Okayama University Medical School

To obtain a useful rat liver cell line for in vitro carcinogenesis, two rat diploid epithelial cell lines were established from a 7-day-old male rat by the repeated colonial clone method. More than 80% of cells from each cell line have maintained normal diploid karyotype for over 30 months in vitro. The diploid cells were identi. fied as normal diploid karyotype by conventional Giemsa and trypsin. Giemsa techniques. They showed little difference in morphology and growth rate between early and late passages. Without cloning, they tended to be heterogenous in cell morphology, became heteroploid in chromosome and showed increased growth potential with time. Highly heteroploid cells which were derived from one of the lines produced ascites and solid tumors when inoculated into syngeneic rats intraperitoneally. Histologically, the tumors were diagnosed as poorly differentiated hepatocarcinomas. One of these diploid epithelial cell lines in early passage contained some activity of tyrosine transaminase and liver type aldolase and .glycokinase. Therefore, it is suggested that these epithelial cell lines represent liver parenchymal cells.

In order to study the viral etiology of human brain tumors, attempts were made to isolate cytopathogenic agents from human brain tumors by the tissue culture of tumor tissues and by the mixed culture of tumor tissues with HeLa cells. Five glioblastomas, a mixed form of glioblastoma and fibrosarcoma, two astrocytomas, two ependymomas, two meningiomas, an oligodendroglioma, a spongioblastoma polare and a choroid plexus papilloma were studied. In the tissue culture, besides the cells which appeared to be the tumor parenchymal cells, varying amounts of fibroblastic cells appeared in all the tumors tested and they increased with the prolongation of the culture period. In any of the tumors tested, no cytopathogenic agents were detected by either the culture of tumor tissues or the mixed culture of tumor tissues with HeLa cells. From the virological point of view, the significance of these negative results was discussed.

By dividing at random 14 normal persons into 7 pairs of two individuals each, lymphocytes were isolated from their peripheral blood and taking one of the pairs as stimulating cells or antigens and the others as responding cells, mixed lymphocyte culture (MLC) was carried out. As for the method of MLC we used our MLC method of unidirectional mixed culture with a small amount of lymphocytes in additition of 1% (v /v) PHA.M and cultured for three days, and a widely used conventional method in which 3H.thymidine uptake was the parameter of the blastformation rate and cultured for seven days. In comparing the results of these two groups of MLC the data in six experiments out of the seven coincided. Namely, with 5x 104 cells each of stimulating cell group and responding cell group, it is possible to achieve satisfactory MLC, the culture can be done only for three days without requiring any special technique and the results can be readily evaluated. Therefore, MLC by our simple method would yield satisfactory results in clinics.

The mode of lymph flow from the breast to the regional lymph node was studied using 1llSAu colloid, to ascertain the concept of lymph node of Groups 1, 2, and 3. Lymph flow through the internal mammary nodes was found to be abundant. Especially so when 1llSAu colloid was injected into the lower half of the breast, there was observed more abundant flow than that to axillary lymph nodes. Therefore, it was concluded that regardless of the site of occupation of a tumor, internal mammary nodes should be con· sidered to be of Group 1. Lymph flow through the subclavian nodes proved to be always less than that to the axillary nodes or internal mammary nodes. The rate of the lymph flow appearance on the scintigram was about 20%. Whether the subclavian nodes should be classified as Group 1 or 2 should be further studied. The rate of supraclavicular node visualization was below 3%. Therefore, the supraclavicular nodes should be considered to be Group 2. When these results are applied to the current rule of handling of breast cancer, discrepancy occurs in the Stage number and R-numer.

The effect of pyridoxine treatment of a homocystinuric patient on the urinary excretion of some sulfur-containing amino acids was studied and the following results were obtained. As a result of pyridoxine treatment, urinary homocystine decreased to a fairly great extent, and its unusual metabolites S.(3-hydroxy-3-carboxyn- propylthio) homocysteine (HCPTHC) and S-C8-carboxyethylthio homocysteine (j3-CETHC) increased to some extent. But its oxidation product (homocysteic acid) showed a tendency to decrease slightly. Urinary methionine and cystine increased to some extent, but cysteinehomocysteine mixed disulfide showed no remarkable change.

Neocarzinostatin (NCS), an antibiotic with a high molecular weight, showed an inhibittory effect on Rauscher mouse leukemia. In normal mice, no significant changes were found in peri· pheral blood pictures except a tendency of lymphocytopenia, when O. 05mg/kg/day and O.50mg/kg/day of NCS were injected intraperi. toneally to two groups of mice for three days. On the other hand, peripheral nucleated cells of Rauscher leukemic mice decreased after intraperitoneal administration of NCS in a dose over O.25mg/kg/day for three days. The cells affected by NCS were mainly erythroblasts and smudged cells. Spleens of Rauscher leukemic mice treated with NCS have been reduced in weight, and histological examinations of livers showed a signicant decrease of infiltrating cells. In three groups treated with 0.25mg/kg/day of NCS for seven days, O.25mg/kg/day for three days and O.50mg/kg/day for three days, the.5()% survival time was longer than in the control group. Particularly, the 50% survival time in Rauscher leukemic mice treated with 0.50 mg/kg/day for three days was over twice that of the control group.

A case of left atrial myxoma accompanied by peculiar symptoms is reported. A 15-year old boy had progressive congestive heart failure and three episodes of acute attacks of panctyopenia. The anemia was accompanied by helmet-shaped, broken red blood cells, erythroid hyperplasia and elevation of indirect bilirubin. The thrombocytopenia gave rise to hemorrhagic tendency of the skin and mucous membrane. The leukocytopenia was seen at the same time. The patient also had general constitutional disturbances showing generalized malaise, persistent fever, elevation of erythrocyte sedimentation rate, positive C-reactive protein, pulmonary infection and anginal attacks. Postmortem examinations revealed a left atrial myxoma and intricated pulmonary changes. There was obliterative endarteritis of the left coronary branch and pulmonary arteries. The interstitial pulmonary fibrosis was also prominent. The pancytopenia should have been induced by the mechanical damage of circulating blood cells by the left atrial myxoma. The pathological findings of the lungs were highly suggestive of pulmonary hypertension, which was assumed to be due to mitral block caused by the atrial myxoma.

T cell concerned with cell mediated immunity and B cell concerned with humoral antibody are classified by scanning electron microscopy of the surface structure of lymphocytes using E binding lymphocytes and EAC (sensitized sheep erythrocytes treated with complement) binding lymphocytes. For the purpose to elucidate morphological differences between T cell and B cell the scanning electron microscope observations were carried out with the blast forming lymphocytes incubated in the presence of PHA. As a result it has been demonstrated that T cells have short microvilli on the cell surface, but the reason for the difference in the number of the microvilli is unclarified. Even T cells have sometimes long microvilli in the younger stage, they are longer and more slender than those of untreated peripheral B cells.

We examined the method for determining microquantities of lipids, including cholesterol, cholesteryl esters and phospholipids. A standard colorimetric procedure of cholesteryl esters was modified to accommodate a quantitative thin-layer chromatography. This method involved the following steps. (1) Separation of lipids by a thin-layer chromatography: Lipids were applied to Silica gel G plates. Plates were developed with petroleum ether-diethyl etheracetic acid (82: 18: 2, vIvIv). (2) Elution of cholesterol and its esters from scraped silica gel: After scraping the silica gel with adhered cholesterol and its esters, they were eluted with chloroform-methanol (4: 1, v,tv). In the case of phspholipids, the silica gel was calcified. (3) Colorimetric determination of the lipids: Cholesterol and its esters eluted from the silica gel were determined by the method of ZAK with ROSENTHAL'S color reagent directly and after saponification, respectively. Phospholipids were calculated from the phosphorous content determined by the method of KATES. On the basis of examination of recovery and analyses of lipids extracted from tissue, it was concluded that this method permitted a reliable estimation of microquantities of cholesterol, its esters and phospholipids from small amounts of biological materials.

Antitumor effects of the combination chemotherapy with hemolytic streptococcus preparation, OK-432, and various anticancer agents were observed on experimental tumors and human cancers. Experimental studies revealed that combined use of OK-432 with Mitomycin C, Nitrogen mustard N-Oxide or Bleomycin was remarkably effective on rodent transplantable tumors such as Ehrlich carcinoma, sarcoma-lOO and rat ascitic hepatoma AH-66. As for the mode of action of OK-432, besides a direct action on cancer cells, a host-mediated action appears to be also involved. Clinical trials were made on 14 cases with various advanced cancers, and favorable response was obtained in 5 with lung cancer. Fever was the major side effect of OK-432 and there was no evidence of bone marrow suppression.

Using a freeze-etching method, the ultrastructure of cell surface of gram-positive cocci was studied by digesting cell wall with lytic enzyme. In M. lysodeikticus, the cell surface revealed a very simplified ultrastructure, i. e. a single cell wall layer and a single plasma membrane layer. On the contrary, the cell surface of S. aureus exhibited a unique structure composed of two cell wall layers and a single ploasma membrane layer. The wall layers were constituted of 160 -180 A particle layer (CWl) which was unsusceptible to the L-ll enzyme and amorphous layer (CW2) which was susceptible. These results suggested that 160-180 A particles in CWl consisted mainly of the teichoic acid.

The streptococcal preparation, OK.432, with predominant host-mediated mode of action, was studied. By giving OK-432 to mice intraperitoneally prior to transplantation of Ehrlich carcinoma, a host-mediated action to increase life-span was clearly confirmed. Pretreatment with OK-432 was also effective against the development of Rauscher leukemia. The host-mediated action of OK-432 varied with the interval between its pretreatment and the inoculation of tumor cells. The effect was most marked when the transplant was performed immediatedly after the pretreatment, and became less marked when the transplant was made one week and two weeks after pretreatment. The host-mediated action can be observed even with a single dose of pretreatment, and becomes more potent as pretreatment was given repeatedly. The host-mediated action was weakened by concomitant pretreatment with cyclophosphamide or roentgen irradiation, and the mechanisms of such action was supposed to be associated with the function of the reticulo-endothelial system.

For predicting median date or incidence rate in the prevalence of japanese encephalitis, the authors considered the 34 factors; namely, the climate, latitude, longitude, and date showing immunological positivity of hemoagglutination inhibiting reaction on 50% the number of swine, etc. To make the mean square residual the smallest which yields, in the case of calculating with the multiple regression equation, the most important and meritorious factors were selected from the factors mentioned above by the voluntary selection rule devised by us. Multiple regression equations were formulated for them. To predict the median date in the prevalence of japanese encephalitis in whole japan, we found that the three factors, i. e. the amount of rainfall in April, the average temperature in March and HI positive rate of swine till the end of july, were essential. And for the foreseeing of incidence rate in Okayama Prefecture, factors concerning mosquitoes were added; this resulted in the useful two factors, namely, common logarithm of the total number of Culex tritaeniorhynchus till july 20th and the rainfall in June.

To observe the possible role of cAMP on the DNA synthesis during specialization-division of myelogenous precursor cells, the authors observed the DNA and RNA synthesis of the cells by in vitro autoradiography. And it is concluded that cAMP or its dibutyryl derivative added to the media penetrated into myelogenous precursor cells and metamyelocytes of mice and enhanced the DNA synthetic capacity of them. cAMP hardly enhanced RNA synthesis. Discussion is made on relation between enhancement of DNA synthesis of metamyelocytes and their possible rejuvenation.

To clarify the ultrastructure of the extended ribosomal RNA molecules, electron microscopic observations were carried out on the RNA molecules extracted from purified ribosomes of mouse ascites sarcoma cells. By the treatment with ethylenediamine-tetraacetate agglomerated rRNA molecules were elongated to thread-like structure by partial unfolding. The lengths of thread-like molecules were measured as less than Iii. The strand of RNA molecules stained with uranyl acetate was observed approximately l5A in width.

Cultured rat liver cells which were cloned from a single cell were transformed into malignant cells by a chemical carcinogen, 4-dimethylaminoazobenzene (DAB). The DAB-transformed cells produced tumors when back-transplanted into new-born rats but the carcinogen-untreated control cells did not. Characteristics of the transformed liver cells were compared to those of DAB-untreated control cells in regard to the morphology, the consumption of DAB from the culture medium by the cells, the incorporation of 3H.DAB into the cells, and the aggregate.forming ability of the cells in rotation culture. The results showed that no significant parameter of malig. nant transformation in culture was detectable except the tumorigenicity of the transformed cells upon the inoculation into animals.

For the purpose to define the mechanism of heavy metal intoxication by inhalation, morphologic observations were made on rat lungs after nasal instillation of iron colloid particles of positive and negative electric charges. Histochemical observation was also made on the liver and spleen of these animals. The instilled iron colloid particles reach the alveolar cavity easily, as can be seen in the tissue sections stained by Prussian blue reaction. Alveolar macrophages do take up them avidly both of positive and negative charges, though much less the positive particles than negative ones. In contrast, the alveolar epithelial cells take up solely positive particles by phagocytosis but not negative ones. Electron microscope observation revealed that the positive particles are ingested by Type I epithelial cells by pinocytosis and by Type II cells by phagocytosis as well. Then the iron colloid particles are transferred into the basement membrane by exocytosis. Travelling through the basement membrane they are again taken up by capillary endothelial cells by phagocytosis. Some particles were found in the intercellular clefts of capillary endothelial cells but not any iron colloid particles in the intercellular spaces of epithelial cells and in the capillary lumen. However, the liver and spleen tissues of the animals given iron colloid showed a strong positive iron reaction. On the basis of these observations, the mechanism of acute intoxication by inhaling heavy metal dusts like lead fume is discussed from the view point of selective uptake of alveolar epithelial and capillary endothelial cells for the particles of the positive electric cha'rge.

Hematological changes and serum iron, vitamin B12 and folate levels after administration of iron plus folic acid were com· pared with changes and levels after supplement of iron alone in 100 pregnant women. No hematological benefits by adding folic acid was revealed. It is concluded that folic acid deficiency in pregnant women is mild and routine supplementation of folic acid is not necessary except for proved cases of folate deficiency. As to the reason why folic acid deficiency is so mild and megaloblastic anemia is so rare in Japan, uniformity of dietary habits, i. e. boiled rice as basic food, was discussed.

By endolymphatic injection of radioisotope 131I.Lipiodol, so.called endolymphatic radiotherapy, we treated 10 cases with malignant lymphoma and found a marked tumor reduction to normal size in all the 10 cases we tried. It seems that this method is one of the most effective therapeutic methods for malignant lymphoma, espe. cially invading into the retroperitoneal lymph nodes.