A method for rapidly measuring the interleukin 2 (IL-2)-responsiveness of allergen-stimulated lymphocytes has been newly developed using propidium iodide (PI) staining and drawing ink quenching of the fluid medium fluorescence. There was a linear correlation between the number of PI-stained cells and the fluorescence intensity. The background was less than 5 percent. This fluorochromasia assay reflected the cell number for the quantitative measurement of lymphocyte proliferation. Antigen-activated patient cells added to IL2, showed greater increase in ^3H-TdR uptake than IL-2-untreated cells, and were capable of acquiring IL2 responsiveness. Increased numbers of the cells were observed by both the PI- and trypan blue-staining methods. In contrast, unstimulated cells also showed increased ^3H-TdR uptake response without increased cell numbers on stimulation with r-IL2 by a 6.8 to 19.3 fold stimulation index compared to the antigen-activated cells. The results indicated that the unstimulated cells in addition with r-IL2 still remained in the initial phase of the DNA-synthetic (S) period through the cell cycle, whereas the activated cells had passed through the post-synthetic gap (C_2) and/or cell division at mitosis (M). ^3H-TdR uptake of cultured cells usually demonstrates the presence of antigen-sensitized lymphocytes by in vitro proliferative response, and shows an increased stimulation index in 4 or 7 day cultured cells on stimulation with allergens. However, proliferation of some cell populations decreased; furthermore no distinct differences in cell proliferation between allergic and normal lymphocytes were observed, as is usual in this assay, although the present method was capable of measuring increased-IL2 responsiveness of patient lymphocytes. The results indicate that the ^3H-TdR uptake method can not be substituted for cell enumeration in the evaluation of the antigen-specificity of induced-IL2 responsiveness of activated cells. Therefore, cell enumeration using the PI-staining method may be preferable in this system. The induced response was observed in lymphocytes from patients with atopic dermatitis, bronchial asthma and/or allergic rhinitis specifically on stimulation with the antigen causing clinical symptoms. The results obtained using the PI-staining method were very similar to those in previous reports where the trypan blue staining method was employed. The present method appears to be capable of rapidly screening etiological antigens for disease and easily monitoring clinical activity.

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Bone marrow cells from BALB/c, C3H/He and WBB6F_1+/+ mice were cultured for 5 wks in the presence of the culture supernatant from prokoweed mitogen-stimulated spleen cells to assess and compare the degree of growth, proliferation and chemical mediator release of the mast cells (BMMC) derived from them. BMMC, which were positive to alcian blue staining, were found in the suspension cells on the culture of the bone marrow cells of either species of mice after 2 wk culture. The percentages of BMMC in the supension cells were increased with time of culture, reaching more than 90% after 5 wks. No differences in the growth and proliferation rate among BMMC from these three species were observed. However, in regard to the amount of anaphylactic leukotriene (LT) and histamine release. BMMC from BALB/c mice were superior to those from other species. From the above results, subsequent experiments were executed with BMMC from BALB/c mice. There was no obvious difference in the releasability of anaphylactic mediators among BMMC obtained at any stages of the passage during 4-12 wk culture. On the other hand, although BMMC cultured for 4 and 5 wks well responded to Ca ionophore A23187 for these mediator release, those for 6 to 12 wks obviously deteriorated with prolongation of the culture. The time couse of the anaphylactic release of immunoreactive (i-) LTB_4, i-LTC_4 and histamine from BMMC revealed that almost maximum release was reached at 10, 20 and 5 min, respectively, after antigen challenge. Several drugs incluidng antiallergics and β-stimulants had no effect on their release. From these results, it is suggested that present BMMC may be inadequate cells for evaluation of antiallergic drugs that can inhibit the anaphylactic mediator release, but may be useful for the research of the mechanism of the release because the cells likely release the mediators without occurrence of complicated subordinate reactions.

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Pathophysiological changes in the airways with aging were examined in 78 patients with bronchial asthma. The FEV1.0% values in patients over the age of 71, and the %MMF, %V^^._<50> and %V^^._<25> values in those over 51 were significantly lower than those of patients between the ages of 10 and 30. The frequency of lymphocytes in bronchoalveolar lavage (BAL) fluid was significantly higher in patients aged between 61 and 70 than in those aged between 41 and 50 (p<0.05). The frequency of each clinical asthma type changed with aging; the number of patients with simple bronchoconstriction type (type Ia) decreased with increasing age in patients under the age of 60, and the number of those with bronchiolar obstruction type (type II) increased with aging. The frequency of patients with bronchoconstriction + hypersecretion type (type Ib) showed a peak between the ages of 51 and 60. Bronchial reactivity to methacholine showed a tendency to decrease with aging. The release of histamine from leucocytes induced by Ca ionophore A23187 was significantly higher in patients between the ages of 10 and 30 than in those between the ages of 51 and 60 (p<0.05) and between 61 and 70 (p<0.01).

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In order to establish a safer and simpler antigen administration method in immunotherapy, we prepared biodegradable microspheres containing antigen and evaluated its safety and efficacy using guinea pigs. Poly (lactic/glycolic acid); (LGA) microspheres containing ovalbumin (OA) were fabricated by solvent evaporation. Over 70% of the OA was released from the microspheres within 3 days, and release was completed within 14 days in vivo. The local tissue reactions to the OA-LGA microspheres were apparently weaker than those to OA-alum. Repeated injections of high dose OA-LGA microspheres to OA-sensitized guinea pigs (high-LGA group) for 8 weeks at intervals of 2 weeks elicited an excellent therapeutic effect, i.e. a significant increase in the threshold value of antigen inhalation test, with a significant increase in IgG2 blocking antibody. The therapeutic efficacy of the high-LGA group was comparable to the conventional immunotherapy model (conventional group) and was superior to the antigen-alum model (alum group). We concluded that administration of antigen-LGA microspheres could become a new immunotherapeutic method for allergic disorders, being safer and requiring a lower frequency of antigen injections than the conventional method.

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From a clinical study it was found that serum C1INH activity increased 6 hours after methylprednisolone (MP) pulse therapy in all the patients we studied with connective tissue diseases. Furthermore plasma C1INH-Cls complex decreased after pulse therapy. These phenomena lead us to investigate the effect of MP on C1s^^-. 1) When a constant amount of C1s^^- (8 μg/ml) was incubated with several concentrations of MP (2-50 mg/ml), the C1s^^- activity of consuming C4 hemolysis was inhibited by MP in a dose-dependent manner. 2) MP inhibited consumption of C2 as well as C4 by C1s^^- in a dose-dependent manner, even when MP had been removed by dialysis following incubation with C1s^^-. These experimental data suggested that the trace amounts of C1s^^- generated by immune complexes could be inhibited by long circulation of MP even at low concentrations in vivo, and resulted in a decrease of C1INH consumption and C1INH-Cls complex formation. These results indicated that the inhibition of C1s^^- activity is one of the most important mechanisms in the process of the anti-inflammatory effect of MP in vivo.

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A statistical analysis of the annual day of onset of Japanese cedar pollinosis was carried out on a total of 305 patients seen at the out-patient clinic for allergic diseases in the Department of Otorhinolaryngology of Kyoto Prefectural University of Medicine between 1989 and 1991 (3 years). The day of onset varied among individuals and was distributed over a period of about one month, in patient number statistics, however, a clear single peak was seen for all 3 years. The day of onset in most patients showed tendency to peak after January 1, i.e., when the maximum temperature integral is approximately 450℃, on warm days where the maximum temperature exceeds 15℃, on days where there is little rain, and on days when there is a strong southerly wind. This peak onset day is about 3 weeks after first day of pollen count, or 3 or 4 days before the first dispersion peak, which corresponds to the day on which pollen dispersion begins in earnest. Furthermore, it was found that there was a drastic increase in the attack rate (from 10% to more than 50%) in pollinosis patients about 1 week before the peak day of onset. By the first dispersion peak, 70-80% of the patients had experienced an attack. The results of the present study may be useful in pollen forecasting and in treating early pollinosis in the dispersion season.

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A six-month old baby, who had been fed for 2 months with casein hydrolysate formula (MA-1) for treatment of milk allergy, developed diarrhea. the baby's RAST score for milk was negative. IgE antibodies to MA-1 but not to MA-1 depleted of lipid (fat-free MA-1), were demonstrated by enzyme-linked immunosorbent assay (ESISA). It was confirmed by the appearance of numerous mast cells and eosinophils in a rectal smear taken after challenge with MA-1 (but not detected after challenge with fat-free MA-1) that some component in the lipid of MA-1 must be an allergen in this patinet.

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A 35-year-old male doctor experienced an anaphylactic reaction of uriticaria, unconsciousness and hypotension one hour after taking a meal including crab meat and alcohol (beer 350 ml + Japanese sake 100 ml). He recovered within several hours after emergency treatment. Another attack occurred 6 months later after taking a meal including crab meat and alcohol. Serum IgE showed 3073 IU/ml. IgE-RAST and skin tests were positive for crab. A crab meat plus alcohol challenge test revealed a slight increase in plasma histamine level. This is a rare case of food (crab meat)-dependent anaphylaxis possibly induced by alcohol.

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The presence of β-endorphin (β-end) was immunohistologically identified in synovial tissue samples biopsied from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The amount of β-end in culture supernatants of synovial tissue explants was also determined by RIA. β-end was strongly detected in mainly superficial synovial cells, vascular endothelial cells and a few synovial interstitial cells in RA patients, but not in OA patients. In RA patients the β-end concentration was significantly higher in the supernatants of tissue explants (26.4±8.3 pg/ml) than in the plasma of the same patients (15.3±2.5 pg/ml) (p<0.01). Using isolated synovial cells, the β-end concentration in the culture supernatants of non-adherent cells (19.4 pg/ml) was higher than that of adherent cells (4.0 pg/ml). It is suggested that β-end is produced by non-adherent cells such as lymphocytes and neutrophils in addition to synovial lining cells and endothelial cells and may play some role in the pathology of RA synovial inflammation.

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To determine whether tazanolast inhibits airway hyperresponsiveness, we studied the effect of this drug on platelet activating factor (PAF)-induced airway hyperresponsiveness in guinea pigs. Inhalation of PAF (1 μg/ml) caused significant airway hyperresponsiveness to acetylcholine (p<0.01) or histamine (p<0.05). Pretreatment with tazanolast (30-300 mg/kg) produced a dose-dependent inhibition of airway hyperresponsiveness induced by PAF inhalation, and significant inhibition (p<0.05) was obtained with the drug (300 mg/kg). Aspirin also inhibited PAF-induced airway hyperresponsiveness, while tranilast produced hardly any inhibition. From these results, it is suggested that tazanolast is effective in inhibiting airway hyperresponsiveness.

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