Nobuaki Akao

Immunoblot analysis of serum IgG, IgA and IgE responses against larval excretory-secretory antigens of Anisakis simplex in patients with gastric anisakiasis.

To increase our understanding of the immune response to Anisakis infection, antigen specific IgG, IgA and IgE responses were identified using an immunoblot technique after polyacrylamide gel electrophoresis of excretory-secretory products from the larval stage of Anisakis simplex. Nine sera were drawn from proven cases of gastric anisakiasis within 3 days after symptoms had developed. The molecular weight of the major antigenic bands were distributed between 50 kDa and 120 kDa of the antigens. In nine cases of gastric anisakiasis, three of them were positive for IgG response, five for IgE, and six for IgA, respectively. None of control sera recognized the antigenic bands in IgA and IgE responses. In contrast, two controls had IgG antibodies against 1-2 proteins in the 65-95 kDa region. The antigenicity of the excretory-secretory products was lost following treatment by 0.2% trypsin, but not by 0.2 M periodic acid. Based on the results of reactivity to lectins, antigenic bands of the ES products possessed mucin type glycoconjugate residues in their protein portion. This indicates that the humoral responses of IgA and IgE antibodies to the larval ES antigens are a more reliable index of infection than that of the IgG response.

Bronchial hyperresponsiveness and airway neutrophil accumulation induced by interleukin-8 and the effect of the thromboxane A2 antagonist S-1452 in guinea-pigs.

Interleukin‐8 (IL‐8) has been shown to be a chemotactic factor for neutrophils, T‐lymphocytes and eosinophils, but it is unknown whether the IL‐8‐induced inflammatory cell accumulation into the airways can cause the bronchial hyperresponsiveness (BHR) characteristic of asthma. IL‐8 at a dose of 0.5 or 5μg/kg was administered intranasally to guinea‐pigs twice a week for 3 weeks. One day after the last administration, animals were anesthetized and artificially ventilated through tracheal cannula and lateral pressure at the cannula (Pao) was measured as an overall index of airway responses to increasing concentrations of inhaled histamine (25, 50, 100, and 200 μg/ml). The IL‐8 treatment significantly enhanced bronchial responsiveness to histamine in a dose‐dependent manner (ANOVA P < 0.01). The provocative concentration of histamine causing a 100% increase in Pao (PC100) at a dose of 0.5 and 5μg/kg of IL‐8 was 68.1 (Gsem 1.12) and 35.6 (Gsem 1.25) μg/ml, respectively. The latter was significantly (P < 0.01) lower than that in control animals treated with PBS (93.3 [Gsem, 1.14] μg/ml)‐ The IL‐8 treatment also induced a significant influx of neutrophils, but not eosinophils, in bronchoalveolar lavage (BAL) fluid (18.3 ± 8.8 and 30.6 ± 8.3% in animals treated with 0.5 and 5 μg/kg, respectively, of IL‐8 vs 3.6 ± 0.7% in phosphate buffered saline‐(PBS)‐treated animals). Furthermore, we examined the effect of the thromboxane receptor antagonist S‐1452 (0.01 or 0.1 mg/kg, i.p. 24 and 1 h before anesthesia) on this IL‐8 induced BHR. S‐1452 significantly inhibited the BHR dose‐dependently (ANOVA P < 0.001). PC100 was 94.0 (Gsem 1.19), 137.4 (Gsem 1.17) and 43.0 (Gsem 1.24) μg/ml with S‐1452 at doses of 0.01 and 0.1mg/ml and saline, respectively. We conclude that IL‐8 causes BHR and airway neutrophil inflammation, and that thromboxane A2 is important in the development of BHR induced by IL‐8.

The effect of the neurokinin antagonist FK-224 on the cough response to inhaled capsaicin in a new model of guinea-pig eosinophilic bronchitis induced by intranasal polymyxin B

Eosinophilic bronchitis without asthma can cause a persistent non-productive cough which is resistant to bronchodilator therapy. To understand the mechanism of the cough in this disorder, an animal model of eosinophilic bronchitis was developed. Guinea-pigs were treated with transnasal administration of polymyxin B or saline twice a week for 3 weeks. The number of eosinophils in bronchoalveolar lavage fluid increased in polymyxin B-treated animals when compared with those treated with saline. In addition, histological examination showed that the number of eosinophils infiltrated into the tracheal epithelium increased; injury to the tracheal epithelium was greater in polymyxin B-treated animals. The numbers of coughs induced by saline and each concentration of capsaicin (10−18, 10−16, 10−14M) were greater in the polymyxin B-treated animals. FK-224 (a neurokinin receptor antagonist) decreased the heightened cough reflex in this animal model of eosinophilic bronchitis. These findings suggest that neuropeptides, and particularly neurokinins, are involved in the heightened cough receptor sensitivity in eosinophilic bronchitis without asthma. This has implications for better understanding of this disorder and its treatment.

Immunoblot analysis of Dirofilaria immitis recognized by infected humans

To help to develop better diagnostic tests for zoonotic pulmonary dirofilariasis, seven patients with histologically confirmed pulmonary dirofilariasis were evaluated using the immunoblot technique. Six patients exhibited a response to the excretory-secretory (AS) antigen proteins with molecular weights of 20-19.5, 17.5-17 and 14 derived from Dirofilaria immitis adult works. However, when adult worm extracts were used as the antigen, there was no qualitative difference in antigen recognition between the Dirofilaria patients, other patients with non-filarial parasitic infections or with lung cancer or tuberculosis, and normal individuals. All the sera, not only those of the Dirofilaria patients but also those of the non-filarial patients and the controls, strongly cross-reacted with the 18-kDa ES antigen. These findings suggest that the ES antigen of D. immitis provides a more sensitive antigen than does the adult somatic antigen; but the ES antigen and the adult somatic antigen have a common antigenic band at 18 kDa.

Changing chemosusceptibility in the second-stage larvae of Toxocara canis by long-term incubation

Second-stage larvae of Toxocara canis were maintained in vitro for one year. Susceptibility of the larvae to drugs was evaluated by means of minimal larvicidal concentration (MLC) and larval bursting percentage. MLCs of citral and decanoic acid were almost constant throughout all stages of incubation. However, bursting percentage markedly varied within the first 20 weeks of incubation. Therefore, while larvae are available for use in the MLC assay at any stage of incubation, those beyond the first 20 weeks after incubation should be used for the bursting assay to obtain reproducible results.