Phagocytosis or the interaction of certain agents with membrane receptors results in greatly enhanced rates of leucocytes oxygen metabolism (respiratory burst) and production of active oxygens such as superoxide (O^-₂) which rapidly dismutes to yield hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH⋅) . Active oxygens produced are used to generate powerful microbicidal agents such as hypochlorous acid (HOCl) . The respiratory burst results from the activation of an enzyme (NADPH oxidase), dormant in resting cells, which catalyzes the following reaction: NADPH+2O^-₂→2O^-₂+NADP⁺+H⁺. The oxidase is considered to be an FAD-requiring enzyme. Cytochrome b₅₅₈ which is found uniquely in phagocytes is also proposed to be an electron carrier. The mechanism by which the oxidase is activated on stimulation of the cells is not clearly understood. Many informations, however, have been gathered concerning the activation event. Calcium ion is essential for the activation and protein phosphorylation by protein kinase C which is activated by diacylglycerol, produced through phosphatidylinositol turnover, appears to be involved in the activation reaction. Cyclic AMP and the products of arachidonic acid cascade regulate the superoxide production.

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MRL mice develops a T cell lymphoproliferative syndrome associated with a systemic lupus erythematosus (SLE) like syndrome and necrotizing arteritis. We detected marked age related variations in major immunoglobulin classes of antibodies to single stranded (ss) DNA in sera of MRL l/l mice. The levels of IgA anti-ssDNA anti-bodies are increased with age, and significantly correlated with the levels of IgA containing circulating immune complexes (IgA-CIC) . IgA-CIC were detected in various diseases including allergic vasculitis and Henoch-Schönlein purpura. IgA-CIC and IgA anti-ssDNA antibodies may play a role in necrotizing arteritis in MRL l/l mice.

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The measurement of leukotriene (LT) C₄ in biological fluid by radioimmunoassay was studied. We have developed a rapid and simple extraction method of LTC₄ from human plasma and synovial fluid for radioimmunoassay. In this extraction method, LTC₄ was recovered in a final methanol fraction by 91% and 76% from plasma and synovial fluid, respectively. The propriety was confirmed by the recovery test and by the dilution method.

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Granules were isolated from sonicated rat mast cells on a Percoll gradient and incubated with [γ³²P] ATP in the presence of Mg²⁺. After stopping the reaction, SDS/PAG electrophoresis was performed and autoradiographs were prepared to determine the incorporation of ³²P into proteins. Intact membrane granules developed two broad and two narrow radioactive bands corresponding to the major granule proteins in 26-40K range. However, almost all of the radioactivity was removed by precipitation with TCA and extraction with acetone. Moreover, when each of the proteins in these bands was acid hydrolysed and analysed, no phosphoserine, phosphothreonine or phosphotyrosine was detected. The results with broken granules differed in that in addition of the reversible major gananule protein labeling, a discreet, relatively prominent labeled band was present at the 44K area both in reduced and unreduced gels. The radioactivity in this area was not removed by TCA and acetone and in Coomassie Brillant Blue stains of electropherogram, a weakly staining band was observed in the 44K area irrespective of the presence of 2-ME. After acid hydrolysis of the 44K protein in broken granules, ³²P was recovered in phosphoserine and phosphothreonine. Intact membrane granules with hypotonic treatment produced ³²P incorporation into the 44K band. Extensive washing of the broken granules did not significantly affect the incorporation of ³²P into the 44K band and addition of cytosol to the granules did not increase the response. Taking these observations, it is apparent that at least one protein in mast cell granules with an apparent molecular weight of 44K in SDS/PAG electrophoresis can be phosphorylated in the presence of ATP and Mg²⁺.

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Ca²+ chylating reagent (Ethylene glycol-bis (aminoethyl ether) tetra acetic acid; EGTA), Voltage dependent Ca²⁺ channel blocker (Verapamil) and calmoduline antagonist (Trifluoperazine; TFP) were tested to evaluate the influence of Ca²⁺ on the O^-₂ production of leukocyte stimulated by formyl-methionyl-leucyl-phenyl-alanine (FMLP), concanavalin A (Con A) and cytochalasin D (Cyt D) . EGTA inhibited O^-₂ production stimulated by Con A+Cyt D and Cyt D+FMLP but did not inhibit significantly it stimulated only by FMLP. EGTA did not inhibit depolarization but inhibited repolarization of membrane potential by the stimulants described above. These facts made us believe the significance of intra-and extra-cellular Ca²⁺ Verapamil inhibited O^-₂ production only with a slight change of depolarization. These facts made us thought that Ca²⁺ might influx into cells depending on the change of membrane potential. And since Verapamil inhibited O^-₂ production only at a high concentration, it could not be used clinically as an anti-inflammatory reagent. TFP inhibited O^-₂ production but had nonspecific action other than a calmoduline inhibitor. So the involvement of calmoduline on O^-₂ production and change of membrane potential was not clear.

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The effects of various kinds of prostaglandins on O^-/⋅₂ production of human PMN stimulated by opsonized zymosan were studied in vitro by a cytochrome C reduction method. O^-/⋅₂ production of PMN was inhibited by a 0.02μg/ml solusion of PGE₁ and a 0.2μg/ml solusion of PGE₂ up to 40% of that of the untreated cells. On the other hand, PGD₂, PGI₂, PGF_2α did not inhibit the O^-/⋅₂ production at a concentration of 0.2μg/ml. Antibody dependent cell mediated cytotoxicity (ADCC) of PMN was also inhibited by both PGE₁ and E₂ at a concentration of 0.2μg/ml up to 50% of cytotoxicity of the untreated cells. The reduction pattern of O^-/⋅₂ production from PMN by treatment with various prostaglandins was not parallel to the increase pattern of cytoplasmic cyclic AMP levels in the similarly treated PMN.

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The immunopotentiating agents including Maruyama Vaccine (SSM), BCG, picibanil and levamisole have been widely used for the treatment of malignant tumor (and autoimmune diseases) . The possible action mechanisms of these drugs have often been investigated in connection with lymphocytes. Recently, it has been shown that active oxygen (AO) generated by polymorphonuclear leucocytes (PMN) display strong antibacterial function and play an important role in the body's defense system. Furthermore, AO has been elucidated to show anti-tumor function, destroying DNA in cancer cells. In addition, the author verified that SSM increases AO generation by PMN in in vitro. In this study, the effect of SSM on nineteen patients with various terminal cancers was investigated, simultaneously with the determination of AO generation by their PMNs. There was no difference of survival rate between the patients with decreased AO generation and those who showed normal AO levels produced their PMNs, before the injection of SSM. However, the cases which responded to SSM by increasing AO generation showed statistically higher survival rate than those who did not increase AO generation by their PMNs following SSM administration. Especially, two patients who were treated with SSM alone could be alive for no less than 9-11 m after the use of SSM. These two cases also well responded to SSM to increase their AO generation. Based on the results obtained in the present study and my previous report, SSM, though its action mechanism against tumor growth has not been completely elucidated, seems to be effective in cancer, by enhancing phagocytic function of primary self defense as well as lymphocytic function of secondary self defense mechanism.

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Since the increase in oxygen intermediate generation by neutrophils and its oxidative damages in the patients with Kawasaki disease were verified, superoxide dismutase (SOD) which quenches oxygen intermediates has been considered to be one of the beneficial therapy for Kawasaki disease. Liposomal encapsulated SOD (L-SOD) which A.M. Michelson developed was reported to be effective in various inflammatory disorders including those to whose pathogenesis an increase in oxygen intermediate generation has close correlation. We applied liposomal SOD injection in the treatment of six Kawasaki disease patients. The drug was effective in five of six patients. Fever faded away within four days after administration. Bilataral hyperemia of the bulbar conjunctiva, nonpurulent swelling of cervical lymph nodes and skin eruption were subsided in a few days after the injection. Formation of aneurysma of arteria coronaria was not observed in three patients out of four in whom coronaria arteria dilatation was confirmed. Unfortunately, one of four was not free of the formation of aneurysma. Liposomal SOD consists of some kinds of lipids and free SOD purified from bovine blood. It has advantages of long span (a harf life: 7.24 hr compared to 6 min of free SOD), better fixation and permeability to the tissues and organs, heat stability and organ specificity when compared to free SOD preparations. Since correlation of the formation of coronaria aneurysma in Kawasaki disease patients to the coagulatory activity activated by oxygen intermediates was reported, L-SOD seems to be applaisable for the treatment of Kawasaki disease and for the prevention of coronaria injury. The drug has the more advantages for its low or no toxicity.

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The effect of a Ca²⁺ -antagonist (Verapamil) on inflammation caused with CdCl₂ (5.0mg/kg) was determined in rat's testis. (1) Concomitant administration of CdCl₂ and Verapamil inhibited testicular hemorrhage and an elevation in tissue level of lipid peroxide in the testis. (2) When rats were administered with CdCl₂ alone, the content of phospholipids in the testis was decreased, while the content of free fatty acids in the testis was increased, whereas these changes were restored to normal after concomitant administration of Verapamil. (3) When rats were administered with CdCl₂ alone, the content of Ca in the testis was significantly increased, while the levels of Mg and K were decreased. These changes were also restored toward normal after concomitant administration of Verapamil. These results suggest that calcium plays an important role in the development of inflammation in the rat testis following the administration of CdCl₂.

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[in Japanese]

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