The development and implantation of the ovum is closely related with the condition of endometrium. It has been considered that the histochemical study on the endometrial change during menstrial cycle is rather important than the pathologiacl investigation. In this connection, Nucleic acid (DNA & RNA), Polysaccharides and Alkaline phosphatase have been determined in 164 cases of nonmal human endometrium. 1) DNA level during menstrual cycle showed a incidence of the cyclic change. 2) RNA were shown markedly in the cytoplasma of the epithelium of endometrial gland and these levels were closely related with the cyclic change of endometrium. 3) Polysaccharides presented mainly in the epithelium and the cavity of endometrial gland, reached a maximum consentration in mid secretory phase. 4) Changes in Alkaline phosphatase level during menstrual cycle were preceded to the Polysaccharide change. 5) Alkaline phosphatase was found to the peak consentration at the ovulation, then decreased gradually.
The author carried out the paperelectrophoresis on 145 cerebrospinal fluid (C. S. F.) specimen taken from neuropsychiatric cases for the protein fractions. From this study correlations were sought between the clinical disease entities and the respective C. S. F. fraction patterns. 1) 50-100 mesh dry Sephadex G-25 was put to the test for the concentration of the C. S. F. The concentrated C. S. F. was dialyzed through a cellophane tube. The new concentration method was found to be simpler and more timesaving than the other contemporaries. Moreover, the Sephadex G-25 prepared for the concentration absorbs and removes low molecule substances from the rest at the same rate as the concentration. The prepared Sephadex can be reused after through cleaning under the running water. Also, it does not denaturize the protein in C. S. F. The author advocates it to be the best for the concentration of the C. S. F. 2) Slight increase of γ-globulin appeared on 2 cases out of 5 Neuro-Behçet syndrome. The fraction of β-globulin was also elevated, parallel to the increase of γ-globulin on 1 case. It is to be noted that although the inflammatory changes were suspected in case of Neuro-Behçet, no marked increase of γ-globulin was observed. My findings simulate allergic process, instead. 3) The author's data on multiple sclerosis contradicted those of the predecessors who had reported an increase of γ-globulin. The author, in place of γ-globulin, found some incrcase in β-globulin fraction. Retrobulbar neuritis also produced an ascent of β-globulin. There seemes to be enough doubts that the increase of γ-globulin in C. S. F. could be a decisive factor for multiple sclerosis and retrobulbar neuritis. 4) Not all the cases of cerebral atrophy resulted in increase of β-globulin in my study. Excluding Alzheimer's disease, the number of years after the onset of an illness tended to influence the amount of β-globulin. Regardless of enlargement of subarachnoid space and ventricles, β-globulin was prone to increase in the cases that passes not less than 3 years after the onset. My three cases of Alzheimer's disease produced marked elevation of β-globulin, regardless of the time after the onset. 5) The author analyzed and discussed in this paper some interesting findings on a few cases, resulted from the corellative study between the fractions and clinical pictures on the neuropsychiatric disorders. In some instances, the C. S. F. electrophoresis can be regarded as an important tool for diagnosing. However, we often obtain arbiturary data from which we yet cannot derive decisive factor. As we pile up more data from similar studies we may obtain enough clues to systematize completely the pathophysiological changes of C. S. F. electrophoresis.
The volume of hepatic blood flow was determined on a total of 100 hepatic diseass patients; acute hepatitis 2, chronic hepatitis 75, hepatocirrhosis 21 and fatty liver 2; by Galactose continual Infusion Method devised by Nakamura et al. At the same time, the correlation between state of hepatic circulation and BSP test was observed stochastically using as index the BSP test findings noted at time of initial out-patient examination and the BSP test find ings obtained within one week both prior to and after hepatic blood flow volume determination made following admission to the hospital. Further, comparisons were made with the hepatic tissue picture obtained by biopsy. The followresults were obtained. 1) The average value of the total hepatic blood flow Volume for the 100 hepatic disease cases was 857±46 cc/min/M2. The 49 cases with wedded hepatic venous pressure (WHVP), but without hepatic shunt flow had an average value of 135±21 mm H2O. 2) A positive correlation with a coefficient of 0.69 was noted between WHVP and BSP test results. Further, an approximate value of 0.64 was demonstrated between the initial out-patient BSP test findings. In cases with WHVP of over 175mm H2O, the change-over rate to negative BSP test results following 3 to 4 weeks of hospitalization, bedrest and treatment was 13.8%, and 71.1% for those with values of less than 175mm H2O. 3) A negative correlation with a coefficient of -0.34 was noted between effective hepatic blood flow volume and BSP test results. Further, a negative correlation of -0.27 was demonstrated between the initial out-patient BSP test findings. In cases with an effective hepatic blood flow volume of less than 500 cc/min/M2, the change-over rate to negative BSP test results following 3 to 4 weeks of hospitalization, bedrest and treatment was 35.0%, while that of cases with volumes of over 500 cc/min/M2 was 48.9%. 4) A positive correlation with a coefficient of 0.55 was noted between rate of hepatic short circuit and BSP test results. A positive correlation of 0.50 was demonstrated between the initial outpatient BSP test findings. In cases with hepatic short circuit, the change-over rate to negative BSP test results following 3 to 4 weeks of hospitalization, bedrest and treatment was 1%, while that in those without hepatic short circuit was 63.3%. 5) Of the 42 cases with more than a moderate degree of hepatic cell degeneration and necrosis (includes 2 cases with negative BSP test findings from the beginning), 9 cases or 22.5% demonstrated normal BSP test findings following 3 to 4 weeks of hospitalization, bedrest and treatment, while of 58 cases with slight degeneration and necrosis (includes 2 cases with normal BSP test findings from the beginning), 24 cases or 82.8% became normal during the same short period of time. 6) A follow-up BSP test survey wase conducted on 30 chronic hepatitis cases whose BSP test findings at time of discharge had become normal, and aggravations were noted in 11. Among those with aggravations, 7 or 63.5% had hepatic circulatory abnormalities during hospitalization. 7) From the above findings, it has been shown that not only degeneration and necrosis of hepatic cells are related to the BSP test value, but a relationship is also noted between the state of hepatic circulation. Therefore, it is necessary to consider both together before making any decision.
The Calactose Continual Infusion Method devised by Nakamura et al and the BSP Continual Infusion Method reported by Bradley et al were used simultaneously on 21 chronic hepatitis and 9 hepatocirrhosis patients, and the hepatic blood flow volume was determined. The results obtained are as follows. 1) The total hepatic blood flow volume as determined by both methods were in agreement in cases with comparatively mild hepatic disturbance, but in such cases as hepatocirrhosis where abvanced cellular degeneration and necrosis were present, the value as determined by BSP method was greater than that obtained by the Galactose method. This is felt to be due to BSP clearance, particularly, the close relationship with the hepatic cell uptake capacity of BSP. 2) There is a definite difference in galactose and BSP elimination rates. The 15 cases that demonstrated a 100% elimination of galactose had BSP elimination rates which ranged from 33.0 to 84.6%, thus, in all cases the rates for the latter were lower. From these reslts, even though the BSP method may indicate a normal liver, it is felt that there may be a hepatic shunt blood flow volume, but this is presumed to be due to the fact that the BSP up take capacity of hepatic cells is not 100% or it may be that although they have such uptake capacity, the BSP which has been taken up is again released into the blood. 3) With the use of dogs BSP in amounts less than the Maximum elimination volume of the liver was administered in succession into the peripheral vein, hepatic vein and portal vein by continual infusion, and the BSP concentration in hepatic venous blood was determined. Results showed that both the portal and hepatic venous areas were involved in the hepatic eimination of BSP. 4) Therefore, it is felt inappropriate to use BSP to determine hepatic blood flow volume in cases with hepatic disturbances.
BSP retention and serum bilirubin level were determined in cases including 51 of acute hepatitis, 19 of extrahepatic obstructive jaundice and 9 of hepatic cancer with hyperbilirubinemia. The following results were obtained. 1) There was a tendency to show some correlation between BSP and serum bilirubin level in cases of extrahepatic obstructive jaundice. On the contrary, no significant correlation between BSP retention and serum bilirubin level was found in cases of hepatocellular jaundice. 2) Hepatic cancer, showing serum bilirubin level to be approximately 2mg/dl in all cases, exhibited BSP retention at 30 min. to be 15 to 30%. 3) there was no significant difference in BSP retentions between hepatocellular jaundice and extrahepatic obstructive jaundice showing same degree of serum bilirubin level in each cases. This indicates that BSP test is not useful for differential diagnosis of these types of jaundice. 4) In the cases with hepatocellular damage, a considerable amount of BSP dye was excreted through the kidney in proportion to its retention in blood.
BSP retention and hepatic blood flow were determined after administration of ATP in liver disease, and the following results were obtained. 1) 14 cases out of 22 with liver disease having, administration of ATP improved in BSP retention, and on the other hand, 8 cases of the remainder showed no changes or aggravation in that test. 2) Considerable grade of hepatic cell degeneration or necrosis was found in liver biopsy tissue of the all aggravating cases. 3) After the intravenous injection of ATP hepatic blcod flow decreased temporarily and was followed subsequently by an increase.
The results of the prognostic survey were given of 79 cases of infectious hepatitis sampled in Kumayama-cho, Akaiwa-gun, Okayama Prefecture where the massive outbreak of the typical severe form occured, and of 82 cases in the Kagato area of Bizen-cho, Wake-gun, Okayama Prefecture where the mild abortive form was mostly seen during the epidemic. In the both areas the epidemics broke out 10 years prior to the author's survey. 1) In Kumayama-cho 65 cases were chosen for a set of liver function tests and the medical examination. Only 41.5% had attained the good health; 24.6% retained chronic hepatitis; 27.7% were diagnosed to be the so-called posthepatitis syndrome; and 6.2% showed hyperbilirubinemia. 2) In the Kagato area 70 cases were checked in the same manner. The results were as follows; recovery in 42.8%; chronic hepatitis in 28.5%; posthepatitis syndrome in 27.1%; hyperbilirubinemia in 1.6%, 3) Nine cases in Kumayama-cho and 6 in the Kagato area were reported dead after sustaining infectious hepatitis. However, excluding a case of hepatic insufficiency, immediate cause of death were devoid of cirrhosis and hepatocarcinoma. 4) Among the 1 year, 5 years and 10 years follow-np study, a reduction of the incidence of chronic hepatitis was observed in 5 years study after the epidemics. The correlation between the incidence of chronic hepatitis and the duration of immunity to infectious hepatitis was discussed in connection with the results, 5) The female and elderly patients had a predilection for chronic hepatitis. 6) At the rate of 50.4% the patients were unaware of their illness, whereas the others had mostly complaints of general malaise and intolerance to fatty meals. Only 4% complained prolonged impaired appetite. 7) Palpable hepatomegaly was observed at the rate of 50.7% in Kumayama-cho, while 55.7% in the area of Kagato did. The incidence of hepatomegaly was reported approximately at the same rate in the acute phase of the illness. Splenomegaly subsided in size on most of the patients later in chronic phase. 8) The incidence of cirrhosis after infectious hepatitis was observed in 3.8% in Kumayama-cho and in 3.7% in the Kagato area.
For the purpose of elucidation on progression of posttransfusion hepatitis to chronic hepatitis, a set of liver function tests was repeated periodically on the patients who had had blood transfusion supplied by blood banks. Liver biopsy was performed on the selected cases of posttransfusion hepatitis for a pathohistological study. The results were as follows: 1) From the second to the fifth day after transfusion the laboratory tests revealed transient signs of liver dysfunction which returned to the normal within 2 weeks. 2) From the third to the seventh day following transfusion, parenchymal damages were observed on the biopsy specimens, whereas the tenth day specimens showed no pathognomonic change. 3) The onset of posttransfusion hepatitis was 3 weeks or more after blood transfusion. Any evidences of liver dysfunction that occured within 2 weeks seemed to have been originated majorly from the transfusion blood itself and minorly from the ill effects of anesthesia, operational interventions and illnesses which necessiated transfusion. The viral infection did not appear to be the case, Shortly after the onset of posttransfusion hepatitis histopathological examination revealed pathohistological findings suggestive of chronic hepatitis in some instances. It was surmised that this illness had strong predilection for chronicity. 5) The parencymal damage in the early stage of the disease and some factors of the preserved blood were thought to be the causes of a propensity to the chronic progression of posttransfusion hepatitis.
Of late clinical assay of transaminase has gained a greater importance and the methods of the assay are now being studied from various angles. As for making the standard curves there are such methods as that of Reitman and Mizobe's, the latter being very similar to the former, but there still remain problems that need to be solved in these methods with respect to the quantity of 2.4-dinitrophenylhydrazine solution to be added. With the purpose to clarify these points, the author conducted a series of experiments and demonstrated theoretically and clinically that the addition of 2.4-dinitrophenylhydrazine solution in the quantity more than twice the amounts employed by various investigators would yield a perfectly straight line curve in contrast to the available reports where the addition of pyruvate in the concentrations up to 0.3 μM as reported by Mizobe and others gives a straight line curve but any quantity above that tends to distort the straight line.
Each of the kidney, the lung, the liver and the mixture of the skin and the muscle of a four-month-old human embryo were separately minced, trypsinized and cultured in test tubes. In the cultures of the kidney and of the liver, both the epithelioid and fibroblastic cells proliferated. In the cultures of the lung and of the skin-muscle, only fibroblastic cells proliferated. Upon successive culture passages, cells of the kidney and the lung ceased the multiplication almost completely at the third passage and the cells of the skin-muscle did so at the fourth passage. The fibroblastic cells of the kidney which proliferated vigorously in the primary culture, decreased markedly at the second culture passage. The skin and muscle of a seven-month-old human embryo was treated and cultured by the same method as that of four-month-old embryo. Similarly fibroblastic cells proliferated, but the cell multiplication was much weaker than in those of the four-month-old embryo and ceased to multiply almost completely at the second culture passage.
Upon inoculation of adenovirus type 12 onto the primary culture cells of each of the kidney, the lung, the liver and the skin-muscle of a four-month-old human embryo, the following changes were observed: 1. In the cells of the kidney and the lung, rounding and aggregation of cells were observed. In those of the liver and the skin-muscle, rounding of cells was observed and, besides this, flat-round or -oval big cells appeared. 2. Such changes appeared earliest and strongest in the culture of kidney cells, next in that of lung cells and were very weak in those of liver and of skin-muscle cells. 3. Of these four human embryonic tissues, good multiplication of the virus was observed only in the culture of kidney cells.
The mechanism of infection and of multiplication of Coxsackie B5 virus was studied using dog kidney cells (to be abbreviated CS cells). As the result it has been found that Coxsackie B5 virus has a high susceptibility to CS cells and proliferates by demonstrating a specific cytopathic effect. The results may be summarized as follows. 1. In the infection pattern of Coxsackie B5 virus to CS cells, the virus is found to proliferate with a high susceptibility to the cells. In addition, morphologioal changes of the cell and the course of virus multiplication parallel relatively well with each other. Namely, both cell degeneration and the virus infectivity reach to their peak on 72 hours after infection, and the infectivity decreases along with gradual detachment of the cells in cultures. 2. In one-step growth experiment conducted after simultaneous infection of a large number of CS cells with increasing amounts of virus, the cycle of the one-step multiplication after a eclipse phase (4-6 hr) is found to be about 16 hours and it is assumed that the cycle is repeated following the infection. Furthermore, it is presumed that the release of regeneratad virus into liquid layer commences within 7 hours and it proceeds very rapidly along with the proliferation of the intracellular regenerated virus.
This paper describes briefly the results of experiments conducted with plaque mutants accidentally obtained in the course of the study of the multiplication pattern of Coxsackie B5 virus with dog kidney cells (CS cell). 1. In the course of successive cultures carried out by having Coxsackie B5 virus multiply in CS cells, a plaque mutant was obtained. In this instance, the plaque mutant could be classified into two kinds, one large and the other small one, and it was possible to form other clones by successive inoculation of each plaque to CS cells in cultures. 2. In the observations of the general properties of each strain obtained after clone formation, the findings suggest on the whole a PS→PL mutation phenomenon. 3. Even judging from the PS→PL mutation phenomenon, properties of Coxsackie B5 virus a, e fairly well preserved, but the characteristics of the virus as confirmed by experiments dealing with the multiplication pattern of virus and by neutralization tests have revealed some changes. Especially with PL strain the mutation is firm, suggesting that a certain fixed condition is necessary under which the virus properties remain intact even in the presence of antiserum and similar mutant does not appear in the other cell strains.
Hematological and cytological studies, including peripheral blood counting, blood picture, myelogram, impressed preparation of spleen and lymph node, and phase contrast microscopic and electron microscopic observations of leukemic cells were performed on the high leukemic strain C58 mice. In the present observation, 61.4% of C58 mice developed leukemia spontaneously after the latent periods varing from 4 to 19 months. In non-leukemic mice, blood counting, peripheralblood picture and myelogram did not show any significant change throughout the each age groups, but lymphocyt/neutrophile ratio decreased gradually after the age of 6 month. Old mice above 12 months of age showed a slight hypochromic anemia and relative neutrophilia. Most of the spontaneous leukemia occurred after the age of 6 month and 61.4% of them occurred between the age of 12 and 16 month. All of the spontaneous leukemias observed in this strain were lymphatic type. White cell count ranged from 4, 500 to 225, 000, averaging 109, 600 with 47.2% lymphoblasts in average. Marked anemia and thrombocytopenia were present. Myelograms showed slight or moderate lymphoblastic infiltrations, but impressed preparation of spleen and lymph node always contained lymphoblasts over 90%. Morphological characteristics of the lymphoblast observed in spontaneous leukemia were narrow basophilic cytoplasm, large nucleus with a few deep indentations, coarse chromatin net work and nucleoles of vague chromatin borders. Phase contrast microscopy of lymphoblast showed nucleal indentations clearly and bright cytoplasm with a few rod-shaped mitocondrias in the Golgi area. Electron microscopic observations of lymphoblast also revealed the characteristic indentations of the nucleus. Spontaneous leukemia could be transplanted easely to the adult C58 mice by cell graft. A transplanted line named OHS-LL, No 1 is being maintained serially up to present. Serially transplanted leukemia mice often showed remarkable myeloid reaction and died after 7-8 days after cell graft. Blastic cells became to show atypical form suggesting of much more primitive and malignant character than that of the original leukemia by serial transplantations. Electron microscopic observations of lymph node and spleen of spontaneous leukemia revealed the presence of numerous virus particles located extracellularly. These particles were morphlolgically identical with the C particle observed in various other leukemic mice.
Isologous inoculations of cell-free extracts from the lymphatic leukemia in the C58 mice were performed using new born and suckling mice. 1. Among the 92 inoculated mice, 31 had died of unknown infectious disease 1 or 2 months after the inoculation. Another 61 survived mice were observed for the development of leukemia thereafter. Forty three of them (70.5%) developed leukemia after the latent periods varing from 6 to 16 months. During the period of 6 to 10 month, 16 mice developed leukemia (37.2% of all leukemias developed). This was higher in incidence comparing with the incidence of leukemia occnrred (13.1%) in the control group within the same period. These fact indicates the accelaration of leukemia development by the isologous inoculation of the leukemic filtrate. 2. Five of the inoculated mice (8.2%) developed myelogenous leukemia which had not been observed in the spontaneous case. Possibility of the transformation of leukemic type from lymphatic to myelogenous by the inoculation of leukemic filtrate was discussed in this paper, although its exact mechanism is not clear as yet. 3. Cytological studies of myelogenous leukemia were performed. Peripheral blood, myelogram and impressed preparations of lymph node and spleen showed numerous appearance of myeloblasts and immature myeloid cells. In May-Giemsa stained preparations, myeloblast showed broad basophilic cytoplasm and eccentrically located nucleus with fine chromatin net work and clearly defined nucleoles. Nuclear indentation which was one of the characteristics of lymphoblast was not seen in myeloblast. Phase contrast microscopy of myeloblast was also lacking of nuclear indentation and showed centrically located large round nucleus. Mitochondrias were abundant and scattered throughout the cytoplasm. In electron microscopy, myeloblast had many well developed endoplasmic reticulums and small spherical mitochondrias in cytoplasm. Nucleus was round and nuclear substance was homogenous with a dense nucleole. 4. Electron microscopic observations of lymph node and spleen of the myeloid leukemia mice revealed numerous extracellulary located virus particles. These virus particles were morphologically indistinguishable from that of lymphatic leukemia mice.
The simplified tissue culture method and the fluorochrominized tissue culture method devised in our laboratory were applied to the bone marrow, spleen and lymph node of the leukemic mice of C58 strain for the purpose of diagnosis and differentiation of leukemia. And following results were obtained; 1. In the case of spontaneous leukemia, tissue culture of the lymph node and spleen showed sharply defined growth zone with increased cell density which is the characteric pattern of acute leukemia. The bone marrow showed rather diffuse border of growth zone with the appearances of many migrating neutrophiles, but cell density was remarkably increased and many leukemic cells appeared in the growth zone. 2. In the case of myelogenos leukemia, the bone marrow culture showed so-called double growth zone which is characteristic of chronic leukemia. On the other hand, the lymph node and the spleen showed diffuse border of the growth zone with increased cell density. Numerouse immature and mature neutrophilic cells appeared in the growth zone. 3. By the fluorochrominized tissue culture method, the lymph node and the spleen of lymphatic leukemia showed typical lekemic growth zone and gave diffuse yellowish-green color due to the cytoplasmic and nuclcar fluorescence of lymphoblasts and other lymphoid cells. On the other hand, the bone marrow, spleen and lymph node of myelogenous leukemia showed relatively diffuse pattern and the inner part of the growth zone gave greenish fluorescence due to the cytoplasm and nucleus of immature leukemic cell, but in the periphery of the growth zone, reddish-orange color was predominant due to the fluoresceuce of cytoplasmic granules of mature neutrophiles. In conclusion, the lymph node and spleen in lymphatic leukemia and the bone marrow in myelogenous leukemia showed typical leukemic growth zone respectively and these findings are useful for the differential diagnosis of mouse leukemia.