth. In the sixth pattern, the vocal folds vibrated partially. Our results support the previous findings on the vibration of the vocal folds. In addition, we identified novel vibratory patterns in the vocal folds. CONCLUSION: We conclude that the production of the whistle register is not just an extension of the falsetto register to the higher fundamental-frequency region; rather, the production mechanism of the whistle register appeared to be diverse as a means of vocalization.

Obesity and eosinophilic inflammation: does leptin play a role.

It has been pointed out that obesity is a risk factor for, and is involved in the exacerbation of asthma. Mounting evidence about adipose tissue-derived proteins (adipokines) gave rise to the current understanding of obesity as a systemic inflammatory disorder. In this review, we summarized the involvement of leptin, focusing on eosinophil functions. Several studies have indicated that leptin can restrain eosinophil apoptosis, enhance migration, increase adhesion molecules and induce cytokine production. Since leptin also acts on a variety of immune cells related to allergic response, increased leptin in obese individuals potentially explains the mechanism by which obesity leads to an exacerbation of asthma. Further studies targeting adipokines will delineate the association between obesity and eosinophil-associated diseases.

The opposing role of two prostaglandin D2 receptors, DP and CRTH2, in human eosinophil migration.

BACKGROUND: Prostaglandin D2 (PGD2) regulates various immunological responses via two distinct PGD2 receptors, prostaglandin D receptor (DP), and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Recent studies have demonstrated that PGD2 induces the migration of eosinophils through CRTH2. Although human eosinophils express both DP and CRTH2, it is unclear whether the function of DP is involved in eosinophil migration. OBJECTIVE: In this study, we investigated the roles of DP and CRTH2 in eosinophil migration by using selective agonists and antagonists. METHODS: Eosinophils were isolated from human subjects with mild eosinophilia by modified CD16-negative selection. After stimulation with or without DP receptor agonist, eosinophil migration was measured by Boyden chamber. The effect of DP agonists on CRTH2-induced eosinophil migration was studied in terms of CRTH2 expression, Ca2+ mobilization, ERK/MAPK phosphorylation, and cyclic AMP (cAMP) production. RESULTS: Treatment with DP agonists inhibited CRTH2-induced chemotaxis of eosinophils. Furthermore, we showed that DP agonists enhanced cAMP production in CRTH2 agonist stimulation without increasing CRTH2 expression. DP mediates eosinophils through the elevation of intracellular cAMP production but does not change CRTH2 expression. CONCLUSION: Taken together, the balance between DP and CRTH2 could influence the degree of PGD2-induced eosinophil migration and DP agonist might regulate eosinophil activation.

Leptin has a priming effect on eotaxin-induced human eosinophil chemotaxis.

BACKGROUND: Tissue eosinophilia is one of the hallmarks of allergic diseases and Th2-type immune responses including asthma. Systemic inflammation caused by adipose tissue in obesity via production of adipokines such as leptin has been attracting attention recently as a contributor to exacerbation of allergic immune reactions. In this study, we examined whether leptin might affect eosinophil chemotactic responses. METHODS: Peripheral blood eosinophils were purified, and the effect of leptin on eosinophil migration was investigated using in vitro systems. RESULTS: High concentrations of leptin induced eosinophil chemotaxis and rapid phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase but not calcium mobilization. We also found that pretreatment of eosinophils with physiological concentrations of leptin amplified the chemotactic responses to eotaxin. This leptin-primed chemotaxis appears to be associated with increased calcium mobilization but not with ERK1/2 and p38 pathways. CONCLUSIONS: These results indicate that leptin has both direct and indirect effects on eosinophil chemotaxis and intracellular signaling. In physiological settings, leptin may maintain eosinophil accumulation at allergic inflammatory foci.

The pathophysiological roles of PI3Ks and therapeutic potential of selective inhibitors in allergic inflammation.

Phosphoinositide 3-kinases (PI3Ks) are known to be involved in a variety of cellular responses such as cell survival, proliferation, differentiation and cell migration. Recently, PI3Ks have been associated with the pathogenesis of asthma because various immune cells regulate allergic responses. Among the three classes of PI3Ks, the roles of PI3K gamma and PI3K delta in allergic responses have attracted particular attention. In a previous report, allergic airway hyperresponsiveness (AHR), inflammation and airway remodeling in an ovalbumin-induced asthma model were decreased in PI3K gamma-deficient mice compared with wild-type mice. In addition, AHR and inflammation were attenuated by administration of a selective PI3K delta inhibitor in a murine model of asthma. These results indicate that PI3K gamma and PI3K delta may be new therapeutic targets for asthma. However, PI3K gamma and PI3K delta may differ in terms of the mechanism of regulation. In this review, we focus on the roles of PI3K gamma and PI3K delta in the pathogenesis of asthma and discuss the mechanistic differences between PI3K gamma and PI3K delta.

Regulation of peroxisome proliferator-activated receptor-gamma expression in human eosinophils by estradiol.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates not only adipogenesis but also immune reaction. We previously demonstrated that human eosinophils expressed functional PPARgamma, although the modulator of PPARgamma expression is less well understood. Because clinical studies have shown that the efficacy of PPARgamma agonists as insulin sensitizers is stronger in women than in men, we investigated whether sex hormones caused any changes in eosinophil PPARgamma expression levels. METHODS: First, purified human peripheral blood eosinophils were cultured with 17beta-estradiol for 24 h, followed by PPARgamma measurement using a flow cytometer. Next, eosinophil PPARgamma expression and serum estradiol were studied in 10 healthy women during the menstrual and follicular phases to identify the physiological significance of estradiol. Eosinophil PPARgamma expression was also compared in 22 men, 21 non-pregnant women, and 15 pregnant women. RESULTS: We observed that PPARgamma protein expression in eosinophils was significantly enhanced by 10(-6)M 17beta-estradiol. Although serum estradiol concentration was increased during the follicular phase, PPARgamma expression levels were not affected by the menstrual cycle. In addition, no significant differences in PPARgamma expression were observed in terms of sex and pregnancy. CONCLUSIONS: These findings suggest that estradiol potentially upregulates eosinophil PPARgamma expression in vitro, although some other mechanisms might be involved in its regulation in vivo.

15-Deoxy-Delta(12,14)-prostaglandin J2 induces IL-8 and GM-CSF in a human airway epithelial cell line (NCI-H292).

BACKGROUND: 15-Deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), a major prostanoid metabolized from prostaglandin D2 (PGD2), plays an important role in various biological processes including inflammatory responses. 15d-PGJ2 exerts its effects through two major receptors, chemoattractant receptor- homologous molecule expressed on Th2 cells (CRTH2) and peroxisome proliferator-activated receptor-gamma (PPARgamma). The 15d-PGJ2/PPARgamma system, in particular, regulates numerous biological processes including adipogenesis, apoptosis, and inflammation. Although our studies have shown that PGD2 (metabolic precursor of 15d-PGJ2) induces IL-8 and GM-CSF production, the role of 15d-PGJ2 (metabolite of PGD2) is unknown in human bronchial epithelial cells. In this study, we investigated the function of 15d-PGJ2 on a human airway epithelial cell line: NCI-H292. METHOD: NCI-H292 cells were cultured in the presence of various stimulants. The resulting supernatants were used for ELISA analysis. RESULTS: We demonstrated that 15d-PGJ2 induced production of IL-8 and GM-CSF from NCI-H292. 13,14-Dihydro-15-keto-PGD2 (DK-PGD2) (CRTH2 agonist) and troglitazone (PPARgamma agonist) failed to increase the production of these cytokines. Pretreatment with ramatroban (CRTH2 antagonist) and GW9662 (PPARgamma antagonist) did not reduce the production of these cytokines. 15d-PGJ2 activated the ERK1/2 signaling pathway in a time-dependent manner. CONCLUSION: These data showed that 15d-PGJ2 is a potent inducer of IL-8 and GM-CSF production through ERK1/2 kinase activation, but this is independent of CRTH2 or PPARgamma activation.

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.

Retinoic acids are potent inhibitors of spontaneous human eosinophil apoptosis.

Retinoic acids (RAs), which are active metabolites of vitamin A, are known to enhance Th2-type immune responses in vitro, but the role of RAs in allergic inflammatory cells remains unclear. In this study, we demonstrated that purified peripheral blood eosinophils expressed nuclear receptors for RAs at the mRNA and protein levels. Eosinophils cultured with all-trans RA (ATRA) and 9-cis-RA showed dramatically induced cell survival and nuclear hypersegmentation, and the efficacy of RAs (10(-6)M) was similar to that of IL-5 (1 ng/ml), the most critical cytokine for eosinophil activation. Pharmacological manipulation with receptor-specific agonists and antagonists indicated that the antiapoptotic effect of RAs was mediated through ligand-dependent activation of both retinoid acid receptors and retinoid X receptors (mainly retinoid acid receptors). Furthermore, using a gene microarray and a cytokine Ab array, we discovered that RAs induced vascular endothelial growth factor, M-CSF, and MCP-1 secretion, although they were not involved in eosinophil survival. RA-induced eosinophil survival appears to be associated with down-regulation of caspase 3 and inhibition of its enzymatic activity. These findings indicate an important role of RAs in homeostasis of granulocytes and provide further insight into the cellular and molecular pathogenesis of allergic reactions.

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.

The synthetic PPARgamma agonist troglitazone inhibits eotaxin-enhanced eosinophil adhesion to ICAM-1-coated plates.

Accumulation and activation of eosinophils in tissue are critical events in the allergic inflammatory response and adhesion molecules play important roles in this process. We previously demonstrated that human eosinophils expressed a nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), and that stimulation with a PPARgamma agonist attenuated cytokine/chemokine-induced eosinophil activation, such as survival, chemotaxis and degranulation. In the present study, we investigated the effect of troglitazone, a synthetic PPARgamma agonist, on adherence to intercellular adhesion molecule-1 (ICAM-1). Eosinophils were purified from human peripheral blood, and the functional adherence to recombinant soluble ICAM-1-coated plates was examined. We found that in the presence of eotaxin, troglitazone inhibited eosinophil adherence in a concentration-dependent manner. This novel activity appears to be associated with modulation of qualitative change of integrins in response to eotaxin, because quantitative reduction of CD11a, CD11b and CD18 expression by troglitazone was not observed using flow cytometry. The PPARgamma agonist troglitazone has a potent inhibitory effect on eosinophil adhesion to ICAM-1, and this may be a therapeutic modality for the treatment of eosinophil-related diseases including bronchial asthma.

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.

[Cross-talk between arteriosclerotic diseases and allergic inflammation].

Arteriosclerosis and allergy are increasingly common diseases. The pathogeneses are both based on changes in lifestyle and chronic inflammation. Herein we discuss cross talk between the two seemingly unrelated diseases, focusing on obesity and various related molecules, including adipokines, peroxisome proliferator-activated receptors (PPARs), C reactive protein (CRP), and soluble adhesion molecules.

Anti- and proinflammatory effects of 15-deoxy-delta-prostaglandin J2(15d-PGJ2) on human eosinophil functions.

The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is recognized as a potent lipid mediator that is derived from PGD(2), which is produced abundantly in allergic inflammatory sites. It is now established that 15d-PGJ(2) negatively regulates cellular functions through its intracellular targets such as peroxisome proliferator-activated receptor-gamma (PPARgamma). However, recent studies revealed that 15d-PGJ(2) appears to possess not only anti-inflammatory activities but also a proinflammatory potential depending on its concentration and the activation state of the target cell. For instance, at low concentrations, 15d-PGJ(2) enhances eotaxin-induced chemotaxis, shape change, and actin reorganization in eosinophils through its ligation with PPARgamma. Moreover, 15d-PGJ(2) itself is a potent chemoattractant, and it induces calcium mobilization, and up-regulates CD11b expression through its membrane receptor--chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Conversely, at high concentrations, 15d-PGJ(2) inhibits eosinophil survival by inducing apoptosis in a PPARgamma-independent manner. Here, we discuss the pathophysiological roles of 15d-PGJ(2) that could act as a paracrine, autocrine, and intracrine substance to regulate eosinophil functions.

Procaterol upregulates peroxisome proliferator-activated receptor-gamma expression in human eosinophils.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates immune reaction. We have previously demonstrated that human eosinophils express PPARgamma and that stimulation with a synthetic agonist for PPARgamma attenuated the factor-induced eosinophil activations. However, the modulator of PPARgamma expression in eosinophils has not yet been studied. In this study, we investigated the effect of procaterol, the synthetic beta2-adrenoceptor agonist widely used as bronchodilators in asthma, on the PPARgamma expression in eosinophils. Purified human peripheral blood eosinophil and the eosinophilic cell line EoL-1 were cultured with procaterol. This was followed by PPARgamma measurement using flow cytometer and quantitative real-time RT-PCR. We observed that PPARgamma was constitutively expressed by EoL-1 and the purified eosinophils and that the therapeutic concentration (10(-9)M) of procaterol markedly enhanced PPARgamma protein expression, which was reversed by the selective beta2-adrenoceptor antagonist ICI-118551. The PPARgamma mRNA expression in EoL-1 and eosinophils was also induced by procaterol. These findings suggest that procaterol could modulate the eosinophil function by increasing the expression of PPARgamma.

Theophylline and dexamethasone induce peroxisome proliferator-activated receptor-gamma expression in human eosinophils.

Eosinophils are major effector cells in allergic diseases including asthma. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates immune reaction. We have previously demonstrated that human eosinophils express PPARgamma and that stimulation with a synthetic agonist for PPARgamma attenuated the factor-induced eosinophil survival and chemotaxis. However, the modulator of the eosinophil PPARgamma expression has not yet been studied. In this study, we investigated the effect of theophylline and dexamethasone (widely used drugs in the treatment of asthma) on PPARgamma expression in eosinophils. Purified human peripheral blood eosinophils were cultured, and therapeutic concentrations of theophylline and dexamethasone were added. Subsequently, PPARgamma was measured using quantitative real-time RT-PCR and flow cytometry. Theophylline and dexamethasone markedly enhanced both mRNA and protein levels of PPARgamma. These findings suggest that the increase in PPARgamma expression on eosinophils may play a role in the anti-inflammatory effects of theophylline and dexamethasone.