A double isotopic enzymatic assay of histamine is sensitive and specific method, and applicable for a variety of samples, viz. plasma, sputa and urine, because the recovery of histamine is determined by addition of known amount of ^3H-histamine. This report concerns the modification of analytical procedure. 1. An enzyme solution of histamine-N-methyltransferase was prepared from pig brains. 2. The incubated solution was washed with ethyl acetate at acidic pH after incubation for 90 minutes. The quantitative limit was reduced to 0.25 ng in 100 or 250 μl of plasma by this procedure. The method using ^3H-S-adenosyl-L-methionine and ^<14>C-histamine was also studied and histamine in plasma was quantitated as little as 0.1 ng of histamine in 100 μl of plasma. 3. Determination of histamine was interfered with a large amount of compound 48/80 (<5μg). It was presumed that compound 48/80 was methylated as a substrate by histamine-N-methyltransferase. 4. Histamine content in one basophil was 0.98±0.28 (mean±SD) ng in a quiescent state of 16 asthmatic patients. Histamine level in urine and sputa was much higher during the attack in an asthmatic patient. In positive cases by inhalation with the allergen, it was observed that the histamine level in plasma increased at 15 and 30 minutes after challenge.

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Immunological tolerance against RGG was induced easily in adult C3H mice by the administration of normal RGG deaggregated by ultracentrifugation. But it could hardly be induced by the administration of deaggregated RGG containing small dose of anti-mouse Ig antibody (ab-RGG), and the threshold of tolerance against ab-RGG was high. The same was observed by the administration of deaggregated RGG containing small dose of anti-mouse serum albumin antibody. To elucidate this fact further, 1 mg of deaggregated RGG containing various amount of specifically purified anti-mouse Ig antibody was used as tolerogen. Though the tolerance occured in all animals by the RGG containing less than 10 μg of antibody, it did not occur in about half of animals by the RGG containing 25 μg of antibody. The tolerance was not induced in almost all of animals by the RGG containing 100 μg of antibody. That is, when the amount of antibody contained in RGG increased, the rate of animals became tolerant decreased. The tolerance occured easily by the deaggregated ab-RGG in animals pretreated by suitable amount of carrageenan which was known to impede the function of macrophage. From these results, it was concluded that the induction of tolerance was inhibited by the presence of antibody against host component regardless of its specificity. So, it was suggested that the immune complex produced in vivo and phagocytosis of it by macrophage played a important role in determining whether tolerance was induced or not.

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For the purpose of studying the response to β-adrenergic stimulation in patients with bronchial asthma, the changes in blood glucose, lactic acid and pyruvic acid were estimated by subcutaneous injection of 7 μg/kg of epirenamine hydrochloride in 37 asthmatics, especially refering the severity and type of asthma. Also the changes in cyclic adenosine monophosphate (cAMP) by epirenamine administration were studied in 5 asthmatics. The following results were obtained. 1) The increasing rates of blood glucose, lactic acid and pyruvic acid in response to epirenamine were less in asthmatics than in normal subjects. As to the severity of asthma, these increasing rates were less in severe asthmatics than in mild and moderate cases. These increasing rates were less in patients with chronic type than with paroxysmal type of asthma. On the other hand, there was found significant relationship between the charges of these metabolites and the suffering duration of asthmatic patients. 2) The increasing rate of cAMP in response to epirenamine was less in asthmatics. This rate was less even in mild cases of asthmatics. From the results above mentioned, it was considered that the response to β-adrenergic stimulation was diminished in asthmatics, especially in severe and chronic type.

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Role of complement system in the release of lysosomal enzymes from chronic myelogenous leukemia cells, mouse Ehrlich ascites tumor cells and mouse Sarcoma-180 cells was investigated in vitro. It was found that activation of the alternative pathway of complement by the addition of inulin, zymosan or bacterial lipopolysaccharide (endotoxin) markedly enhanced the release of lysosomal enzymes, β-glucuronidase and acid phosphatase, from these cells. Addition of EDTA prevented the release of lysosomal enzymes from these cells, while EGTA-Mg^<++> did not, indicating the enhanced release of the lysosomal enzymes was due to activation of the alternative pathway of complement. It was also found that the labilization of lysosome by the alternative pathway of complement did not led the cell destruction. It is concluded that activation of the complement system, especially the alternative pathway, plays a role in the release of lysosomal enzymes from tumor cells without cell injury.

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A few improvements on 2-step LIF agarose assay were tried, and the fundamental details investigated. Lymphocytes from the healthy were suspended in MEM with 20% autologous plasma at a cell concentration of 3×10^6/2 ml. Supernatants with LIF activity were harvested by centrifugation after the culture was done for 3 days under the stimulation of PHA-P (20 μg/2 ml). Healthy human peripheral leukocytes were used as migrating indicator cells for evaluation of LIF activity. In this method, it seemed that the changes of pH and residual PHA of low concentration of culture supernatant could be excluded as influencing factors on the migration area of leukocytes. It was disclosed that LIF activity was contained in fractions of albumin when analyzed by gel filtration. The trials, which appeared to have resulted in stability and reproducibility of the experiments, were as follows: (1) Leukocyte migration plates (Sterilin) were used instead of glass Petri dish. (2) Uniformly 500×10^4 leukocytes were added in 1 ml of culture supernatant. (3) After 30 minutes incubation, accurately 150×10^4 leukocytes from a centrifugation sediment were placed in a well of agarose plate.

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One hundred and seven samples of colostrum were obtained from healthy mothers on the 1st to 6th postpartal days. Paired samples of serum and colostrum were obtained from 13 healthy mothers. IgE levels in colostrum and serum were measured by radioimmunosorbent test (RIST) and paper radioimmunosorbent test (PRIST). At the same time, immunoadsorption of IgE from colostrum with the immunoadsorbent containing anti-human IgE (AHIgE) was carried out. The results were as follows. 1) The mean IgE levels in colostrum by RIST for the postpartal six days were respectively as follows: 538.9±324.4, 496.5±430.8, 465.5±505.5, 434.9±515.1, 304.2±90.8 and 392.9±175.8 unit/ml. 2) IgE in colostrum by PRIST was not detected in 13 out of 19 samples. The detected IgE level in colostrum ranged from 0.5 unit/ml to 1.7 unit/ml. In maternal serum, IgE level obtained by PRIST was correlated to IgE level by RIST. 3) Immunoadsorption ratio of IgE from colostrum with the immunoadsorbent containing AHIgE ranged from 0 to 44.5%, with a mean of 23.3% of IgE level obtained by RIST. IgE in colostrum was adsorbed with AHIgE, but IgE level by RIST was still high. This suggested the possibilities of interference by some unknown factors other than IgE. 4) The ratio of colostral IgE level by PRIST to serum IgE level was 0.0079±0.0043. These results suggested that RIST might be nonspecifically interfered by some unknown factors in colostrum. IgE level in paired samples of colostrum and serum by PRIST suggested that little, if any, IgE is selectively synthesized or secreted in the mammary gland. IgE in colostrum would be expected to be passively diffused from the serum.

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