Haruhisa Mita

Isolation and Characterization of Two Allergens from Dermatophagoides farinae

Two purified allergens, designated as DF1 and DF2, were isolated from the extract of the whole culture of Dermatophagoides farinae by a combination of ammonium sulfate precipitation and ion exchange, hydrophobic, chelate and gel chromatography. DF1 was isolated as a heat-sensitive acidic protein with an apparent molecular weight of 25,000 and an isoelectric point of 4.6-7.2. DF2 was isolated as a heat-stable basic protein with an apparent molecular weight of 15,000 and an isoelectric point of 7.8-8.3. No allergenic cross-reactivity was seen between DF1 and DF2. Both DF1 and DF2 were shown to be the major allergens of D. farinae by the results of radioallergosorbent test and histamine release assay.

Comparative Analysis of Physicochemical and Immunochemical Properties of the Two Major Allergens from Dermatophagoides pteronyssinus and the Corresponding Allergens from Dermatophagoides farinae

Two major allergens, DP1 (Der p I) and DP2 (Der p II), were isolated from the whole culture extract of Dermatophagoides pteronyssinus, and the physicochemical and immunochemical properties of these allergens were compared with those of the corresponding allergens from Dermatophagoides farinae, DF1 (Der fI) and DF2 (Der fII). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polyacrylamide gel isoelectric focusing and amino acid analysis, both DP1 and DP2 were demonstrated to have close physicochemical similarity with DF1 and DF2, respectively. On immunodiffusion with the use of rabbit antisera, the two Der I allergens showed the reaction of typical partial identity, while the two Der II allergens showed the reaction of almost complete identity. Radioallergosorbent test (RAST) and RAST absorption experiments with the use of sera from mite-allergic patients showed that human IgE antibody response to the Der I allergens was directed against both cross-reactive and species-specific determinants. In contrast, IgE antibodies to the Der II allergens were demonstrated to react almost completely to cross-reactive determinants.

Detection of IgE antibody to a radiocontrast medium

Background Very little is known about the mechanistns underlying adverse reactions to radiocontrast medium. On the basis of the clinical features of the adverse reactions, il has generally been considered that an IgE‐dependent mechanism is not involved in these adverse reactions, and only a few studies have demonstrated the presence of IgE antibody to radiocontrast medium iti patient sera.

Release of Leukotriene C4 from Human Eosinophils and its Relation to the Cell Density

Human eosinophils and neutrophils were isolated from 20 ml of peripheral blood by dextran sedimentation, centrifugation with lymphoprep, and density gradient centrifugation with Percoll. The incubation of neutrophils of purity higher than 99% (density: 1.080-1.085 g/ml) with calcium ionophore A23187 for 20 min resulted in the release of a little amount of leukotriene C4 (2.3 +/- 0.6 ng/10(6) cells) as measured with a commercial radioimmunoassay kit. On the other hand, the release from eosinophils was over 10 times that from neutrophils. When the releasability of leukotriene C4 from eosinophils was examined in relation to the cell density, the eosinophils with lower density were observed to produce greater amounts of leukotriene C4.

Increased activity of 5-lipoxygenase in polymorphonuclear leukocytes from asthmatic patients

The formation of 5-lipoxygenase products of arachidonic acid, 5-HETE and 5,12-diHETE, was determined in 100,000 X g supernatant of polymorphonuclear leukocytes from 17 healthy subjects, 17 patients with extrinsic asthma and 15 patients with intrinsic asthma. After the supernatant was incubated with 14C-arachidonic acid in the presence of calcium and indomethacin, the lipoxygenase products of arachidonic acid were separated by thin layer chromatography. The results were expressed as the percentage conversion of 14C-arachidonic acid into the product per 10(7) cells. The formation of 5,12-diHETE, but not of 5-HETE, was significantly increased in the cells from the group of patients with extrinsic asthma (4.38 +/- 0.78%, mean +/- S.E.; p less than 0.01) and intrinsic asthma (6.09 +/- 1.11%; p less than 0.01), when compared to normal subjects (1.74 +/- 0.30%). Both extrinsic and intrinsic asthmatics had significantly enhanced 5-lipoxygenase activity, which was expressed as the sum of percentage conversion of 14C-arachidonic acid into 5-HETE and 5,12-diHETE. The percentage conversion in normal subjects was 4.19 +/- 0.39%, 6.24 +/- 0.84% for 17 patients with extrinsic asthma (p less than 0.05), and 8.59 +/- 1.29% for 15 patients with intrinsic asthma (p less than 0.01). There was no significant difference between these asthmatic groups. These results indicate that 5-lipoxygenase activity is increased in patients with bronchial asthma.

Allergenicity of acid protease secreted by Candida albicans

We have previously reported the cases of Candida albicans (C. alb) acid protease (CAAP)‐induced atopic asthma. In this study, the allergenicity of the released enzyme CAAP was examined among asthmatic patients with positive immediate skin response to crude C. alb antigen. Among 49 patients with positive skin response to crude C. alb, anti‐crude C. alb IgE antibodies were detected in 40 and anti‐CAAP IgE antibodies were detected in 18. Moreover, anticrude C. alb IgE antibodies were detected in all of the patients in whom anti‐CAAP IgE antibodies were detected. No correlations between IgG antibodies to both antigens or between IgE and IgG antibodies to CAAP were observed. CAAP induced significant T‐cell proliferation in 20/28 patients showing positive T‐cell proliferation response to crude C. alb antigen. Most of the patients showing positive conjunctival response to crude C. alb antigen also showed positive response to CAAP. Most of the patients showing high levels of serum IgE antibody and positive histamine‐release response of peripheral blood leukocytes to CAAP showed positive conjunctival response. The results indicate that CAAP is an important allergen in C. alb‐related mucosal allergy.

Anti-Allergic Activity of Formoterol, a New Beta-Adrenoceptor Stimulant, and Salbutamol in Human Leukocytes and Human Lung Tissue

Formoterol and salbutamol were compared for in vitro inhibition of allergen‐induced histamine release from allergic leukocytes and human lung tissue passively sensitized with allergic serum. Formoterol inhibited the release of histamine from leukocytes but salbutamol showed little inhibiting effect. When combined with theophylline, formoterol was a more potent inhibitor of the release of histamine from leukocytes in allergic patients than salbutamol. In fragments of human lung sensitized with allergic serum, the concentration required to inhibit histamine release by 50% was 2 × 10−11 M for formoterol and 8.5 × 10−9 M for salbutamol. The potency of salbutamol and formoterol in blocking specific 3 H‐dihydroalprenolol binding to beta‐adrenoceptors on guinea pig lung membranes revealed that formoterol had higher affinity for beta‐adrenoceptors than salbutamol, and the concentration required for half‐maximum stimulation of adenylate cyclase was approximately 200‐fold higher for salbutamol than for formoterol.

Increased Generation of Leukotriene C4 from Eosinophils in Asthmatic Patients

Human eosinophils with a density of over 1.095 were isolated from peripheral blood by dextran sedimentation, centrifugation with Lymphoprep and density gradients with Percoll. After the cells (1 × l05ml) were incubated with 1 μg/ml calcium ionophore A23187 for 20 min, leukotriene C4 (LTC4) content in the supernatant was measured by radioimmunoassay. The generation of LTC4 was significantly higher in the cells from extrinsic asthmatics (23.5 ± 14.8 ng/l06 cells, mean ± SD, n= 26, P < 0.01) and intrinsic asthmatics (24.6 ± 20.6 ng/106 cells, n= 27, P < 0.01) as compared with normal healthy subjects (8.3 ± 7.7 ng/106 cells, n= 10). There was no significant difference in the generation of LTC4 between intrinsic and extrinsic asthmatics. These observations indicate that eosinophils from asthmatic patients have increased ability to release LTC4.

Effect of AA‐861, a 5‐Lipoxygenase Inhibitor, on Leukotriene Synthesis in Human Polymorphonuclear Leukocytes and on Cyclooxygenase and 12‐Lipoxygenase Activities in Human Platelets

AA‐861, a selective inhibitor of 5‐lipoxygenase of arachidonic acid, was tested for ability to inhibit leukotriene C4 and leukotriene B4 synthesis in human polymorphonuclear leukocytes after calcium ionophore stimulation. AA‐861 dose‐dependently inhibited leukotriene B4 and leukotriene C4 generation in human polymorphonuclear leukocytes; the concentration required to inhibit generation by 50 % (IC50) was 3 × 10−7 M for leukotriene B4 and 1 × 10−8 M for leukotriene C4. BW‐755C inhibited the generation of leukotriene C4 with an IC50 of about 10−5 M, indicating that AA‐861 is about 1000 times more potent than BW‐755C. AA‐861 did not affect the activity‐ of either cyclooxygenase or 12‐lipoxygenase at a concentration up to 10−5 M in human platelets. AA‐861 did not inhibit histamine release from human basophils. These results indicate that AA‐861 selectively inhibits 5‐lipoxygenase but not cyclooxygenase or 12‐lipoxygenase in human specimens.

Nasal blockage and urinary leukotriene E4 concentration in patients with seasonal allergic rhinitis

Background: Cysteinyl‐leukotrienes have been reported to have a primary role in the induction of nasal blockage of allergic rhinitis. However, there has been little experimental evidence that substantiates the relationship between nasal blockage severity and urinary leukotriene E4 (U‐LTE4) concentration in patients with seasonal allergic rhinitis (SAR).