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We established an enzyme-linked immunosorbent assay (ELISA) for determining moth-related antigens in house dust. Samples, biotinated anti-moth antibodies, and horseradish peroxidase labeled streptavidin were reacted with the microplate on which anti-moth rat polyclonal antibodies were absorbed. The concentration of moth antigens was correlated with absorbance in the range from 0.1 to 100 micrograms/ml. Cross-reaction with other insect antigens was observed; 10 micrograms of midge antigens and 100 micrograms of mosquito antigens corresponded to 1 microgram of the moth antigens, respectively. With this method, moth-related antigens can be detected in house dust. However, there was no remarkable difference in antigen levels among the three locations examined: a large city, a small town, and a rural area.

In our clinic we have encountered nasal allergy due to fungi such as Candida, Aspergillus, and Alternaria. We therefore conducted a retrospective study of patients who consulted our Department between 1984 and 1991. A clinical review of patients who showed a positive skin test for Candida, Aspergillus, or Alternaria was performed. Our study revealed that Alternaria showed the highest ratio of strong positivity upon both RAST and skin test among the three fungi examined. These results led us to reconsider Alternaria as an important fungal antigen.

In Japan, pollen of Cryptomeria japonica and Chamaecyparis obtusa are a yearly source of distress for many people suffering seasonally from allergic rhinitis. To study common epitopes shared by the two species, two monoclonal antibodies (moAbs) were raised against Cry j 1, which is the most predominant allergen in C. japonica. One of the moAbs was found to be reactive even to the major allergen of C. obtusa, demonstrating that the moAb (J1B01) can detect an epitope shared by both species J1B01 strongly inhibited the binding of the major allergens of C. japonica and C. obtusa to IgE of patients who are sensitive to C. japonica and C. obtusa. This finding signifies the importance of the epitope recognized by J1B01.

To determine the importance of silkworm moth allergens, we tested 267 patients with allergic rhinitis with CAP-RAST, a new assay system for detecting specific IgE in sera. We used the following allergen sources: house-dust mite, Candida, Alternaria, cat dander, orchard grass, ragweed, mugwort, Japanese hops, Japanese cedar, Japanese cypress, cotton, silk, larval chironomid midge, adult chironomid midge, and silkworm moth. As expected, the highest positive rate of reaction, as determined by CAP-RAST, was 73.8% for Japanese cedar, followed by 53.2% for mite and 50.6% for Japanese cypress. Although the positive rate for silkworm moth was not as high as for cedar pollen, one-third of patients had specific IgE against silkworm moth. The positive rate of reaction to silkworm moth was much higher than that to chironomid. It was interesting to note that the patients reported had not been documented as having frequent contact with silkworm moth allergen. Correlations between silkworm moth and silk, and silkworm moth and chironomid midge were found to be significant (P < 0.001). Silkworm moth allergen showed the third highest reaction rate in patients with severe symptoms. These results suggest that silkworm moth allergen should be considered to be important in patients with allergic rhinitis in Japan.

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.

Conventional sectioning of frozen and fixed tissues is usually performed using the glass knife. Although the glass knife is economical, it has several disadvantages. In this study, the Authors have examined the sectioning quality of the sapphire knife in frozen tissues as an alternative to the glass knife. It was concluded that the sapphire knife has better quality and durability than the conventional glass knife in cutting frozen tissues.

The significance of inhibitors in the coagulation-fibrinolysis system was examined by inducing experimental inflammation with carrageenin. Fast-acting plasmin inhibitor, alpha 2-plasmin inhibitor (alpha 2-Pl), was recently discovered in human blood and it was stated that such alpha 2-Pl represents the most sensitive and specific inhibitor of plasmin. The possible role of alpha 2-Pl has been studied in detail in vitro and in vivo in relation to the thrombolytic condition and bleeding based on hyperfibrinolysis. However, the significance of alpha 2-Pl has not been clarified for the inflammatory state and diseases. In the present study in order to clarify the possible role of alpha 2-Pl in inflammatory responses, the antiplasmin activity in the plasma and local fluid (exudate) was investigated using an animal model of inflammation. After subcutaneous injection of carrageenin into the dorsum of rats, on day 7 and 21, the antiplasmin activity in the plasma was significantly higher than that in the plasma of non-treated rats (p less than 0.001). However, the antiplasmin activity in the plasma on day 7 was not significantly different from that on day 21. On the other hand, the antiplasmin activity in the exudate on day 21 was significantly lower than that on day 7 (p less than 0.001). Based on ;these results, it is suggested that the antiplasmin in the inflammatory exudate was consumed during the inflammation to form the hard wall of the carrageenin pouch.

In order to clarify the pathophysiological significance of the fibrinogen (FgDP)-protamine complex, the interaction between fibrinogen and protamine sulphate was studied in vitro and in vivo. Using the electrophoretic technique, an attempt was made to determine whether the complex could be detected in vitro and in the circulating blood. From the present studies, it has been clarified in in vitro experiments that the slower mobility of two peaks on immunoelectrophoresis represented a complex of protamine sulphate and fibrinogen (FgDP). Furthermore, the peak of slower mobility on crossed immunoelectrophoresis appeared in the circulating blood of a rabbit receiving administration of excess protamine sulphate. That is, in vivo experiments showed that a complex of protamine sulphate and fibrinogen (FgDP) could be detected in the circulating blood.

Previous reports have shown that fibrinolytic activity was increased in the circulatory blood of rabbits with Arthus tonsillitis. In this study, t-AMCHA was administered intravenously to rabbits, in order to clarify its role as an antiplasminic agent in the control of Arthus tonsillitis. First, to establish the proper method of administration of t-AMCHA and the quantity to be administered, a preliminary estimation was made of the requisite concentration of t-AMCHA in the circulating blood and tonsillar tissue. Then macroscopic observations and histopathological studies were performed on the tonsils of rabbits with Arthus tonsillitis after t-AMCHA administration at various doses. Finally, after injection of the antiplasminic agent, estimations were made of the plasma fibrinolytic activity in the blood of the rabbits under study. It was decided that administration of t-AMCHA at 3-hourly intervals could maintain a constant concentration of the substance in the tonsillar tissue. The marked hyperaemia and bleeding which were observed macroscopically and histopathologically at 1-2 days after the onset of tonsillitis, disappeared from the parenchyma of the tonsils and pharyngeal tissue surrounding the tonsils in the group receiving regular injections of t-AMCHA at 3-hourly intervals. After estimating certain parameters of the fibrinolytic system in the blood, it was found that fibrinolytic activity decreased and whole plasmin was not consumed as a result of the administration of t-AMCHA during the early stage of tonsillitis. The process of Arthus tonsillitis can thus be controlled by the administration of the antiplasminic agent.