Ryuta Haraguchi

Dual-phase response model for bronchial asthma.

The authors have successfully developed an animal model of dual-phase bronchial responses and very high IgG titer by sensitizing Hartley-strain male guinea pigs. Specific airway resistance, which was determined in a two-chamber body plethysmograph, was elevated to sevenfold during immediate response, followed by a late phase response with a smaller but marked elevation in resistance. Furthermore, hematological and histological examinations revealed that the total cell count increased in BAL obtained during both immediate and late bronchial responses as compared to pre-OVA challenges. A significant increase in BAL eosinophils was present only for the late bronchial samples, and this finding was supported by histological examination.

Role of Chemical Mediators in Airway Hyperresponsiveness in an Asthmatic Model

Background: Airway hyperresponsiveness (AHR) is one of the characteristic features of human asthma. The presence of AHR and the precise mechanisms immediately after establishment of sensitization in guinea pigs are unclear, although there are many reports showing allergen exposure that causes an increase in bronchial responsiveness associated with eosinophil influx into the airway in sensitized guinea pigs. Objective: We investigated the inhibitory effects on AHR to histamine of ONO-1078, a leukotriene antagonist; indomethacin, a cyclooxygenase inhibitor; S-145, a thromboxane A2 (TXA2) antagonist, and Y-24180, a platelet-activating factor (PAF) antagonist, to assess the involvement of chemical mediators in AHR employing ovalbumin (OA) sensitized guinea pig models. Methods: Male Hartley guinea pigs were used. Each group comprised 4–7 animals. The animals were sensitized to OA, injecting intraperitoneally 30 mg of cyclophosphamide and 2,000 µg of OA together with 100 mg of aluminum hydroxide as the adjuvant. The guinea pigs were artificially ventilated via a cannula using a small-animal respirator after intraperitoneal anesthesia with pentobarbital sodium for tracheotomy. The pressure at the airway opening (PAO) was measured using a differential pressure transducer, and a differential pressure of peak PAO (peak ΔPAO) at inspiratory phase as an overall index of bronchial response to bronchoactive agents was used. While being artificially ventilated, the animals were exposed to physiological saline solution containing various concentrations of histamine (4.9, 9.8, 20, 39, 78, and 156 µg/ml) by inhalation for 30 s at 3-min intervals. Determinations were made at 1 min after each inhalation. The chemical mediators were each (30 mg/kg of ONO-1078, 3 mg/kg of S-1452, and 1 mg/kg of Y-24180) administered orally to sensitized guinea pigs, and the airway response to histamine was assessed. Each group comprised 4–7 animals. Results: The airway response to histamine was significantly greater in the sensitized group than in the nonsensitized group at histamine concentrations of 36 (p < 0.05), 78, and 156 mg/ml (p < 0.01). Leukotrienes C4 and D4: 30 mg/kg of ONO-178 did not show any inhibitory effect on airway response to inhaled histamine. Cyclooxygenase: 5 mg/kg of indomethacin did not show any inhibitory effect on the airway response to inhaled histamine. TXA2: the AHR to inhaled histamine at doses of 9.8, 39, 78, and 156 µg/ml was significantly inhibited by prior administration of 3 mg/kg of S-1452. PAF: the AHR to inhaled histamine at doses of 9.8, 39, and 78 µg/ml was significantly inhibited by prior administration of 1 mg/kg of Y-24180. Conclusions: S-1452 (3 mg/kg) and Y-24180 (1 mg/kg) significantly inhibited AHR to histamine, while ONO-108 (30 mg/kg) and indomethacin (5 mg/kg) did not. The results suggest that TXA2 and PAF are involved in AHR in OA-sensitized guinea pigs.

Continued inhalation of lidocaine suppresses antigen-induced airway hyperreactivity and airway inflammation in ovalbumin-sensitized guinea pigs

It is unclear whether inhaled lidocaine is effective against airway hyperreactivity and inflammation in asthma. The aim of this study was to investigate the effects of inhaled lidocaine on airway hyperreactivity and inflammation. Airway reactivity to inhaled histamine, cellular composition of bronchoalveolar lavage (BAL) fluid, plasma substance P (SP), and isolated lung tissue were evaluated in ovalbumin (OVA)-sensitized guinea pigs 7 days after OVA challenge. The effects of inhaled lidocaine on this model were also evaluated. Treatment with lidocaine was administered in two fashions: as single inhalation or inhalation bid for 7 consecutive days, for comparison with a saline-inhaled control group. Airway hyperreactivity to histamine, increase in number of total cells and increased proportion of eosinophils in BAL fluid, and marked eosinophil infiltration in airway walls were noted even 7 days after OVA challenge in the control group. Plasma SP level was also significantly increased. Although treatment with single lidocaine inhalation did not affect airway hyperreactivity, continued inhalation (bid for 7 days) attenuated airway hyperreactivity. Continued, but not single, inhalation of lidocaine also suppressed infiltration of eosinophils in BAL fluid and in airway walls. In addition, plasma SP levels were significantly reduced by continued but not by single inhalation. It appears possible that lidocaine when inhaled suppresses eosinophilic inflammation of the airway and SP-induced neurogenic inflammation, leading to alleviation of airway hyperreactivity.

Role of muscarinic acetylcholine receptors in a guinea pig model of asthma.

We examined the density of muscarinic acetylcholine receptor (mACh-R) subtypes (M1R, M2R and M3R) in guinea pig lung. The density of M3R in the lung tissue of ovalbumin (OA)-sensitized guinea pigs was higher than that in the control group. However, no difference was observed in the affinity of M3R between the sensitized and the control lungs. No difference was observed in the density and affinity of M1R and M2R in sensitized and control lungs. Pilocarpine, which is an M2R stimulant, increased the density of M3R in the lung tissue and the rate of the increase in sensitized guinea pigs was less than that in the control group. In contrast, methoctranine, which is an M2R antagonist, decreased the density of M3R and the rate ofthis decrease was the same in the sensitized and control groups. These results suggest that, in OA-sensitized guinea pigs, a dysfunction of M2R leads to the abnormal density of M3R.

Effects of suplatast tosilate (IPD Capsules®) on the production of active oxygen by neutrophils and of IL-8 by mononuclear cells

In bronchial asthma, eosinophils and neutrophils are activated, so that the production of active oxygen species increases, causing airway epithelial injury. Suplatast tosilate (IPD Capsules) is a novel immunomodulating antiallergic drug that acts against bronchial asthma through a new mechanism. To evaluate the effects of suplatast tosilate on mononuclear cell-mediated IL-8 production, and neutrophil-mediated active oxygen species production at sites of inflammation, we collected peripheral blood from healthy subjects and separated the neutrophils as well as mononuclear cells. Suplatast tosilate was added at a concentration of 1 x 10(-6), 1 x 10(-7) or 1 x 10(-8) M, and cells were incubated for 10 min at 37 degrees C. Then, the neutrophils were stimulated with fMLP, and luminol-dependent chemiluminescence (LDCL) was measured, while IL-8 production was determined with an ELISA kit. Suplatast tosilate (1 x 10(-6) M) inhibited neutrophil-mediated active oxygen species production by 12.4% in terms of the peak, and by 16% in terms of the integral value. Moreover, it significantly inhibited mononuclear cell-mediated IL-8 production at concentrations of 1 x 10(-6), 1 x 10(-7) and 1 x 10(-8) M, in a concentration-dependent manner. This study indicated that suplatast tosilate may inhibit neutrophil infiltration by suppressing monocyte-mediated IL-8 production, and it may also inhibit the activation of neutrophils at sites of inflammation. These results suggest the possibility that suplatast tosilate may not only be of benefit for asthma, but may also prevent or control pulmonary fibrosis or emphysema, for which no effective treatment is presently available.

Roles of histamine receptor in a guinea pig asthma model

Histamine plays an important role in bronchoconstriction mediated by histamine receptors which provoke bronchial asthma attack. In this study, we measured H1 and H2 receptors in the guinea pig lung membrane fraction and obtained the following results. The maximum binding (Bmax) of H1 receptors in the guinea pig lung membrane fraction was significantly higher in the OA-sensitized group than that in the non-sensitized group, but affinity (Kd) did not differ between the groups. Otherwise, the maximum binding (Bmax) of H2 receptors in the guinea pig lung membrane fraction was significantly lower in the OA-sensitized group than that in the non-sensitized group. But affinity (Kd) did not differ between the groups. These findings suggest a close association of Histamine receptors both H1 and H2 in the pathology of asthma.

A scoring algorithm for predicting the presence of adult asthma: a prospective derivation study

Background: To predict the presence of asthma in adult patients with respiratory symptoms, we developed a scoring algorithm using clinical parameters. Methods: We prospectively analysed 566 adult outpatients who visited Kinki University Hospital for the first time with complaints of nonspecific respiratory symptoms. Asthma was comprehensively diagnosed by specialists using symptoms, signs, and objective tools including bronchodilator reversibility and/or the assessment of bronchial hyperresponsiveness (BHR). Multiple logistic regression analysis was performed to categorise patients and determine the accuracy of diagnosing asthma. Results: A scoring algorithm using the symptom-sign score was developed, based on diurnal variation of symptoms (1 point), recurrent episodes (2 points), medical history of allergic diseases (1 point), and wheeze sound (2 points). A score of ≥3 had 35% sensitivity and 97% specificity for discriminating between patients with and without asthma and assigned a high probability of having asthma (accuracy 90%). A score of 1 or 2 points assigned intermediate probability (accuracy 68%). After providing additional data of forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) ratio <0.7, the post-test probability of having asthma was increased to 93%. A score of 0 points assigned low probability (accuracy 31%). After providing additional data of positive reversibility, the post-test probability of having asthma was increased to 88%. Conclusions: This pragmatic diagnostic algorithm is useful for predicting the presence of adult asthma and for determining the appropriate time for consultation with a pulmonologist.

Usefulness of QVAR for the treatment of bronchial asthma--with and without use of an inhalation device.

The treatment of bronchial asthma with QVAR (hydrofluoroalkane-134a BDP; 3M Pharmaceuticals, St. Paul, MN, USA) is usually conducted without an inhalation assistance device. However, Japanese patients who experience difficulty in coordinating activation with inspiration of inhaled steroid drugs are instructed on the use of such a device. We therefore examined the necessity of using an inhalation assistance device (INSPIR-EASE, IE) and its effect on quality of life (QOL) in the treatment of patients with bronchial asthma taking QVAR. Hence, lung function and QOL associated with taking QVAR plus IE or QVAR alone were examined by a cross-over method in 44 bronchial asthma patients (STEP 2 or 3) over 20 years of age. In all patients, lung function tests conducted 12 weeks after start of treatment indicated significant improvements of forced expiratory volume in 1 second (FEV1) with QVAR alone compared with QVAR plus IE (p < 0.05). In patients less than 70 years of age, significant improvements of forced vital capacity (FVC) and nitric oxide (NO) were also observed with QVAR alone compared with QVAR plus IE (p < 0.05). Examination of QOL the Living with Asthma Questionnaire indicated that medication usage was significantly improved with QVAR alone compared with QVAR plus IE. Significant improvement of FEV1 was observed with QVAR alone compared with QVAR plus IE, and additionally in patients less than 70 years of age improvement of FVC and NO was also marked. This study confirmed the usefulness of QVAR alone in patients with bronchial asthma.

In Vitro Effects of Saiboku-to on Cytokine Production from Peripheral Blood Mononuclear Cells of Bronchial Asthma Patients

AbstractObjective: This study aimed to evaluate the efficacy of Saiboku-to, a Japanese traditional herbal medicine (Kampo), on the production of interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interferon-γ (IFN-γ) and interleukin-2 (IL-2) by peripheral blood mononuclear cells in bronchial asthma patients following antigenic stimulus by the house dust mite. Design: Twelve bronchial asthma patients with IgE RAST (radio-allergosorbent test) scores to mites of 3 and over were enrolled in the study. Separated mononuclear cells from the patients’ blood were suspended in a 24-hole plate with 10% fetal calf serum (FCS) and RPMI-1640 medium. Mite antigen was added to the above cell-suspension (final concentration 10 mg/ml) and cultured for 72 hours at 37°C in 5% CO2. Saiboku-to (TJ-96, Tsumura & Co.) was added to the above sample system at the same time as mite antigen and cultured for 72 hours under the same conditions. After 72 hours of incubation, the amount of various cytokines (IL-2, IL-3, IL-4, IL-5, INF-γ) was measured using enzyme-linked immunoabsorbent assay kits. Results: Saiboku-to 500 mg/L significantly inhibited the production of IL-3 and IL-4 from peripheral blood mononuclear cells. Moreover, it significantly increased the production of INF-γ from peripheral blood mononuclear cells. Conclusion: These preliminary findings suggest that Saiboku-to could be considered as one of a new class of anti-asthmatic drugs because of its influence on cytokine production.

Inhibitory Effect of an Anti‐allergic Agent, TMK‐688, on Interleukin‐2 and Interleukin‐4 Release by Mite Antigen‐stimulated Monocytes From Peripheral Blood of Asthmatic Patients

The effect of an anti-allergic agent, TMK-688, which inhibits 5-lipoxygenase, on cytokine release by antigen-stimulated monocytes from peripheral blood of atopic asthmatic patients positive for mite antigen was studied. Monocytes were stimulated by mite antigen and then incubated for 72 h. The levels of interleukin-2 and interleukin-4 in the supernatant treated with TMK-688 (1 times 10−7 M) were significantly lower (P < 0.05 and P < 0.01, respectively) than levels in the untreated control. The findings suggest that TMK-688 is effective in treating bronchial asthma, as it not only inhibits 5-lipoxygenase but also has effects on T lymphocytes and inhibits various types of cytokine release.