With the purpose to determine the fluctuation of pH. of the stool after drinking “Yakult”, cow's milk fermented by some sort of “Family Lactbacillaceae”, the authors made normal adults and patients with lung tuberculosis drinking one bottle (60 cc) a day of Yakult successively, and estimated the pH. of their stool colorimetrically and obtained following results. 1. In the case of normal adults taking a bottle (60 cc) of Yakult a day successively, no marked fluctuation can be recognized. 2. In the case of patients with lung tuberculosis receiving drug treatment (chemotherapy) pH. range was 6.07.0 namely marked difference from normal adults can be recognized. 3. In the case of patients with lung tuberculosis receiving chemotherapy, after drinking Yakult, pH. of their stool become equal with that of normal adults'. By drinking Yakult marked effect was recognized.
In order to determine the fluctuation of vitamin B1 in the urine after drinking “Yakult”, cow's milk fermented by some sort of “Family Lactbacillaceae”, the authors made normal adults and patients with lung tuberculosis drink one bottle (60 cc) a day of Yakult successively, and estimated their content of vitamin B1 in their urine polarographically and with paper chromatography. Results are as follows. 1. In the case of one bottle (60 cc) of Yakult successively, there occurs a little increment of vitamin B1 content in their urinc of normal adults comparing with tuberculous patients receiving chemotherapy. 2. In the case of taking one bottle of Yakult, no marked effect can be recognized in their urine of tuberculous patients receiving chemotherapy. However, when three bottles (180 cc) of Yakult are given every day, marked increase of vitamin B1 can be observed even in their urine of the patients, and also impeding factor in polarographical estimation disappears, proving that Yakult is considerably effective in improving vitamin B1 metabolism.
The relationship between the ulceration in the glandular stomach of rats, induced by adrenalectomy, and the substitution therapy or the changes in blood components has been investigated. As agents for the substitution therapy after adrenalectomy physiologic saline solution, cortisone and DOCA were used. During the substitution therapy, non-protein nitrogen in whole blood, protein and sodium-potassium concentration in serum were determined. When the abnormality of sodium-potassium balance in the serum after adrenalectomy was corrected by the administration of physiologic saline solution or cortisone (0.1-0.15/mg/day/100g B. W.), ulceration could be prevented. However, when the excessive substitution therapy by cortisone (5 mg/bay/animal) was done, it was failed to prevent the induction of ulceration. When DOCA was used excessively and sodium-potassium balance in serum was under the state of disharmony, no ulceration was found. And it was recognized that the ulceration and the change in the none protein nitrogen or protein was not intimately correlated. Through these experiments, it has been, therefore, surmized that the gastric ulceration following adrenalectomy was mainly influenced by the sort of substitution therapy, rather than by the alteration of certain specific factor in the blood components.
In this part, the changes in the concentration of water, sodium and potassium in the wall of the fore-stomach, glandular stomach, intestine and muscle of the rat, treated in the same manner as stated in the Part 1, were determined. And the relationship between these changes and the ulceration of the glandular stomach was studied. Water content in the every tissue of the rat, in either non-treated or treated by physiologic saline solution after adrenalectomy, was increased, but not in the rat treated by the other agents. And it was recognized that the ulceration and the change of Water content in the gastric wall were not intimately corelated. The changes of sodium and potassium contents in these digestive organ were not identiccal, and no regulation was observed there. And it was recognized that the ulceration and the change of sodium and potassium content in the gastric wall after adrenalectomy were not intimately corelated.
About on hemorrhagic tendency following massive transfusions of preserved blood, dynamics of mesenteric capillaries, changes in capillary resistance and platelets, and bleeding time were studied experimentally and clinically. Following transfusions, peripheral capillaries were dilated and the blood stream become slow and pooling. Endothelial damages, increase in the permeability and escaping of red blood cell through the wall were observed. Capillary resistance was decreased in the early stage of transfusions and was closely related to the injected blood volume. Striking decrease in number of platelets and prolongation of bleeding time were also recognized in almost all of the cases. It was surmizd that the main factor of hemorrhagic tendency was increase in the permeability or the fragility predisposed by changes in capillary wall with reduction in platelet counts due to blood transfusion itself.
Concerning the vascular disorders caused by massive transfusions of preserved blood, the influence of several treatments on the vascular system was estimated by the capillary resistance, hyaluronidase diffusion ratio, platelet count and bleeding time. There was a striking effect on the improvement of the vascular disorders as continuous injection of intravenous hydrocortisone. Fresh blood and platelet transfusions improved the vascular impairments, same as in adrenochrome. And an effectiveness was observed even in alternated transfusion of the fresh and the presrved blood. ε-Aminocapronic acid was useful for the correction of vascular disorder due to fibrinolysis. Moreover thromboplastin preparations, such as Manetol, and Naphthionic acid were also effective. It was proposed that those measures described above should be performed as prophylaxis before or during blood transfusions, rather than as a therapeutics after transfusions.
Rabbits bearing Brown-Pearce tumor in liver or muscle were examined and the following results were obtained. 1. As the growth of the tumor progressed, catalase activity of the liver was remarkably depressed and albumin and α-globulin decreased and β-, γ-globulin and Φ-fraction increased in plasma electrophoresis. 2. Injury of reticuloendothelial system of the liver by x-ray irradiation with 1000r on the liver depressed catalase activity of the liver and decreased albumin and β-, γ-globulin and increased α-globulin in plasma electrophoresis. Transplantability of the tumor and mortality were elevated. 3. Carcinolytic agents played the same role on catalase activity of the liver and plasma protein fraction as x-ray irradiation with 1000r on the liver. They are accounted for a gift to the reticuoloendothelial system of the liver. Carcinolytic effect was not observed in Carcinophylin, Mitomycin and Azan. 4. Activation of reticuloendothelial system of the liver by x-ray irradiation with 60r showed carcinolytic effect and the same pattern as that of natural healing was observed in plasma electrophoresis, but catalase activity of the liver was slightly depressed. 5. In cases of natural healing or intransplantable cases, catalase activity of the liver was elevated and plasma protein fraction showed the change from the pattern of tumor growth to the pattern of its recovering process. γ-globulin increased in early stadium. 6. Immunity inhibited the tumor growth markedly and elevated catalase activity of the liver and showed the same pattern in electrophoresis as the cases of natural healing. 7. γ-globulin is considered to play an important role in carcinolytic process, while it does not show carcinolytic effect by itself.
Three acetanilide- and two pyrazolene derivatives were tested for anti-inflammatory effect on the edema of rat's hind-paws induced by the local injection of dextran, hyaluronidase, formaldehyde, histamine and 5-hydroxyptamine, and for histamine-release inhibitory effect on the rat receiving an intraperitoneal injection of ovomucoid which increases urinary output of histamine as a result of histamine release in the whole body. Phenacetin, acetanilide, aminopyrine, aminopropylone and butazolidine served as controls. GP-I, one of the pyrazolone derivatives (for formula cf. table 1), showed fairly marked supression on all 5 kinds of edema with effects comparable to aminopyrine and butazolidine. Acetanilide derivatives tested, also exerted marked inhibition on the edema other than by hyaluronidase or formaldehyde. All the compounds inhibited the histamine release due to ovomucoid injection, but no graded difference could be seen.
Of late many investigators have come to study blood cells with fluorescence microscope, but most of these works are carried out by supravital staining method only. Therefore, the author has attempted vital observations of bone marrow cells under the fluorescence microscope by adding fluorescent dye, acridine orange, to the medium of the simple tissue culture, the method devised in our department. For this study some fundamental studies have been conducted concerning the toxicity of dye, the selection of barrier filters, the secondary fluorescence of cells, and the influence of exciting rays on the cell growth; and obtained the following results. 1. After studying the relative growth rate, the cell density index, and the wandering velocity of neutrophils it has been found that tissue culture is possible at low concentration, under 10-4 of the medium with acridine orange. 2. Barrier filter, 0G5, is the most suitable one for the observation of the secondary fluorescence of the cell. 3. The concentration of the acridine orange medium at which the most distinct picture of fluorescence obtainable is at 10-4, and the concentration at 10-5 is the minimum at which fluorescence can be recognized. 4. The ill effect on cells due to exciting rays may be eliminated by avoiding successive exposure to the rays. 5. The author has devised a medium for the bone marrow tissue culture, consisted of a drop of serum and a drop of physiological saline solution containing 80 γ/cc vitamin B12 and 0.2mg/cc acridine orange, and has designated this as the simple method of fluorochrominized bone marrow tissue culture.
In the present experiment the author conducted the bone marrow tissue of normal persons by the simple method of fluorochrominized bone marrow tissue culture as established in Report 1, and observed the growth zone and various bone marrow cells appearing in the zone. 1. The cells are dense in the central part of the growth zone, and the density gradually decreases towards the periphery, giving off reddish orange fluorescence diffusely with yellow and green fluorescent spots scattered in this zone. 2. Cytoplasma is green, while mitochondria and various granules give off yellowish green or reddish orange fluorescence. However, the fluorescence of mitochondria is quite trivial, and the nuclei and nucleoles are green but nucleoles give off fluorescence earler than the nuclei. 3, In young cells green fluorescence of cytoplasma is marked, and it grows less distinct as they mature whereas reddish orange granules tend to increase along with the maturation of cells and their size is variegated. 4. In myelogenous cells reddish orange fluorescence of granules is characteristic while yellowish green fluorescence of the nuclei in lymphocytic cells. In mouocytic cells a variety of fluorescence such as green cytoplsama and reddish orange granules agglomerating around the nucleus can be recognized, and likewise fluorescence specific to each cell type can be observed in other cells. 5. The green fluorescence of cytoplasma of erythroblasts is gradually lost along with maturation, and no fluorescence can be recognized in erythrocytes, while substantia reticulofilamentosa of reticulocytes present red fluorescence. 6. When two barrier filters, GG 4 and OG 4 are used together, it is easy to differentiate cells of the erythroblast series from cells of the leucocyte series.
By performing the simple method of fluorochrcminized bone marrow tissue culture the author observed the degeneration picture of leucccytes, and also studied changes in fluorescence in the cells cultured under an adverse condition but added with acridine orange. The following are the results of the present experiment. 1. It has been found that the majority of commonly-known findings by supravital staining are proven to be degeneration picture of cells. 2. As for the degeneration signs, the swelling and fading of reddish orange granules can be pointed out, while in cytoplasma vacuoles either stained or unstained with acridine orange, abnormal processes, the reddening or green swelling of cytoplasma can be recognized: The nuclei have agglutinated chromatin and have become yellow, red, and swollen in green color, and in nucleoles green swelling or reddening can be observed. 3. In the degeneration picture likewise each cell type present different, fluorescence. 4. When cells become shrunk, the fluorescence is red, while when cells are swollen, it is green. 5. In the cells with decreased function reddish orange granules fade quickly and their nuclei turn yellow. 6. Those cells that have lost the fluorescence of their granules, and those that have red cytoplasma and red nuclei as well as those with their nuclei green and swollen as alldead cells.
(1) By painting the skin of 84 mice of uncertain origin with 20-methylcholanthrene dissolved in the 0.5-1.5% benzene solution, it was possible to observe that lymphoid tumors developed in 16.7% of the test animals. The latent period was 87 days in average. The life span of the mice with lymphoid tumors was 29 days in average, and the lymphoid tumors were consisted of lymphocytic leukemia and lymphocytic leukosarcomatosis. (2) As for the changes of the peripheral blood pictures of the mice with lymphocytic leukemia, the author recognized leucopenia, lymphopenia, an increase in the number of large lymphocytes, eosinophilia, and appearance of erythroblasts in the preleukemic stage, and also leucocytosis, lymphocytosis, and appearance of lymphoblasts in the leukemic stage. (3) The leukemic infiltration was observed in various organs including spleen, lymph nodes, thymus, bone marrow, etc. (4) The author succeeded in transplanting the lymphoid tumors also to other group of mice of uncertain origin.
After inducing lymphocytic leukemia by painting the skin of the mice of dba strain and other group of uncertain origin with 20-methylcholanthrene, the author carried out tissuccultures of bone marrow, lymph nodes, and spleen of these test animals and also cytological and histological examinations for purpose of clarifying the origin and development of lymphocytic leukemia. On the basis of the peripheral blood pictures, the author classified the development of lymphocytic leukemia into the following three stages, i. e., (1) preleukemic stage (with leucopenia but no lymphoblasts in the peripheral blood), (2) initial stage of leukemia (with slight leucocytosis and a few lymphoblasts in the peripheral blood), and (3) advanced stage of leukemia (with marked leucocytosis and many lymphoblasts in the peripheral blood). The following are the results and conclusions of the present experiment. (1) The changes in the peripheral blood pictures observed during the preleukemic stage as described in Part I, that is, leucopenia and lymphopenia, are caused by the arrest of maturation in lymph follicles of the spleen and by hypoplasia of lymph nodes. Eosinophilia can be observed in bone marrow of every single case and the stimulation of the reticuloendothelial system is believed to be one of its causes. Lymphoblasts of the peripheral blood during the initial stage of leukemia are mainly derived from the spleen. The distribution curve of the greatest diameters of lymphocytes in the peripheral blood gradually shifts to the right as lymphocytic leukemia develops. This phenomenon coincides with the increase in large lymphocytes during the preleukemic stage and with the appearance of lymphoblasts during the initial stage of leukemia. (2) In tissue cultures, the bone marrow, spleen, thymus, and lymph nodes of leukemic mice show the growth patterns specific to acute lymphocytic leukemia, though these ars not so typical in mice as in men. Namely, the margin of growth zone is sharply defined, the density of cells is very high, and the growth zone is consisted mainly of lymphoblasts with some mature lymphocytes. In the preleukemic stage, the spleen shows relatively leukemic growth pattern, but the lymph nodes show hypoplasia or slight hyperplasia, In the initial stage of leukemia, the spleen shows the leukemic growth pattern, but the lymph nodes and thymus show hyperplasia or leukemic growth pattern. In the advanced stage of leukemia, the spleen, lymph nodes, thymus, and the bone marrow begin to show the leukemic growth pattern. (3) Cytomorphologically, leukemic cells appear first of all in the lymph follicles of the spleen, and they increase in the lymph nodes and bone marrow along with progress of leukemia. The lymphoblats in the lymph nodes during the preleukemic and initial stages of leukemia are little atypical, but they show prominent nucleoli and lobulation of the nucleus in the advanced stage and also mitochondria increase in number. The distribution curve of the greatest diameters of lymphocytes in the lymph nodes shifts gradually to the right through the development of lymphocytic leukemia as in the case of peripheral blood. (4) In autopsy findings, there can be observed a slight enlargement of spleen in the preleukemic stage, moderately enlarged spleen and slightly enlarged lymph nodes in the initial stage, and marked enlargement of spleen, lymph nodes, and liver in the advanced stage. (5) Histologically, the lymph follicles of spleen are enlarged due to the proliferation of reticulum cells and lymphoblasts in the preleukemic stage. Lymph nodes show hypoplasia or slight hyperplasia. Namely, lymphocytic leukemia can be understood to be circumscribed to the spleen in this stage. In the initial stage, lymphoblasts infiltrate diffusely through the whole spleen and lymph nodes show hyperplasia or gradual enlargement of germinal centers due to the proliferation of lymphoblasts with the formation of lymph follicles.
With lymphoid tumors (lymphocytic leukemia and lymphocytic leukosarcomatosis) induced in the mice of uncertain origin by smearing with 20-methylcholanthrene, the author tried to clarify the relationship between lymphocytic leukemia and lymphocytic leukosarcomatosis by performing bone-marrow and lymph-node tissue cultures in combination with cytological examinations, and obtained the following results. (1) Lymphocytic leukosarcomatosis is mostly aleukemic, and the percentage of lymphoblasts in the peripheral blood is low. Some cases of lymphocytic leukosarcomatosis reveal a marked increase of large lymphocytes in the peripheral blood. (2) The lymph nodes and bone marrow of aleukemic lymphocytic leukosarcomatosis consist mostly of lymphosarcoma cells with the greatest diameters of more than 15.3μ and marked lobulation of the nucleus. These cells are thought to be derived from Amano's “lymphogonia”. In addition, in the lymph nodes and bone marrow of aleukemic lymphocytic leukosarcomatosis, there can also be found little atypical lymphoblasts with the greatest diameters of less than 15.3μ, what may be considered as leukemic cells. and it seems that those lymphoblasts found in the peripheral blood of leukosarcomatosis are derived from these leukemic cells. (3) The lymph nodes and bone marrow of aleukemic lymphocytic leukosarcomatosis are consisted mostly of lymphosarcoma cells, while, in leukemic lymphocytic leukosarcomatosis and lymphocytic leukemia, the percentage of leukemic cells increases and the percentage of lymphosarcoma cells decreases. (4) In the bone-marrow and lymph-node tissue cultures, aleukemic lymphosarcomatosis generally presents the growth pattern resembling tumor, while leukemic lymphocytic leuko-sarcomatosis shows the growth pattern exactly similar to that of acute lymphocytic leukemia. (5) In autopsy findings, lymphocytic leukosarcomatosis shows more remarkable enlargement of lymph nodes than lymphocytic leukemia. Histologically, in aleukemic lymphocytic leukosarcomatosis there is little tendency of lymphoblast infiltration and metastasis to the liver and kidneys. (6) Since aleukemic and leukemic leukosarcomatosis can transform progressively to lymphocytic leukemia, all these are believed to be essentially the same disease.
The effect of unsaturted fatty acid fraction from the liver of irradiated animal on fertilization and cell division of sea urchin egg (Hemicentrotus tulcherrimus) have been observed. The sperms exposed to the snbstance for 5 minutes decrease in their mortility and lost the fertilization acivitity resuting in the lose of their ability for the formation of fertilizing membrae in eggs. The eggs exposed to this substance after fertilization present the stop or the delay in their cleavage. Such an action on eggs becomes marked when the concentration of this substance increases. The most susceptible stage seems to be the metaphase at which the chromosoms are arrested on the equatolial plate loosing the tendency to move toward the poles. These effects of this substace can be seen even after removing the peroxide as equally in the original substance.
There are many investigations on the disturbances caused by x-rays but these works are consisted mainly of histo-morphological researches and rerely from the viewpoint of the acting mechanism of cells. More recently Lea, Barron et al, however, have studied the histochemical changes in the animals irradiated with x-rays and they have clarified the direct action of x-ray irradiation to a certain extent. In our laboratory Dr. Yamamoto has succeeded in isolating a certain cytotoxin from unsaturated fatty acid fraction, an extract of the liver of the rabbits exposed to x-rays. In the subsequent studies with this cytotoxin it has been found that this substance has an inhibitory action on the mitosis of tumor cells. Therfore, we have carried out a series of experiments to see the effect of this substance, in the presence of succinic dehydrogenase, on the respiration of Ehrlich-ascites-tumor cells. As the result it has been confirmed that this cytotoxin possesses an action as to depress both the respiration of the cancer cell and that of normal liver cells.
The influence of the various extracts of various bacteria and organs of animals on the infections aspect of hepatitis virus was studied by the histopathological findings in the inoculated mice. According to the results of experiments, some extracts showed inhibitory action and someone revealed promoting effect, but also others were no influence on the course of infection. It was nucleic acid and nucleo protein that were extracted from cholera vibrio which was inhibitory effect. The Proteins which were extracted from Typhoid bacillus showed raising effect on the sensitivity of the mouse against the infectious hepatitis virus. It will be found more effective substances which have inhibitory action, if further studies are performed on the other various substances.
Change of blood sedimentation rate before and after therapy were examined in 50 patients of cancer. 1) Before therapy, only 6 patients (12%) were normal extent. 2) It was cancer of lung which tendency to increase was much more stronger than otlrers. 3) Cases in which couse after therapy (Operaeion or X. ray therapy) was good, blood sedimentation rate returned to normal extent. 4) It will be find out complication, if periodic examination of blood sedimentation rate after operation was performed.
The mechanism of fibrinolysis following massive transfusion of preserved blood was investigated, clinically and experimentally. Results obtained were as follows: 1. Fibrinolytic activity was not seen in the preserved blood, and the fibrinolysis observed in the transfusion of preserved blood was derived from preserved blood itself which acted upon as a stress, but it was slight in the degree. On the other hand, the fibrinolysis was relatively remarkable in the cases undertaking sugical procedures, especially a pulmonary operation, and etc. However, it was not considered that either one of above cases would become to develop a hemorrhagic tendency. 2. Fibrinogen level did not show a remarkable change with a massive transfusion of preserved blood, but was decreased in the cases operated upon the organs being high in the thromboplastin level and became a cause of a hemorrhagic tendency. 3. A hemorrhagic tendency was not always observed even in the cases showing highly positive fibrinolysis, but it was thought to be cased by the defects in platelet, vascular and coagulation factors of many kinds, in conjuction with the changes in general and local conditions of patients, and with surgical procedures. The fibrinolysin, however, promoted the development of hemorrhagic tendency following the disturbances in vascular and various kinds of coagulation factors.
Therapeutic and prophylactic measures against a fibrinolysis following massive transfusion of preserved blood was studied, and following results were obtained. 1. Administration of ε-aminocaplonic acid, ACTH, and cortisone prevented a fibrinolysis following massive transfusion of preserved blood. 2. These therapeutics, especially, showed a marked preventive effect when they were applied together at early stage. 3. At the same time in the cases administed together with thromboplastin and adrenochrome preparations of every kinds as well as fresh blood, fibrinolysis was not completely prevented, but those were expected to inhibit other disturbance in coagulation mechanism accompanied with the above phenomenon.
Convulsions were repeatedly caused by electrical stimulation in rabbits. Total free amino acids contents in these rabbits showed no fluctation, while glucose metabolism decreased like as in those with latent cerebral local anaphylaxis and human epileptics. The glucose utilization was cured to normal level by continuing injection of Glutamic acid, GABA, and Asparagine, but total amino-N changed scarecely. It is an important fact, that free amino-N content increased by GABA injection in rabbits with convulsion.
It has been found that when the animals are exposed to X-rays there will be produced in vivo a substance that is highly hemolytic, and when this hemolytic substance extracted from the liver of the X-ray irradiated animals is injected into animals, the histological findings of such animals resemble very closely those of X-ray disturbances. Moreover, the quantity of the hemolytic substance produced by X-ray irradiation in vivo seems to be one of the most important factors thatsways the severity of the X-ray disturbances. It is, therefore, presumed that hemolytic anemia is one of the factors that plays an important role in the X-ray blood disturbances and that the difference in the concentration of the hemolytic substance brings about the changes in the histological picture.
After transplanting Yoshida sarcoma cells under the skin of the thigh of hybrid mice, the author injected 0.2cc of 2% unsaturated fatty acid fraction (denominated as OX substance) intramuscularly and observed the changes in the histological picture, in the mitotic phase as well as the changes in the quantity of DNA per cell, along with the lapse of time, and obtained the following results. 1. The histological pictures revealed the phenomena such as an increase in necrotic foci, a general diminution in stainability and indistinct cell boundary. 2. In counting the number of those cells with a relatively strong activity in cancer foci at various mitotic phases, it has been found that from 6 to 12 hours after the OX injection the numher of the cells in metaphase is decreased and from 24 hours on the number of such cells approaches the normal. 3. In the estimation of DNA content per cell, from 6 to 12 hours after the OX injection the number of the cells undergoing a relatively active proliferation but with a decreased DNA content tends to increase but after 24 hours the content per cell approaches the normal level.