Kimiko Sakai

Origin of intracellular Ca2+ elevation induced by in vitro ischemia-like condition in hippocampal slices

Microfluorometry was used to investigate the origin of intracellular Ca2+ ([Ca2+]i) elevation in field CA1 of gerbil hippocampal slices perfused with a glucose-free physiological medium equilibrated with a 95% N2/5% CO2 gas mixture (standard in vitro ischemia-like condition). Large [Ca2+]i elevation was detected about 4 min after the beginning of standard in vitro ischemia-like condition, which was accompanied with a negative shift of extracellular DC potential. When slices were perfused with Ca(2+)-free in vitro ischemia-like medium, large [Ca2+]i elevation was observed about 3.5 min after the beginning of Ca(2+)-free in vitro ischemia-like condition, however, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the standard in vitro ischemia-like medium that contained Ca2+. When slices were perfused with the Ca(2+)-free in vitro ischemia-like medium that contained dantrolene (50 microM) which is known to prevent Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the Ca(2+)-free in vitro ischemia-like medium that did not contain dantrolene. These results indicate that large [Ca2+]i elevation induced by in vitro ischemia-like condition in field CA1 of the hippocampus was caused by both Ca2+ influx from extracellular space and Ca2+ release from intracellular Ca2+ stores, and that a part of the Ca2+ release was due to Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores.

Effect of different sensitizing doses of antigen in a murine model of atopic asthma

The dose of antigen is assumed to be one of the important factors in the polarized development of helper T cell subsets, i.e. Th1 or Th2 cells. We investigated the effect of the sensitizing antigen dose in a murine model of atopic asthma, which involved sensitization with ovalbumin (OVA) followed by repeated exposure to OVA aerosols. BALB/c mice were primed with varying doses of OVA (0, 10, 100 and 1000 μg) plus Al(OH)3 on days 0, 7 and 14, and were challenged with OVA aerosols (50 mg/ml for 20 min) on days 15–20. There were striking antigen dose‐related differences in OVA‐specific antibodies: high IgE and low IgG2a titres were found in mice sensitized at 10 μg, while low IgE and high IgG2a titres were seen at 1000 μg. The sensitizing dose was inversely correlated with the total cell count and the eosinophil count in bronchoalveolar lavage fluid (BALF), as well as with the extent of histological changes such as goblet cell hyperplasia of the bronchial epithelium and cellular infiltration into bronchovascular bundles. Antigen‐induced bronchial hyper‐responsiveness (BHR) to methacholine was observed with sensitization at 10 μg but not at 1000 μg. Splenic mononuclear cells (SMNC) obtained from mice sensitized at either dose showed proliferation in response to OVA. Production of IL‐4 and IL‐5 by OVA‐stimulated SMNC was inversely correlated with the dose of sensitizing antigen. High‐dose sensitization resulted in general suppression of cytokine production by SMNC, including interferon‐gamma (IFN‐γ). The BALF levels of IL‐4 and IL‐5 were increased by low‐dose sensitization, whereas IFN‐γ and IL‐12 levels were increased by high‐dose sensitization. These results suggest that the dose of sensitizing antigen defines the phenotypic changes in the present murine asthma model, presumably by influencing the pattern of cytokine production.

Prolonged antigen exposure ameliorates airway inflammation but not remodeling in a mouse model of bronchial asthma.

Background: In naive rodents, repeated exposure to aerosolized antigen induces suppression of the Th2 response to the antigen. We hypothesized that more prolonged exposure of established asthma model to antigen aerosols may downregulate asthmatic phenotype. Methods: After establishing an ovalbumin (OVA)-induced asthma model, mice were further exposed to OVA (prolonged exposure group) or phosphate-buffered saline (positive controls) 3 days per week for 6 weeks. During week 7, the mice of both groups were finally challenged with OVA. Results: Prolonged OVA exposure resulted in marked suppression of serum OVA-specific immunoglobulin E (IgE) antibody levels, eosinophilia of the airway, and airway hyperresponsiveness (AHR). However, airway remodeling characterized by goblet cell hyperplasia and airway fibrosis was observed to the same degree in both groups. These effects were accompanied by diminished production of Th2 cytokines such as interleukin-4 (IL-4), IL-5 and IL-13 in bronchoalveolar lavage fluid (BALF) and cultured supernatant of splenocytes. Furthermore, prolonged exposure markedly increased IL-12 levels in BALF. Conclusions: Prolonged antigen exposure has inhibitory effects on eosinophilic inflammation, AHR and IgE response to antigen, but not on airway remodeling, presumably via inhibition of Th2 cytokines and increased IL-12 production in the lungs.

Effect of Suplatast Tosilate (IPD-1151T) on a Mouse Model of Asthma: Inhibition of Eosinophilic Inflammation and Bronchial Hyperresponsiveness

Background: Suplatast tosilate (IPD) is a newly developed ‘anti-allergic’ drug. It seems to be a unique compound because of its ability to suppress IgE but not IgG or IgM production in vivo and cytokine production from type 2 helper T cells (Th2) in vitro. However, information on its in vivo effect on an animal model of asthma is limited. Method: BALB/c mice sensitized to ovalbumin (3 times, 2-week interval) were challenged with ovalbumin by inhalation (50 mg/ml for 20 min, once a day for 6 days). In this study, we explored the influence of IPD on eosinophil infiltration into the airways, bronchial hyperresponsiveness (BHR) to methacholine, specific IgE antibody production, and cytokines in bronchoalveolar lavage fluid (BALF) using this murine model. Results: Treatment with IPD significantly reduced the number of total cells and eosinophils in BALF (around –40%) and almost completely inhibited the development of antigen-induced BHR. Histological findings confirmed the reduction of submucosal cell infiltration in the lung, and disclosed the marked inhibition of bronchial epithelial cell damage. Ovalbumin-specific IgE was slightly but significantly reduced. The levels of IL-4, IL-5 and IL-13 in BALF were significantly decreased in mice treated with the compound compared to those in untreated mice. Conclusion: These results suggest that IPD is capable of inhibiting the production of Th2 cytokines, which inhibit eosinophil infiltration into the murine airway, IgE synthesis, and development of BHR, in a murine model of asthma.

Effect of aerosolized docosahexaenoic acid in a mouse model of atopic asthma.

Background: Docosahexaenoic acid (DHA) found in fish oil is known to depress inflammation-related mediators. We investigated a novel delivery method of tridocosahexaenoyl-glycerol (DHA-TG). Methods: BALB/c mice (6–8 weeks old) were primed intraperitoneally with ovalbumin (OVA) and Al(OH)3 on days 0 and 7, and with aerosolized OVA on day 7. Primed mice were challenged by repeated exposure to aerosolized OVA on days 15–17. Just before each exposure to aerosolized OVA, the mice were also exposed to an aerosol of emulsified DHA-TG or soybean oil, or saline (days 7 and 15–17). Bronchial hyperresponsiveness (BHR) to methacholine was measured, and bronchoalveolar lavage fluid was obtained 24 h after the last challenge (day 18). Lungs were histologically examined. Results: Bronchoalveolar lavage fluid of saline-treated mice showed an increased cellularity with predominant eosinophils. Exposure to DHA-TG significantly reduced the total cell number and the eosinophil percentage in lavage fluid, whereas soybean oil did not. Conclusion: DHA but not soybean oil exposure reduced BHR and cell infiltration to bronchovascular bundles. This type of DHA administration could be studied in clinical trials.

Effect of Pranlukast, a Leukotriene Receptor Antagonist, in Patients with Severe Asthma Refractory to Corticosteroids

We investigated the effect of pranlukast (ONO-1078), a cysteinyl leukotriene receptor antagonist, in 11 patients with severe bronchial asthma. The patients had been treated with 1600 micrograms/day of beclomethasone or 800-1600 micrograms/day of beclomethasone plus 2.5-20 mg/day of prednisolone, but remained symptomatic. After a 2-week baseline period, the patients received 225 mg of pranlukast twice daily for 8 weeks. Morning and evening peak expiratory flow rate (PEF) and symptom scores (cough, dyspnea, sleep) were recorded in an asthma diary. Ten patients completed the study. Symptom scores, especially dyspnea and sleep scores, and the number of rescue beta 2-agonist inhalations were significantly decreased. The morning PEF significantly improved from a mean baseline value of 311 to 341 L/min by the end of the study period. The evening PEF also improved, from 328 to 348 L/min, although the difference was not significant. These results suggest that pranlukast may be effective in treating patients with severe asthma who are refractory to corticosteroid therapy.

T‐helper 1 cells induce alveolitis but do not lead to pulmonary fibrosis in mice

T‐helper (Th)1 cells have a pivotal role in the pathogenesis of hypersensitivity pneumonitis. Continued low-level exposure to the antigens may induce chronic hypersensitivity pneumonitis with lung fibrosis. Although such exposure may activate Th1 cells in the lung, it is not known whether activation of Th1 cells per se can lead to pulmonary fibrosis. To determine this, the lung pathology induced by Th1 clones was investigated. Mice (BALB/c) were injected intraperitoneally with Th1 clones 1–4 times. Each injection was performed 4 days apart and was followed by repeated exposure to aerosolised ovalbumin (OVA) once a day for 5 days. The number of macrophages and lymphocytes in bronchoalveolar lavage fluids (BALF) increased as the number of Th1 transfers increased. The number of neutrophils also increased but peaked in the second transfer and then decreased following further transfers. Increased cell infiltration, thickness of alveolar walls and number of type II cells in the lung occurred. However, histological findings showed no evidence of fibrosis and hydroxyproline levels did not increase. Findings of histology and BALF were ameliorated 2 weeks after the discontinuation of OVA exposure, indicating the reversibility of the Th1-induced pathology. In conclusion, adoptive transfer of T‐helper 1 cells results in reversible alveolitis but does not lead to pulmonary fibrosis.

Adoptive transfer of T-helper cell type 1 clones attenuates an asthmatic phenotype in mice.

T-helper cell type 1 (Th1) cells have been postulated to have a significant role in protective immunity against allergic diseases. However, recent studies using polarised Th1 cells showed conflicting effects on both airway responsiveness and eosinophilic inflammation in a mouse asthma model. The current study explored the effects of adoptive transfer of established Th1 clones on a murine model of atopic asthma. Mice (BALB/c) were sensitised with ovalbumin (OVA) and challenged with aerosolised OVA (5%, 20 min) for 5 days. Just before starting the first challenge, Th1 clones (5×106·body−1) or PBS alone were injected via the tail vein. After assessment of airway responsiveness to methacholine, bronchoalveolar lavage fluid (BALF) was obtained. Histological examination, including morphometric analysis, measurement of cytokines in the BALF and Northern blotting of lung chemokines, was also performed. Adoptive transfer of Th1 clones showed a significantly increased total number of cells, whereas significantly decreased eosinophils were found in the BALF, when compared with mice with injection of vehicle alone or splenic mononuclear cells. Administration of Th1 clones significantly decreased the infiltration of eosinophils but increased mononuclear cells in the peribronchial area. Goblet cell hyperplasia and peribronchial fibrosis were also suppressed by Th1 clones. The transfer of Th1 cells significantly decreased airway responsiveness. Th1 injection significantly increased interferon gamma in the BALF, but significantly decreased interleukin (IL)-5 and IL-13. Eotaxin mRNA was predominantly expressed in the lungs of asthma model mice, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) predominates in such mice with Th1 transfer. In conclusion, results suggest that the adoptive transfer of T-helper cell type 1 clones can suppress both lung eosinophilia and airway responsiveness, but increase noneosinophilic inflammation in a mouse model of asthma.

Myeloperoxidase antineutrophil cytoplasmic antibody‐positive bronchiectasis in an elderly patient

A 76‐year‐old man with bronchiectasis was hospitalized for a 2‐month history of continuous fever due to exacerbation of the disease. The patient had suffered from bronchiectasis for approximately 50 years. Pseudomonas aeruginosa was persistently detected in his sputum. The patients serum was positive for myeloperoxidase antineutrophil cytoplasmic antibody (MPO‐ANCA), but there were no symptoms of vasculitis. Antibiotic therapy effectively resolved the disease exacerbation leading to a decrease in the level of MPO‐ANCA.

Secretion of Adrenocorticotropic Hormone Induced by Allergen Inhalation in Patients with Atopic Asthma

Allergen inhalation in atopic patients results in cytokines production or release of preformed cytokines, some of which are known to induce adrenocorticotropic hormone (ACTH) secretion in experimental conditions. We examined whether allergen inhalation can induce ACTH secretion in vivo. A significant elevation of ACTH levels was observed in 2 and 24 hr after allergen inhalation challenge. However, methacholine challenge with the same degree of airflow limitation did not induce ACTH elevation, indicating that this may not be due to bronchoconstriction per se. Our results indicate that allergen inhalation can trigger ACTH secretion in patients with atopic asthma.