1. Antibodies specific to antipyrine were detected by gel diffusion, PCA reaction, passive hemagglu-tination and radioimmunoelectrophoresis in the sera of rabbits and guinea pigs immunized with antipyrine conjugated to some kinds carriers. 2. Divalent hapten, bis-(antipyrin-diazo)-ε-amino-caproic acid, did not stimulate antibody production, but elicited PCA reaction as the challenging antigen. 3. It was noticed that the method of labelling isotope to the carriers of antigens was easy and effective for detection of antibodies to haptens. 4. Circulating antibodies were not demonstrated in the sera of pyrazolone sensitive individuals by PCA reaction, passive hemagglutination, radioimmunoelectrophoresis and radioimmunodiffusion techniques.

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A study was made of the effect of disodium cromoglycate on the anaphylactic mediator release from lungs of three species in vitro models of the immediate hypersensitive reaction and the following results were obtained. 1) The release of anaphylactic mediator, histamine and SRS-A, from the lung of guinea pig sensitized with egg albumin was neither inhibited by disodium cromoglycate, nor by the incubated soltion of it with the liver homogenate of guinea pig. 2) Disodium cromoglycate did not inhibit the Schultz-Dale reaction in tracheal muscle preparation of guinea pig. 3) The amount of mediator release was slightly decreased by disodium cromoglycate from the lung of rat sensitized with egg albumin in complete Freund's adjuvant. 4) Disodium cromoglycate caused a significant reduction in the release of anaphylactic mediator from portions of chopped monkey lung passively sensitized with human sera sensitive to house dust.

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Changes of basophil leucocyte counts of the peripheral blood in 181 patients with bronchial asthma have been investigated and it has been shown that basophil leucocytes have a strict relation to asthmatic attacks; the basophils show a normal level in non-attacks as in healthy controls, an increase in pre-attacks, and tend to decrease in attacks. The observation of changes of peripheral basophils appears to make possible an early detection of the following attack, and it is suggested that the future attack will appear in almost cases when the basophil count is higher than the average upper limit (65/cmm). Similarly during the administration of glucocorticoids, the basophil count over the average upper limit is a sign of the incipient attack. Although basophils show high levels only in pre-attacks, eosinophils in asthmatic patients tend to be higher than healthy controls whether they are in non-attacks, or in preattacks, and so eosinophils has hardly a close relation to asthmatic attacks.

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Plasma kinin was studied from the clinical stand point in our lavoratories and the results presented. 1) The value of urinary kinin excretion ranged 5-35 μg per day in healthy persons, and no difference found between healthy persons and patients with various diseases. 2) The circulating plasma kinin was not excreted into the urine, and urinary kinin seemed to be produced in kidney tissue. 3) The kallikrein-kinin system is considered to play a physiological role in regulation of renal function. 4) Bradykininase activity was very strong in circulating blood, especially in liver diseases and in hyperthyroidism. On the other hand, this enzyme activity was markedly low in bronchial asthma. 5) Plasma kinin activity could not be detected in healthy persons. However, this activity was definitely increased in some cases of bronchial asthma, dermatological diseases and pancreatic diseases. 6) Plasma kallikreinogen levels in healthy persons ranged 3.5-4.4 Frey units per ml. In some patients with hyperthyroidism and with hepatitis, low kallikrenogen levels were observed. In some patients with bronchial asthma, however, high levels were found. 7) The levels of plasma kininogen in 10 healthy persons ranged 4.1-6.9 (mean 5.63±0.76)μg/ml. Kininogen I and II were 2.0-2.9 (mean 2.48±0.28)and 2.0-4.0(mean 3.15±0.55)μg/ml. 8) The kinin-forming activity in subcutaneous fluid of exanthema area of dermatological patients elevated more than that of normal control.

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Investigation of selective adsorvents for kinins from bilogical materials was carried out. Inorganic polymer consisting of silicic acid, such as silica gel florisil, kaolin or acidic terra abla was a suitable adsorbent for kinin condensation. Among them, silica gel was the best one which adsorbed kinins tightly from aqueous or alcholic solution while albumin, acidic or neutral amino acids, urea or salts were not adsorbed. The kinins on silica gel was easily eluated with the mixture of acetic and pyridin. Recovery rate of added 50 ng of bradykinin in body fluids was 40-80%. The method was applied for the analysis of urinary kinins plasma kinins or kinins in amniotic fluid or lymph. The values obtained were 10-20 ng/ml (human urine), 5-10 ng/ml (human amniotic fluid) and below 1 ng/ml (human plasma and dog lymph). Kinin fraction from human urine was further purified by ion-exchange chromatography. Bradykinin and kallidin were identified by DNS-method.

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Inhibiting effect of glutathione (GSH) on kininase in various tissues kinin effect potentiation of it were investigated. Kininase activity in various tissue homogenate (lung, kidney, intestine, pancreas, adrenals, testis, heart, spleen, liver, brain etc.) of rabbit was inhibited at the concentration above 10 μM. Fifty per cent inhibition of kininase activity in serum was at concentration of 60 μM. In contrast, the inhibition by cysteine and thioglycollate were at the concentration of 300 μM and that by o-phenan-throline was at that of 6 μM. Kinetic study showed that GSH inhibited the binding of kinin to kininase in competitive manner. Dose-response curve of bradykinin shifted to left in the presence of GSH. Phatmscological actions of kinin and kinin forming enzyme were potentiated; i. e., the hypotensive effect of bradykinin was strengthened by the pretreatment of GSH or their simultaneous administeration to the animal. Other kininase inhibitors such substances as o-phenanthroline, cystein and Ca-EDTA had almost no potenciative effect. There was , therefore, no parallelism between kinin effect potentiation and the inhibiting power for kininase. GSH was pharmacologically inctive unless kinin was present; i. e., the hypotensive effect of GSH appeared only in the condition that kallikrein was present in active state. When it was inactivated by Trasylol, GSH could not show any effect. Pharmacological actions of kinin such as vasodilatation, capillary permeability increment smooth muscle stmulation and blood pressure lowering were potentiated significantly by GSH and not by the other kininase inhibitors. GSH specifically modified the effect of kinin but not the effects of eledoisin, ATP, Ach, 5-HT and histamine. Accordingly, GSH can be used as a tool to determine whether kinin partcipiates in a certain physiological and pathological event in the body or not.

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The degree of the degradation of tosyl-arginine methyl ester (TAME) by kallikreins can be deter-mined by means of the measurement of methanol liberated from TAME. Methanol was oxidized to form-aldehyde by KMnO, and the amount was determined colorimetrically after the formation of a stable chro-motropic acid-formaldehyde complex. This method is very easy to perform and is 10 times more sensitive than the usual method which bases on the measurement of the residual TAME. Good agreement was obtained in comparison of the results of the both methods. Using this new method, the esterolytic activities of guinea pig coagulating gland kallikrein (CGK) and the same fractions of the prostate gland, pancrease and submaxillary gland were assayed and the most potent activity was found in the coagulating gland. The same observation was obtained in the comparison of the other biological activities of those fractions. The kinin released by CGK was recognized as bradykinin through its property on paper, CM-cellu-lose column and thin-layer chromatographies. This was confirmed by the finding as the N-terminal amino residue of the kinin. Besides kallikrein, the coagulating gland contains a potent kininase, too. The kininase formed insoluble precipitate and was mostly removed through dialysis against distilled water, but not against saline solution. Following this simple procedure for the separation of kininase, the isoelectrofocusing frac-tionation was carried out for the purification of CGK, and CGK was eluted out between pH 3 to 4. Further purification is mow undergoing.

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It has recently been shown that the contractile action of bradykinin on isolated smooth muscles is potentiated by vatious substances, such as dimercapral, cysteine, thioglycolic acid, glutathione and some fibrinopeptides. Proteolytic enzymes, such as chymotrypsin and trypsin sensitize smooth muscle to bradykinin. We isolated the bradykinin-potentiating factors from the venom of Agkistrodon halys blomooffii (trivial name "Mamushi") and studied on the structure of one of the potentiators. Partial structure of one of the potentiators is shown as follows; (Glu, Gly, Leu, Pro_3, Arg)-Lys-Ile-Pro-Pro. The structural relation between potentiators and bradykinin seems interesting, because of their high proline content.

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The effect of bradykinin to allergic reaction was examined. As the allergie reaction, Arthus reaction in actively sensitized rabbits with egg-albumin, PCA (passive cutaneous anaphylaxis) in guinea pigs with egg-albumin and anti-rabbit egg albumin system, Schwartzman phenomenon (Schwartzman filtrate isolated from E. coli filtrate) in robbits and Thomas reaction in rabbits were tested. Synthesized bradykinin was usually intravenously administered within 5 minutes after the challenging injection except in the cases of Arthus phenomenon 2 hours after the challenging injection. The results show that the administretion of bradykinin potentiate the allergic or allergy-like reaction such as Arthus reaction, PCA, Schwartzman phenomenon and Thomas reaction. These results may suggest the role of bradykinin in allergic reaction.

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1) The mast cells isolated from the peritoneal fluid of rats are easily degranulated following the incubation with either α-chymotrypsin (10-300γ/ml), sinomenine (3-100γ/ml), dextran (100-1, 500γ/ml)or compound 48/80 (0.1-1 γ/ml). The activity is approximately equal to α-chymotrypsin in a concentration of 300γ/ml, sinomenine in that of 100γ/ml and compound 48/80 in that of 0.5γ/ml and some what weaker in 1,500γ/ml of dextran. 2) Preincubation of the mast cells with either diphenhydramine (10-1,000γ/ml), cyproheptadine (1-100γ/ml), homochlorcyclizine(1-100γ/ml) or dexamethasone (0.1-10γ/ml) antagonizes the effect by the histamine liberators. The activity of cyproheptadine, homochlorcyclizine and desamethasone is approximately the same in potency, while that of diphenhydramine is somewhat weaker than that of the others.

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Recently it has focused the attention of investigators that a certain kinds of polypeptides which were looked to provoke pain sensation, might take some common or similar pathways to be developed, to agents of blood coagulation or fibrinolysis, but their virtual and detailed mechanism was not cleared up yet. The authors tried to scrutinize the relationship between them, following up the shift of coagulation-fibrinolysis system in typical diseases or syndromes in which pain sensation could be invited relatively simple maniplations and particularly the correspondence of the activation of Hageman factor (Factor XII) with the enhancement of fibrinolysis. In a patient with Raynaud's syndrome who began to feel severe pain together with violet colouration at his left forearm shortly after it was braced tightly with a cuff at the pressure of 140 mmHg. Blood was drawn by venipuncture from cubital veins of both forearms before and at 5 minutes after the bracement, and the non-contact and contact partial thromboplastin times (PTT) aed euglobulin lysis time (eug LT) were measured to be found that the non-contact PTT was shorter at the braced side than the other, but the fibrinolytic activity measured by eug LT did not show any correspondence with this shift of Factor XII activity. At the same time, however, the Rumpel-Leede test for capillary fragility was proved to be higher positive at the braced side. This poor causal relationship between PTT and eug LT was also found in blood of many cases with Raynaud's disease or syndrome. Eisen proposed a schema for activation mechanism of plasmakinin and its allied materials in 1966, in which plasmin should play active role, and in our cases pain sensation and elevated capillary permeability were invited by the ischemic muscle contraction test, but the participation of fibrinolysis agents was not proved so eloquently. This interruption of causal relation between activation of fibrinolysis and enhancement of coagulation which was observed in blood of various cases, such as with thromboangitis obliterans (Buerger's disease), thrombophlebitis acuta, blood vessel trauma, etc. This discrepancy was further observed in vitro experiments in which contact factors were activated by contact with celite, kaolin, glass powder, etc., and was also supported by data of Suzuki et al. and Fujii et al. who applied the most purified agents sofar in their reaction systems.

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Nine phlogogenic gubstances such as Eledoisin-like substance, Physalaemin and Bradykinin were in-jected intradermally and the inhibitory effect of Homoclomin-given orally on the intracutaneous reaction caused by them was observed in 30 subjects. The intradermal reaction caused by 8 of them including Eledoisin-like substance was suppressed was by internal use of Homoclomin-tablet. On the other hand, swelled eruption caused by intracutaneous injection of Physalaemin was not inhibited by oral use of Homo-clomin-in all of the 4 subjects examined. In this case swelled eruption has a strong resemblance to spon-taneous one, though accompanied no subjective symptoms such as itchihg sensation. The state of degra-nulation in mast cells and changes in blood vessels and peripheral nervous elements were compared hitopathologically immediately after an injection of these substances in 0 subjects, in 12 of whom intracu-taneous reaction had been observed. In the four subjects given intradermal injection of Physalaemin the intensity of degranulation was not parallel with changes in peripheral nervous elements. Regarding the intensity of degranulation Physalaemin held the sixth rank in 9 substances. On the contrary, it was ranked first with regards changes in blood vessels and peripheral nervous elements. At least in those 4 cases, therefore, it was hard to consider that degranulation of mast cells played a so important role in genesis of swelled eruption by Physalaemin. The networks of peripheral vegetative and viscero-sensory nervous elements with reactive capillaries in the papillae of the skin was regarded as rather important.

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Bradykininase activity in plasma was estimated in 20 healthy subjects and 42 patients with bronchial asthma. The assay method consists of the incubation of synthetic bradykinin with venous blood plasma at 37℃ for 15 minutes and assay of remaining bradykinin on the dog systemic blood pressure. Bradyki-ninase activity was expressed by the percent destruction of bradykinin after incubation. Twenty healthy subjects were subjected to this study. The bradykininase activity ranged 26-64% (mean 47.2 ± 8.3%) in these normal controls. Forty-two asthmatic patients were subjected to this study. The bradykininase level ranged 16-57% (mean 36.4 ± 10.0%). Because bradykininasel eve lin bronchial asthma seems to be lower in the patients with severe attacks than ones with mild clinical signs and symptoms, all the patients were divided into three categories according to the "Ito's classification". Bradykininase activities were 25-57% (mean 41.3 ± 9.2%) in mild cases, 16-47% (mean 32.0 ± 8.0%) in moderate cases, 17-31% (mean 24.7 ± 7.1%) in sever cases. On the other hand, higher than normal bradykininase activities were observed in asthmatic patient under steroid therapy.

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