Takao Shida

Measurement of allergens associated with dust mite allergy. II. Concentrations of airborne mite allergens (Der I and Der II) in the house.

Assays of mite allergens (Der p I, Der f I and Der II) in the air of houses became feasible with the use of a low-noise air sampler and a sensitive radioimmunoassay described previously. The levels of the airborne allergens Der I (Der p I + Der f I) and Der II in the living room of 10 houses during usual domestic life were very low, 29.5 and 6.3 pg/m3, respectively, with a Der I: Der II ratio of 4.7:1. At the time of bedmaking, they greatly increased, about 1,000-fold, to 30,900 and 12,600 pg/m3, respectively, with a Der I: Der II ratio of 2.5:1. The amounts of Der I and Der II in the floor dust of the living room were 2,040 and 2,690 ng/g of fine dust, respectively, with a Der I: Der II ratio of 0.8:1. Der I seemed more prone to become airborne than Der II.

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.

Suppression of IgE production by IPD-1151T (suplatast tosilate), a new dimethylsulfonium agent: (1). Regulation of murine IgE response.

The effect of IPD-1151T, a new dimethylsulfonium compound, on the IgE response was investigated in the mouse system. The oral administration of IPD-1151T to immunized BALB/c mice suppressed the primary IgE antibody response and depressed the elevation of serum IgE levels, whereas the same treatment did not affect the IgG antibody response. The enhanced expression of low-affinity IgE receptor (Fc epsilon RII/CD23) on the spleen cells of immunized mice was also inhibited by IPD-1151T administration. It was further demonstrated from the adoptive transfer experiment that IPD-1151T, administered to hapten-primed B cell donors, but not to carrier-primed T cell donors, exerted its suppressive influence on the hapten-specific secondary IgE antibody response in irradiated syngeneic recipients. Interestingly, IPD-1151T concentration-dependently inhibited the production of interleukin 4 (IL-4) by D10G4.1, known to be a typical Th2 clone. However, IPD-1151T did not suppress the production of IgE and IgG1 by normal splenic B cells stimulated with lipopolysaccharide and IL-4. Moreover, IL-4-induced expression of Fc epsilon RII on normal spleen cells was not inhibited by the agent. These results strongly suggest that the IgE-suppressive activity of IPD-1151T is most likely due to the inhibition of IL-4 production at the T cell level.

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.

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.

Urinary excretion of leukotriene E4 and 11-dehydrothromboxane B2 in patients with spontaneous asthma attacks.

BACKGROUND Cysteinyl leukotrienes (LTs) and thromboxane A2 (TXA2) are known to play an essential role in the pathogenesis of atopic asthma. However, their role in nonatopic asthma has not as yet been clarified. The objectives of this study were to define (1) the participation of LTs and TXA2 in nonatopic asthma and (2) the relationship between LTs and TXA2 in asthma attacks. METHODS Urinary excretion of leukotriene E4 (LTE4) and 11-dehydrothromboxane B2 (11DTXB2) was measured in 10 atopic and 10 nonatopic asthmatics who were admitted to hospital with either an acute asthma attack or status asthmaticus. RESULTS In atopic asthmatics, urinary excretion of LTE4 and 11DTXB2 was significantly higher on admission with an asthma attack, and returned to control levels when the patients were in the improved state (179+/-29 to 65+/-16 ng/day in LTE4, 1,085+/-250 to 440+/-90 ng/day in 11DTXB2). Similar findings were observed in nonatopic asthmatics (148+/-13 to 61+/-11 ng/day in LTE4, 1,089+/-206 to 457+/-60 ng/day in 11DTXB2). However, when the individual data during the attack were analyzed, there was no correlation between urinary excretion of LTE4 and that of 11DTXB2 in both types of asthma. CONCLUSION Both LTs and TXA2 may be implicated in the pathogenesis of the nonatopic as well as the atopic type of asthma, but no correlation between these two metabolites was observed in the individuals.

Effect of Butyric Acid on Induction of Differentiation into Eosinophil-Like Cells in Human Eosinophilic Leukemia Cells, EoL-1 Cell Line: Possible Role of Granulocyte-Macrophage Colony-Stimulating Factor as an Autocrine Differentiating Factor

EoL-1 cells differentiated into mature eosinophil-like cells when incubated with butyric acid (BA). The differentiated cells possessed many granules that stained with Luxol fast blue and acquired the ability to produce leukotriene C4 and eosinophil cationic protein (ECP), and migrated toward some eosinophillotactic attractants, including PAF, IL-5, IL-3 and GM-CSF. Monoclonal antibody to GM-CSF and IL-5, especially GM-CSF, strongly decreased the ability of BA to induce differentiation, whereas antibody to IL-3 did not. Recombinant human (rh) IL-3, rhGM-CSF and recombinant mouse (rm) IL-5, by themselves, did not have the ability to induce differentiation. On the other hand, the presence of these cytokines significantly (p < 0.01) augmented BA-induced cell differentiation. Other cytokines including rhIL-1 alpha/beta, rhIL-2, rhIL-4, rhIL-6, rhIL-8, rhTNF-alpha and rhIFN-gamma had no effect on differentiation. Expression of GM-CSF and GM-CSF receptor mRNA was detected in cellular RNA obtained from BA-stimulated EoL-1 cells. Moreover, about 5-10 pg/ml of GM-CSF was detected in the culture supernatant of BA-stimulated EoL-1 cells. These findings suggested that BA-induced EoL-1 cell differentiation was elicited under the influence of GM-CSF endogenously produced in these cells. GM-CSF also seemed to be an autocrine differentiating factor in EoL-1 cells.

Seasonal Changes in Mite Allergen (Der I and Der II) Concentrations in Japanese Homes

BACKGROUND There has been no report on seasonal changes in Der II allergen in floor and bedding dust. OBJECTIVE The purpose of this study was to examine the extent of seasonal changes in Der I and Der II allergens in the floor and bedding dust found in houses. METHODS We measured the absolute concentrations of mite allergens in dust collected monthly for 1 year from both the floors and bedding of eight houses in Tokyo. Dust samples were obtained from eight families without regard to their allergy histories. The concentrations of the Der p I, Der f I, and Der II allergens were measured by fluorometric sandwich ELISA. RESULTS We found seasonal changes in the concentrations of these mite allergens. The highest concentrations of Der I (Der p I plus Der f I) and Der II (Der p II plus Der f II) were present from August to October, and the lowest ones from March to April. In floor dust, the mean highest concentrations of Der I and Der II (35.0 and 20.2 microgram of fine dust) were sevenfold and fivefold respectively, times the mean lowest concentrations. In bedding dust, the mean highest concentrations of Der I and Der II (51.3 and 29.6 microgram/g of fine dust) each were fourfold times the mean lowest concentrations. CONCLUSIONS The patterns of seasonal changes in Der II in floor and bedding dust were similar to those of Der I.

Quantitation of platelet-activating factor by high-performance liquid chromatography with fluorescent detection.

Platelet-activating factors, 1-O-hexadecyl- and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-AGEPC and C18AGEPC), were measured by reverse-phase high-performance liquid chromatography with fluorescent detection. C16AGEPC, C18AGEPC, and 1-O-hexadecyl-2-propionyl-sn-glycero-3-phosphocholine, which was suitable for use as an internal standard, were hydrolyzed with phospholipase C, and then the resulting hydrolyzed products were derivatized with 7-methoxycoumarin-3-carbonyl chloride or 7-methoxy-coumarin-4-acetic acid to form 7-methoxycoumarin ester derivatives which permit a fluorometric detection. The lower limit of detection of the derivatives was about 100 pg at a signal-to-noise ratio of 5:1. A commercial platelet-activating factor was demonstrated to contain C16AGEPC (70%) and C18AGEPC (12.8%) by the present method. The present method was also applicable to the measurement of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activity in a lysate of human polymorphonuclear leukocytes.