[Steroid resistance of severe asthma - mechanisms and therapeutic targets].

Asthma therapy in general has improved a lot in recent years, but it is still a major problem that severe asthma, which accounts for 10 to 20%, still suffers from strong symptoms on a daily basis despite all therapeutic agents used in combination. American SARP and European ENFUMOSA started in 2000 to advance pathophysiological insights of severe asthma. Clinical usage of antibodies and inhibitors against IgE, TNF, IL-5, IL-4, IL-13, and TSLP are also accumulating. Some of these molecular-targeted drugs improve respiratory function and reduce acute exacerbations in patients with severe asthma. Until now, cytokines have been assumed to be involved in chronic inflammation, but it is also interesting to elucidate the pathways of how cytokines are involved in respiratory function and acute exacerbations. We registered approximately 100 steroid-dependent asthma patients in Japan. Although long-lasting poor control of the disease was considered the cause of severe asthma in the past, steroid dependence in one third of the cases occurred within 2-3 years after the onset. Steroid resistance seems a key process from the early stage of the disease. Steroid resistance of T cell level was induced by extracellular co-stimulation and cytokine signals. The inhibition may improve steroid sensitivity and treat steroid-resistant asthma. Therefore, we established a steroid-resistant asthma model for the first time by transferring steroid resistant T cell clones, and analyzed the steroid sensitivity recovery effect of CTLA4-Ig. In addition, a multicenter, double-blind, placebo-controlled exploratory trial was performed as a POC study investigating the efficacy of abatacept in treatment-resistant severe asthma. Elucidation of the pathophysiology and mechanism by which steroids do not work is expected to be a breakthrough for the prevention and treatment of severe asthma.

[Usefulness of an anti-mouse cerebellar tissue-derived antigen antibody test in predicting immunotherapy efficacy in patients with idiopathic cerebellar ataxia].

BACKGROUND: Autoimmune cerebellar ataxia (AICA) is a general term for diseases in which the cerebellum is damaged by an autoimmune mechanism. For the diagnosis of the AICA, anti-thyroid antibodies (anti-thyroid peroxidase antibody and anti-thyroglobulin antibody), anti-glutamic acid decarboxylase (GAD) antibodies, and anti-gliadin antibodies are measured. Immunotherapy is known to be effective for AICA, but some patients with effective immunotherapy lack autoantibodies associated with cerebellar ataxia. The purpose of this study was to clarify whether the effectiveness of immunotherapy in patients with suspected AICA could be predicted by anti-mouse cerebellar tissue-derived antigen antibody tests. METHODS: This study was conducted on 25 patients with idiopathic cerebellar ataxia (excluding multiple system atrophy, hereditary spinocerebellar degeneration, cancer-bearing patients, and patients taking phenytoin) who received immunotherapy from 2005 to 2016 at Tokyo Medical University Hachioji Medical Center. The patients were suspected of having AICA because they were positive for cerebellar ataxia-related autoantibodies (anti-thyroid antibody, anti-GAD antibody, anti-gliadin antibody, or anti-transglutaminase 6 antibody) or other autoantibodies. Antibodies that bind to mouse cerebellar tissue-derived antigens were defined as "anti-mouse cerebellar tissue-derived antigen antibodies" in this study, and their IgG-class antibodies were comprehensively measured using a slot blot. RESULTS: Anti-mouse cerebellar tissue-derived antigen antibody test results were correlated with immunotherapy efficacy. Furthermore, the combination of anti-mouse cerebellar tissue-derived antigen and anti-GAD antibody tests could predict the effectiveness of immunotherapy with 83% sensitivity and 100% specificity, while the combination of the anti-mouse cerebellar tissue-derived antigen, anti-GAD, and anti-gliadin (IgA class) antibody tests could predict the effectiveness of immunotherapy with 94% sensitivity and 86% specificity. CONCLUSION: Anti-mouse cerebellar tissue-derived antigen antibody tests could help to provide useful information for immunotherapy administration to patients with idiopathic cerebellar ataxia suspected to be AICA.

Syntaxin 17 promotes lipid droplet formation by regulating the distribution of acyl-CoA synthetase 3.

Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. In the present study, we show that syntaxin 17 (Stx17), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein whose expression in the liver is regulated by diet, participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase (ACSL)3, a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum (ER) to LDs during LD formation. Stx17 interacts with ACSL3, but not with LD formation-unrelated ACSL1 or ACSL4, through its SNARE domain. The interaction occurs at the ER-mitochondria interface and depends on the active site occupancy of ACSL3. Depletion of Stx17 impairs ACSL3 redistribution to nascent LDs. The defect in LD maturation due to Stx17 knockdown can be compensated for by ACSL3 overexpression, suggesting that Stx17 increases the efficiency of ACSL3 redistribution to LDs. Moreover, we show that the interaction between Stx17 and ACSL3 during LD maturation may be regulated by synaptosomal-associated protein of 23 kDa.

Chronic high-fat feeding impairs adaptive induction of mitochondrial fatty acid combustion-associated proteins in brown adipose tissue of mice.

Since brown adipose tissue (BAT) is involved in thermogenesis using fatty acids as a fuel, BAT activation is a potential strategy for treating obesity and diabetes. However, whether BAT fatty acid combusting capacity is preserved in these conditions has remained unclear. We therefore evaluated expression levels of fatty acid oxidation-associated enzymes and uncoupling protein 1 (Ucp1) in BAT by western blot using a diet-induced obesity C57BL/6J mouse model. In C57BL/6J mice fed a high-fat diet (HFD) over 2-4 weeks, carnitine palmitoyltransferase 2 (Cpt2), acyl-CoA thioesterase (Acot) 2, Acot11 and Ucp1 levels were significantly increased compared with baseline and control low-fat diet (LFD)-fed mice. Similar results were obtained in other mouse strains, including ddY, ICR and KK-Ay, but the magnitudes of the increase in Ucp1 level were much smaller than in C57BL/6J mice, with decreased Acot11 levels after HFD-feeding. In C57BL/6J mice, increased levels of these mitochondrial proteins declined to near baseline levels after a longer-term HFD-feeding (20 weeks), concurrent with the accumulation of unilocular, large lipid droplets in brown adipocytes. Extramitochondrial Acot11 and acyl-CoA oxidase remained elevated. Treatment of mice with Wy-14,643 also increased these proteins, but was less effective than 4 week-HFD, suggesting that mechanisms other than peroxisome proliferator-activated receptor α were also involved in the upregulation. These results suggest that BAT enhances its fatty acid combusting capacity in response to fat overload, however profound obesity deprives BAT of the responsiveness to fat, possibly via mitochondrial alteration.

Detection and counting of a submicrometer particle in liquid flow by self-mixing microchip Yb:YAG laser velocimetry.

We observed intermittent modulation by scattered light from a single submicrometer particle moving in the flow channel using a self-mixing microchip Yb:YAG laser Doppler velocimeter (LDV) under lateral beam access. The Doppler-shift frequency chirping (i.e., velocity change) was identified in accordance with a particle passage through the beam focus. Single particle counting, which obeys the Poisson distribution, was performed successfully over a long period of time. The experimental results have been reproduced by a numerical simulation. The LDV signal was increased over 20 dB for a 202-nm particle without chirping by collinear beam access with the laser beam axis aligned along the flow direction.

Identification of acyl-CoA thioesterase in mouse mesenteric lymph nodes.

Acyl-CoA thioesterases (ACOTs) are a group of enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA, with the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, ACOT7, is expressed at a significant level in the mesenteric lymph nodes (MLNs) of mice. While crude preparations of the mesenteric visceral fat contained significant levels of palmitoyl-CoA thioesterase activity, enzyme activity was concentrated 6.9-fold in MLNs compared with the residual adipose portion after excision of MLNs. When MLN homogenates were centrifuged, 82% of the enzyme activity was recovered in the cytosolic fraction, concomitant with almost exclusive recovery of ACOT7. Immunoprecipitation using anti-ACOT7 antibody estimated that 87% of enzyme activity in the homogenates was accounted for by ACOT7. On MLN sections, the germinal centers of secondary lymphoid follicles were immunostained with the antibody. In MLNs of mice fasted for 16 h, ACOT7 levels were induced 1.8-fold, which reflected a 1.5-fold increase in enzyme activity. These findings suggest that ACOT7 may be involved in dietary intake-associated responses in fatty acid metabolism in MLNs.

Expression and distribution of acyl-CoA thioesterases in the white adipose tissue of rats.

Acyl-CoA thioesterases (Acots) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and coenzyme A, and have the potential to regulate the intracellular levels of these molecules. In this study, we show that a cytosolic isoform, Acot1, is expressed and distributed in immature adipocytes located in the perivascular region of the white adipose tissue (WAT) of rats. Immunoblot analyses detected Acot1 in all of the WATs examined, while immunohistochemistry revealed positively stained layered structures surrounding the adventitia of blood vessels in the subcutaneous WAT. When the subcutaneous WAT was digested with collagenase and centrifuged, Acot1 was recovered in the stromal vascular fraction (SVF), and not in the large mature adipocytes. In the SVF, undigested cells attached to short tubular fragments of blood vessels showed positive immunostaining, as well as a proportion of the dispersed cells. These fibroblast-like cells contained fine particulate lipid droplets, stained by oil-red O dye, in their cytoplasm, or expressed fatty acid-binding protein 4, an adipocyte marker. After induction of adipocyte differentiation following a 15-day preculture without insulin, the dedifferentiated cells showed increased Acot1 expression with a diffuse distribution throughout the cytosol. These findings suggest that Acot1 expression is transiently upregulated at an early stage of adipocyte maturation, possibly to maintain cytosolic acyl-CoAs below a certain level until the cells acquire their full capability for fat storage.

Murine T cell-derived contractile activity for bronchial smooth muscle cells.

BACKGROUND: Adoptive transfer of helper T (Th) cells conferred a late asthmatic response upon antigen challenge. A possible production of a contractile activity for bronchial smooth muscle (BSM) by activated Th cells was examined. METHOD: Murine Th clones were stimulated with immobilized anti-CD3 antibody in the presence or absence of anti-CD28 antibody. Culture supernatants were dialyzed and then applied to the collagen gels containing cultured human BSM cells. RESULTS: Culture supernatants of activated but not resting murine Th clones that conferred a late asthmatic response, induced the contraction of human BSM cell-containing collagen gels. CONCLUSION: Activated Th cells produce a contractile activity for BSM in vitro.

Analysis of molecular dynamics of colloidal particles in transported dilute samples by self-mixing laser Doppler velocimetry.

Colloidal particles in a liquid medium are transported with constant velocity, and dynamic light scattering experiments are performed on the samples by self-mixing laser Doppler velocimetry. The power spectrum of the modulated wave induced by the motion of the colloidal particles cannot be described by the well-known formula for flowing Brownian motion systems, i.e., a combination of Doppler shift, diffusion, and translation. Rather, the power spectrum was found to be described by the q-Gaussian distribution function. The molecular mechanism resulting in this anomalous line shape of the power spectrum is attributed to the anomalous molecular dynamics of colloidal particles in transported dilute samples, which satisfy a nonlinear Langevin equation.

Hypoglycemic effects of clove (Syzygium aromaticum flower buds) on genetically diabetic KK-Ay mice and identification of the active ingredients.

Clove (Syzygium aromaticum flower buds) EtOH extract significantly suppressed an increase in blood glucose level in type 2 diabetic KK-A(y) mice. In-vitro evaluation showed the extract had human peroxisome proliferator-activated receptor (PPAR)-γ ligand-binding activity in a GAL4-PPAR-γ chimera assay. Bioassay-guided fractionation of the EtOH extract resulted in the isolation of eight compounds, of which dehydrodieugenol (2) and dehydrodieugenol B (3) had potent PPAR-γ ligand-binding activities, whereas oleanolic acid (4), a major constituent in the EtOH extract, had moderate activity. Furthermore, 2 and 3 were shown to stimulate 3T3-L1 preadipocyte differentiation through PPAR-γ activation. These results indicate that clove has potential as a functional food ingredient for the prevention of type 2 diabetes and that 2-4 mainly contribute to its hypoglycemic effects via PPAR-γ activation.

CD44 is critical for airway accumulation of antigen-specific Th2, but not Th1, cells induced by antigen challenge in mice.

CD44 is a cell adhesion molecule involved in lymphocyte infiltration of inflamed tissues. We previously demonstrated that CD44 plays an important role in the development of airway inflammation in a murine model of allergic asthma. In this study, we investigated the role of CD44 expressed on CD4(+) T cells in the accumulation of T-helper type 2 (Th2) cells in the airway using CD44-deficient mice and anti-CD44 monoclonal antibodies. Antigen-induced Th2-mediated airway inflammation and airway hyperresponsiveness (AHR) in sensitized mice were reduced by CD44-deficiency. These asthmatic responses induced by the transfer of antigen-sensitized splenic CD4(+) T cells from CD44-deficient mice were weaker than those from WT mice. Lack of CD44 failed to induce AHR by antigen challenge. Expression level and hyaluronic acid receptor activity of CD44, as well as Neu1 sialidase expression on antigen-specific Th2 cells, were higher than those on antigen-specific Th1 cells. Anti-CD44 antibody preferentially suppressed the accumulation of those Th2 cells in the airway induced by antigen challenge. Our findings indicate that CD44 expressed on CD4(+) T cells plays a critical role in the accumulation of antigen-specific Th2 cells, but not Th1 cells, in the airway and in the development of AHR induced by antigen challenge.

Comparative analysis of steroid sensitivity of T helper cells in vitro and in vivo.

BACKGROUND: Glucocorticoid (GC) action on asthma has been partly explained by the inhibition of T cell activation. We analyzed the steroid sensitivity of ovalbumin (OVA) reactive helper T (Th) cell clones both in vitro and in vivo. METHOD: For in vitro experiments, Th clones were cultured with antigen-presenting cells, OVA, and various concentrations of dexamethasone (DEX). The proliferative response of each Th clone was measured by (3)H-thymidine uptake. For in vivo experiments, unprimed BALB/c mice were transferred with Th clones, challenged with OVA, and administered DEX subcutaneously. The number of infiltrating cells in bronchoalveolar lavage fluid (BALF) was measured. RESULTS: Six Th clones were classified as steroid-sensitive or steroid-resistant clones in terms of the effects of GC on the proliferative responses analyzedin vitro. Airway infiltration of eosinophils and lymphocytes of mice transferred with steroid-sensitive clones were effectively inhibited by the administration of DEX. In contrast, those of mice transferred with steroid-resistant clones were not significantly inhibited by DEX; the number of eosinophils in the BALF of mice transferred with 1 steroid-resistant clone, i.e. T5-1, was only partially reduced. CONCLUSION: The steroid sensitivity of Th clones measured in vitro was consistent with that of an adoptively transferred asthma model measuredin vivo. Steroid-sensitive and resistant asthma models seem valuable for understanding the mechanisms of steroid resistance in severe asthma.

Sustained downregulation of YY1-associated protein-related protein gene expression in rat hippocampus induced by repeated electroconvulsive shock.

YY1AP-related protein (YARP) is a structural homolog of YY1-associated protein (YY1AP), which has a YY1-binding domain. During perinatal development, YARP mRNA expression is increased at a late stage of embryonic neurogenesis. It is not known whether YARP expression is regulated during adult neurogenesis. Electroconvulsive shock (ECS), a model for a highly effective depression treatment, is known to induce hippocampal neurogenesis after repeated treatment, so we employed ECS to measure the expression of YARP mRNA. Northern blots revealed significantly decreased expression of the YARP gene after repeated ECS but not single ECS. In situ hybridization clearly demonstrated a reduction of YARP mRNA expression in the CA (CA1, CA2, and CA3) subfields. Although clonic-tonic seizure was induced not only by ECS but also by injection of kainic acid to the striatum, the regulation of YARP mRNA expression was different between ECS and kainic acid. YARP mRNA was decreased only by the ECS method, suggesting that YARP expression is different at embryonic and adult neurogenic stage.

Prevention of allergic asthma by vaccination with transgenic rice seed expressing mite allergen: induction of allergen-specific oral tolerance without bystander suppression.

This study tested the feasibility of oral immunotherapy for bronchial asthma using a newly developed subunit vaccine in which a fragment (p45-145) of mite allergen (Der p 1) containing immunodominant human and mouse T cell epitopes was encapsulated in endoplasmic reticulum-derived protein bodies of transgenic (Tg) rice seed. Allergen-specific serum immunoglobulin responses, T cell proliferation, Th1/Th2 cytokine production, airway inflammatory cell infiltration, bronchial hyper-responsiveness (BHR) and lung histology were investigated in allergen-immunized and -challenged mice. Prophylactic oral vaccination with the Tg rice seeds clearly reduced the serum levels of allergen-specific IgE and IgG. Allergen-induced CD4(+) T cell proliferation and production of Th2 cytokines in vitro, infiltration of eosinophils, neutrophils and mononuclear cells into the airways and BHR were also inhibited by oral vaccination. The effects of the vaccine were antigen-specific immune response because the levels of specific IgE and IgG in mice immunized with Der f 2 or ovalbumin were not significantly suppressed by oral vaccination with the Der p 1 expressing Tg rice. Thus, the vaccine does not induce nonspecific bystander suppression, which has been a problem with many oral tolerance regimens. These results suggest that our novel vaccine strategy is a promising approach for allergen-specific oral immunotherapy against allergic diseases including bronchial asthma.

Neuronal expression, cytosolic localization, and developmental regulation of the organic solute carrier partner 1 in the mouse brain.

Organic solute carrier partner 1 (OSCP1) is a mammalian, transporter-related protein that is able to facilitate the uptake of structurally diverse organic compounds into the cell when expressed in Xenopus laevis oocytes. This protein has been implicated in testicular handling of organic solutes because its mRNA expression is almost exclusive in the testis. However, in this study, we demonstrated significant expression of OSCP1 protein in mouse brain, the level of which was rather higher than that in the testis, although the corresponding mRNA expression was one-tenth of the testicular level. Immunohistochemistry revealed that OSCP1 was broadly distributed throughout the brain, and various neuronal cells were immunostained, including pyramidal cells in the cerebral cortex and hippocampus. However, there was no evidence of OSCP1 expression in glia. In primary cultures of cerebral cortical neurons, double-labeling immunofluorescence localized OSCP1 to the cytosol throughout the cell body and neurites including peri-synaptic regions. This was consistent with the subcellular fractionation of brain homogenates, in which OSCP1 was mainly recovered after centrifugation both in the cytosolic fraction and the particulate fraction containing synaptosomes. Immunoelectron microscopy of brain sections also demonstrated OSCP1 in the cytosol near synapses. In addition, it was revealed that changes in the expression level of OSCP1 correlated with neuronal maturation during postnatal development of mouse brain. These results indicate that OSCP1 may have a role in the brain indirectly mediating substrate uptake into the neurons in adult animals.

Upregulation of fatty acyl-CoA thioesterases in the heart and skeletal muscle of rats fed a high-fat diet.

In rodent models of diet-induced obesity, prolonged high-fat feeding increases the cellular uptake of fatty acids and causes lipotoxicity in the heart and skeletal muscle, where substrate overload to beta-oxidation generates mitochondrial stress. We examined the hypothesis that, because of its catalytic properties, acyl-CoA thioesterase (ACOT) would counteract these detrimental situations by modulating intracellular acyl-CoA levels. Rats were fed a low- or high-fat diet for up to 20 weeks, and the expressions of ACOT isoforms and fatty acid beta-oxidation enzymes were analyzed by western blotting. The expressions of ACOT1, ACOT2 and ACOT7 proteins in the heart and soleus muscle were significantly increased, by 2.0-7.6-fold, in rats fed the high-fat diet as compared with the low-fat diet group. These effects were accompanied by increases in carnitine palmitoyltransferase and acyl-CoA oxidase expression. However, ACOT was not induced in the extensor digitorum longus muscle or the liver. Subcellular fractionation of heart and soleus muscle homogenates confirmed expression of both the cytosolic and mitochondrial ACOT isoforms. These results underscore the functional relationship between ACOT and fatty acid oxidation, and suggest adaptive upregulation of ACOT to protect against fatty acid oversupply in the heart and skeletal muscle.

Regulated expression of acyl-CoA thioesterases in the differentiation of cultured rat brown adipocytes.

Acyl-CoA thioesterases (ACOTs) are enzymes that catalyze the hydrolysis of fatty acyl-CoAs to free fatty acids and CoA-SH. In this study, we show that the expression profile of the ACOT isoforms changes remarkably during the differentiation of cultured rat brown adipocytes. Immunocytochemistry suggested that cytosolic ACOT1 was present in the preadipocytes, while mitochondrial ACOT2 was additionally expressed as the cells differentiated, concurrent with the accumulation of lipid droplets in the cytoplasm. Western blotting confirmed that, in contrast to ACOT1, the ACOT2 expression level was very low in the preadipocytes. However, after differentiation, the ACOT1 level fell to one-half of the baseline level and ACOT2 increased 18-fold. ACOT2 expression in the differentiated adipocytes was further enhanced by treatment with lipids or troglitazone. These changes in the ACOT2 expression level correlated well with changes in the expression of carnitine palmitoyltransferase 2, a mitochondrial ß-oxidation enzyme. These results indicate that, in differentiating brown adipocytes, cytosolic ACOT1 becomes downregulated as the cellular use of acyl-CoA increases, while mitochondrial ACOT2 is upregulated as the ß-oxidation capacity increases. ACOT isoform expression may be regulated during brown adipocyte differentiation to support the fat storage and combustion characteristics of this cell type.

Eosinophils are required for the induction of bronchial hyperresponsiveness in a Th transfer model of BALB/c background.

BACKGROUND: Helper T (Th) cells are deeply involved in the pathophysiology of bronchial asthma, such as eosinophilic inflammation, bronchial hyperresponsiveness (BHR), airflow limitation and remodeling. It is still unclear whether Th cells contribute to BHR independently of eosinophilic inflammation. The double GATA (dblGATA) site is a high-affinity GATA-binding site in the GATA-1 promoter. dblGATA site-deficient (Delta dblGATA) mice lack eosinophils. METHOD: Ovalbumin (OVA)-reactive Th clones were transferred into Delta dblGATA and wild-type (WT) mice of BALB/c background. The number of eosinophils in the bronchoalveolar lavage fluid (BALF) and bronchial responsiveness to methacholine were examined after OVA challenge. RESULTS: The number of BALF eosinophils was significantly increased in WT mice, but not detectable in Delta dblGATA mice. BHR was also induced in WT mice, but significantly attenuated in Delta dblGATA mice. CONCLUSION: Eosinophils are involved in T-cell-mediated BHR.

Determination of velocity of self-mobile phytoplankton using a self-mixing thin-slice solid-state laser.

We present an analysis of the shoulder-shaped power spectrum observed in the modulated laser output due to feedback light scattered from dynamic changes in self-mobile phytoplankton with flagella in seawater performed using a self-mixing laser Doppler vibrometry system. The power spectrum occasionally has shoulder-shaped broad frequency components superimposed on a Lorentz-type spectrum. This reflects the translational motion of phytoplankton moving across the beam-focus area. The velocity of phytoplankton in the focus area can be obtained by applying a curve fitting procedure to the power spectrum. Moreover, the average velocity and the velocity distribution of phytoplankton can be determined from curve fitting of the long-term power spectrum.