Okayama University Medical School

Histochemical evaluations of human sarcomas such as reticulum cell sarcoma, fibrosarcoma, lymphosarcoma and neurofibrosarcoma, were carried out with five hydrolytic enzymes and eight oxidative enzymes. The activities of acid phosphatase and beta-glucuronidase were slightly positive in the neoplastic cells observed. Beta-esterase activity was also positive but varied according to the kind of sarcomas. Alkaline phosphatase activity was faint or negative in sarcoma cells, though positive in capillary walls. Leucine aminopeptidase activity was negative giving not any appreciable coloration of the cell as far as the method employed is concerned. Among the activities of dehydrogenases, the most intense activity was observed in lactic dehydrogenase. The activities of succinic and beta-hydroxybutyric dehydrogenases were slight. The activities of alpha-glycerophosphate, glutamic and betahydroxybutyric dehydrogenases were faint or slight. The activities of NADPlinked dehydrogenases, glucose-6-phosphate and isocitric dehydrogenase were all faint or slight in these sarcoma cells.

With the purpose to prevent the dissemination and consequent metastasis of cancer cells at the time of operation we gave 10 mg of Mitomycin C per day for four consecutive days prior to surgical operation of gastric cancer (total of 322 patients), and this so-called adjuvant chemotherapy proved to be effective on the cases with serosal involvement and infiltrating type of cancer, irrespective of histological types. It also gave five-year survival rate of 35 per cent. However, to lymph nodes already metastasized, the adjuvant chemotherapy proved to be not effective.

DNA synthesis and cell renewal of mouse intestinal epithelium were studied with radioautography after injection of thymidine-H³ to know the difference of the mode of epithelial cell generation relating to the different frequency of cancer developement in several parts of small and large intestines. Succinic dehydrogensase activity was also observed by histochemical method. Renewal time of the intestinal epithelium of mouse is about three days throughout the intestine with somewhat longer time in rectum and anus, and relatively shorter one in ileum compared to the other parts of the intestine. Daily regenerating rate was low in large intestine, especially in rectum and anus. Strong activity of succinic dehydrogenase appeared in the bottom of crypt and seems to be correlated to the active cell division. Epithelial cells in large intestine move very slowly upward and few of them seem to move to the opposite side or stay long time at one place. Intermitotic time is about 27 hours in small intestine and about 40 hours in large intestine. These suggest some relations between the mode of the epthelial cell renewal and cancer development. Because in human the frequency of cancer development is very high in large intestine, rectum and anus, and the epithelial renewal of these areas is supposed to be delayed similarly as in mice.

Large doses of adenovirus type 12 were injected intraperitoneally into adult hamsters, and development of tumors and other pathological findings were studied in comparison with those in hamsters injected when newborn. Doses of 38~47 TCID60 per gram body weight produced tumors in 3 of 12 hamsters injected at 37~57 days of age. A dose of 170 TCID60 per gram body weight produced tumors in one of 18 hamsters injected at 61~71 days of age, but in none of 18 hamsters injected at 147~174 days of age, while the same dose per gram body weight produced tumors in 24 of 26 hamsters injected when newborn. In hamsters injected at adult ages, the number of tumors per animal decreased and the latent period for tumor development became very long as compared with those in hamsters injected when newborn. Regardless of the age at the time of injection, acute inflammatory change was observed in the peritoneum which later developed into various degrees of peritoneal adhesion. Adenovirus type 3 also induced the peritoneal adhesion. Histology of tumors was studied and discussed.

With gastric carcinomas the activities of eight dehydrogenases; succmlC, lactic, malic, α glycerophosphate, glutamic, β-hydroxybutyric, glucose-6-phosphate and isocitric dehydrogenase were statistically estimated. Principal findings may be briefly summarized as follows. These enzymatic activities differed considerably even in the same classification of carcinomas and generally ranged from strong to weak in the following order: lactic, malic, glucose-6-phosphate, isocitric, succinic, α-glycerophosphate, glutamic and β-hydroxybutyric dehydrogenase. The activities of adenocarcinomas were stronger than those in simple ones, and these were not related appreciably to cell differentiation in adenocarcinomas except succinic, glutamic, glucose-6-phosphate and isocitric dehydrogenase. As for succinic dehydrogenase and NAD-linked dehydrogenases except for lactic dehydrogenase, the activities were strongest in intestinal metaplasia and early mucosal carcinomas, the next being in benign adenomatous polyps and weakest in the other carcinomas. As for NADP-linked dehydrogenases and lactic dehydronase, the activities were also strongest in intestinal metaplasia and early carcinomas, the second in the other carcinomas and the third in the benign polyps. Generally, these dehydrogenase activities were strongest in free carcinoma cells in blood and lymph vessels and in actively growing part of several carcinomas and weakest in the central area of tumors, especially almost negative in the central necrotic area.

1) Three different types of muscle fibers were clearly distinguished in the intercostal muscles and the diaphragm of human, cat and rat by histochemical demonstration of oxidative enzymes, phosphorylase and glycogen. 2) The intercostal muscles and diaphragm each presented dissimilar patterns of the muscle fibers. The diaphragm did not show any definite correlation between the diameter and the histochemical reaction of muscle fibers but its red fibers indicated a more intense uptake of Nitro-BT formazan deposits in the subsarcolemmal region. In this conection, the relationship between the motor innervation and histochemical evaluation of the diaphragm was described. 3) Phosphorylase and glycogen showed reciprocal reactions to the oxidative enzyme activity. They were generally high in large fibers but low in small fibers, and moderate in intermediate fibers.

1. Isovaleric acid-1-C14, -4-C14, or C14-CaC03 with or without non-isotopic isovaleric acid was orally administered to rats and the incorporation of these isotopes into liver cholesterol, fatty acid, or urinary isovalthine was examined. 2. Isopropyl group of isovaleric acid was more efficiently utilized for cholesterol synthesis than carboxyl group, and also for cholesterol synthesis than for fatty acid. These results indicate that isovaleric acid is cleaved into two fragments before it is utilized for cholesterol synthesis. 3. Carbon dioxide was used for the synthesis of liver cholesterol and of liver fatty acid. Isovaleric acid seems to enhance the incorporation of carbon dioxide into cholesterol. 4. All the experimental rats received isotopic or non-isotopic isovaleric acid excreted isovalthine, but no radioactivity was found in it. Thus, isovaleric acid residue of urinary isovalthine molecule is not derived from isovaleric acid administered, and carbon dioxide is not the carbon source of urinary isovalthine. 5. Suspicious metabolism of isovaleric acid or of carbon dioxide was discussed. 6. Isotopic isovalthine which was synthesized from (± ) α-bromoisovaleric acid-4-C14 is administered to rat and it was found that the isotope did not incorporate into cholesterol or fatty acid of liver and of brain. About 15% of isotopic isovalthine was recovered in urine up to the next day after injection. The large part of isovalthine was missing.

Summing up the above problems we may group them as follows: 1) Szirmai's angio-myograph and myotonometer furnish us with means to evaluate the author's successful method or medical treatment in thrombophlebitic, postthromboembolic (ulcer, etc···) states on the basis of the blood circulation through the muscles, clearly registered by the angio-myograph. 2) Szirmai's medical preparation "HAH" serves as a quick and effective cure for thrombophlebitis. Results are very often reached within a few days. The patient's health is restored so as to make him able to work. 3) The above preparation assures full success in the cure of thrombotic esp. thromboembolic states of the lower limbs-cases of ulcus cruris includedwhich up to now could not be favourably influenced by any other method of treatment. Description of varieties of above problems and other types of cases of peripheral circulation (Endangitis, etc.) and their relationships with the subject will be given in additional papers. The author reports on registering and controlling thrombophlebitis, postthromboembolic states, including ulcus cruris, origin~ting either in above morbid conditions or in independent causes by means of the angio-myograph and myotonometer devised by the author. The reader is made familiar with the author's (Szirmai's) preparation "HAH" (Heacrin) and with the results achieved by applying it for the cure of acute thrombophlebitis and thrombotic states. Results are often showing up remarkably soon (2 to 6 days).

The activities of five hydrolytic enzymes, alkaline and acid phosphataSe, beta-esterase, leucine aminopeptidase and beta-glucuronidase, of human gastric carcinomas from 180 patients were investigated histochemically. Alkaline phosphatase activity was almost negative in the carcinoma but was weakly positive in this tumor at times (about 10 to 20 per cent). Acid phosphatase activity which displayed a slightly increasing tendency of the reaction in poorly differentiated tumor was variegated and mainly from feeble to moderate in activity. Beta-esterase reaction was in varying degrees with each case, but more malignant the carcinomas, the weaker was the activity. Leucine aminopeptidase was positive in about 30 to 60 per cent of the specimens observed but the reaction was founded to be localized often in some areas and generally similar to alkaline phosphatase reaction. The activities of leucine aminopeptidase, alkaline phosphatase and beta-esterase were positive at a higher rate in mucinous carcinomas than in non-mucin producing one. Beta-glucuronidase activity was slight or moderate in general but rather strong in the early stage of carcinomas.

In the present communication the recent works done by the Rheumatism Research Group of Department of Orthopedic Surgery, Okayama University, are described. The principal findings may briefly be summarized as follows. 1. Pathohistological pictures of the synovial membrane are classified into six types. Among them, Fibrinoid type and Follicular-Fibrosis type are the representative ones of chronic rheumatoid arthritis. 2. For the evaluation of the systemic as well as the local activities in rheumatoid arthritis and for judging the therapeutic effect, some indices have been established. 3. Injection of steroid hormones into the local joints fails to give satisfactory results in advanced, chronic rheumatoid arthritis. In such instances the flushing of the joint with physiological saline solution is effective. 4. In the case of chronic rheumatoid arthritis where the inflammation of hand and phalangeal joints is marked, RA-test gives rapid and more intense reaction, and most of such cases are of Follicular-Fibrosis type. 5. When lymph follicles appearing in the synovial membrane are stained when methyl green pyronine, the arrangement of lymphoid cells and plasma cells becomes distinctly clear. By micro-autoradiographic observations it can be seen that ³H-thymindine injected into the joint cavity is mostly ingested by the lymphoid cells in lymph follicles. 6. In the observation by the fluorescent antibody method multinuclear leucocytes found in the joint fluid and in the peripheral blood react with 19S and 7S-gamma-globulins. 7. When the serum and the joint fluid of the patient with rheumatoid arthritis are fractionated, they separate into three peaks at 19S, 7S, and 4S. Both S. S. C. A.-test and L. F. T. tests reveal the peak at 19S. The serum of chronic hepatitis positive to RA-test and the serum of rheumatoid arthritis are found to react immunologically the same to anti-β2 M globulin sheep serum. 8. When the reticulo-endothelial system of rat is blocked by 900,000 molecules of poly-vinyl-pyrroridon, the ability of antibody production is diminished. 9. Chemical synovectomy of injecting osmic acid is effective to FibrinoidCoating type. Its action mechanism lies in the complete cleaning of the surface of synovial membrane. 10. By radiating synovectomy with 193Au a fairly good result can be expected. 198Au is ingested by those cells in the surface layer of the synovial membrane and also by histiocytes in the synovial membrane. When 5 mc of 198Au are injected into the knee joint, a marked necrosis of the synovial membrane occurs. When 198Au is added to the ascites cells of rabbit during the tissue culture, in the concentration of over 14 μC degeneration of these cells can be recognized. 11. From the examination results of prognosis on those 25 cases with 41 rheumatoid knee joints after surgical synovectomy, it is considered that this method is indicated for Follicular-Fibrosis type. Ones with rheumatoid knee joint of Fibrinoid-Coating type gold sol treatment should be resorted to. In the cases of hand joints, surgical synovectemy is to be recommended at a relatively early stage.

The distribution and activities of five hydrolytic and eight oxidative enzymes were histochemically studied in 60 different tumors of the human intestines. Benign polyp showed similar activities of most enzymes as those in normal crypt cells of large intestine with exception of higher activity of succinic dehydrogenase in benign polyp than in crypt cells. Malignant polyp had higher activities of most oxidative enzymes. Reticulo-sarcoma had weak activities of all enzymes. Carcinoid had strong activities of glucose-6-phosphate dehydrogenase and isocitric dehydrogenase while very weak of succinic dehydrogenase. Carcinoma showed varying degrees of the activity of all enzymes. Alkaline phosphatase and aminopeptidase were almost negative in all cells but in the stromal elements their weak activities were sporadically observed. Most enzymes were decreased in the central area of the carcinoma cell nestle, while in the infiltrating area or in the margin of cell nestle they were not decreased and sometimes increased.

1. The ratios of free 5α-cholestan-3β-ol and cholesterol and esterified 5α-cholestan-3β-ol were higher in pylorus than in cardia. 2. Esterified cholesterol level was higher in cardia than in pylorus. 3. Among the stomach cancer tissues examined free cholesterol level was higher than in the non-cancerous. 4. Esterified 5α-cholestan-3β-o1 and cholesterol levels were lower in the cancerous tissues than in the non cancerous.

The present report describes the findings on the infectivity of DNA partially purified from SV-40 which was propagated in the monkey kidney cells (BSC-1) in vitro and the importance of nucleic acids as oncogenic factors, particularly the induction of tumor by DNA in newborn hamsters. 593 newborn hamsters in total were used in the present experiments, and cannibalism among them posed as a serious problem. On 30 days postinoculation, very remarkable changes occurred in the liver, lung and subcutaneous areas. Cellular responses of the perivascular cells were predominant. and they were distributed in the interstitial tissues of the liver (liver cirrhosis in primates) and lung. Three hamsters of those subcutaneously inoculated with nucleic acids developed tumors and two tumors appeared in the subcutaneous tissues on 130 days postinoculation, which were identified to be the ones induced by intact SV-40 virus. Other tumors appeared in the liver, lung, intestinal ducts and abdominal surface at 126 days after subcutaneous injection. The cytological observations revealed multiple hemangiosarcoma combined with proliferation of the perivascular cells. On the other hand, cellular responses to nucleic acids were more marked by inoculation of the cell-free filtrate of BSC-1 infected by DNA than of DNA, and essential histologic findings were similar to the respo.nse to infectious DNA. Thirty-nine hamsters (30 per cent) developed tumor within about 200 days postinoculation of the filtrates. Sarcomas were common and they were confined to the subcutaneous tissues in 35 hamsters and to the peritoneum in two others by subcutaneous inoculation of the filtrates. The intestinal gland-cell carcinomas, however, could be induced at 37 and 59 days postinoculation in two hamsters of one litter (7 newborn hamsters) and in the other three newborn hamsters subcutaneous sarcomas were induced by inoculation of the same agent. These results suggest that the observation on the oncogenic capacity of nucleic acids would give us a clue to resolve the course of cancer from the view point of the infectious nucleic acid.

For the purpose to study in vivo changes of the mice bearing Ehrlich ascites tumor with special emphasis on the problems of cellular antibody and serum antibody, a series of experiments such as neutralization tests in vivo and in vitro study of the effect of lymph-node cells from the tumor bearing animals on target cells were carried out, and the findings thus obtained are briefly summarized as follows : 1. Regional lymph-node cells from the mouse transplanted with Ehrlich ascites tumor cells show a marked cytotoxic action on their cultured target cells, JTC-11, synergistically with serum from mouse bearing Ehrlich cancer. 2. The tumor cells inoculated with lymph-node cells from the tumor bearing animals showed a retardation in growth and finally regressed. 3. Spleen and lymph nodes of tumor bearing animals showed a marked increase in weight.

Adenovirus type 12 is of human origin and it shows carcinogenic activity in experimental animals. With negatively stained particles of this virus electron microscopic observations were carried out. As the result it was demonstrated that its capsid, like other adenoviruses, is an icosahedron and each capsomere is of a hexagonal shape with a hollow in its center, each of which is surrounded by 6 adjacent capsomeres and is composed of numerous small subunit-like-particles.

Human brain turnors removed from 126 patients were histochemically examined and following results were obtained. 1. In general, alkaline phosphatase activity is decreased in poorly differentiated gliomas, but is not related to the tumor cell infiltration. 2. All the cases of alkaline phosphatase negative gliomas have poor reconvalescent course and most of the positive cases show good reconvalescence. 3. Alkaline phosphatase, leucine aminopeptidase and acid phosphatase activities are remarkable in fibroblastic meningioma, moderate or feeble in meningocytic meningioma, and negative in malignant meningioma. 4. The activities of alkaline phosphatase, β-esterase, leucine aminopeptidase and acid phosphatase are decreased in most of meningocytic meningiomas when the duration of symptoms and signs is short. 5. Succinic dehydrogenase, malic dehydrogenase, isocitric dehydrogenase and β-glucuronidase are strongly reactive in malignant meningioma; from strong to moderate in meningocytic meningioma and from moderate to feeble in fibroblastic meningioma. 6. There is a slight increasing tendency of the activities of succinic dehydrogenase, malic dehydrogenase, isocitric dehydrogenase in fibroblastic meningioma and p·glucuronidase for a short duration of symtoms and signs. 7. In the case of acoustic neurinomas the higher the alkaline phosphatase activity, the longer is the duration of symptoms and signs.

There is as yet no plausible and convincing explanation for the etiology of rheumatoid arthritis. The authors investigated anti-synovial tissue antibody in the body fluid of rheumatoid arthritis by means of indirect immunofluorescent technic using non-affected synovial tissues as antigen. As the result the anti-synovial tissue antibody was detected in 7 cases of the 15 synovial fluid samples of rheumatoid arthritis and in two out of the six serum samples. The site of the localization of this antibody was demonstrated to be in the synovial membrane, especially in synovial cells and in the small blood vessel walls situated immediately adjacent to the synovial surface, but it was found in no connective tissues other than synovial membrane. It seems that this anti-synovial tissue antibody should be considered as an independent factor from rheumatoid factor, and that rather than the rheumatoid factor it is more actively associated with the localization and progression of chronic inflammation within the rheumatoid arthritis joint.

Macromolecular PVP was introduced intravenously into rabbits for a long period of 3 months, 10 g of PVP in total, and the observations were carried out to see disturbances in hematopoiesis, lymphopoiesis, and immune reaction with special reference to the histologic changes of marrow, liver, spleen and lymph nodes. The results were as follows: 1. A mild anemia was induced by the PVP injection. RE cells of liver and bone marrow were swollen moderately but otherwise no significant histologic changes were induced in bone marrow and liver. 2. A severe lymphocytopenia resulted: the RE cells of lymph follicles were blocked by PVP, the follicles collapsed into a homogeneous mass with fibrosis and minimized lymphopoietic tissues. These results suggest that RE cells of the so-called germinal centers are important for the reproduction or the differentiation of lymphocytes. 3. Intravenous injection of egg albumin caused the serum antibody formation with a marked proliferation of plasma cells around small vessels in lymph nodes and spleen as in the case of control animal. The data indicate that plasma cells are solely responsible for the serum-antibody formation and plasma cell may differentiate from adventitial cells of small vessels but not from lymphocyte or reticulum cell.

For the purpose to clarify the control mechanism of erythroid cell differentiation, the author observed morphologic changes in bone-marrow cells and circulating red cells in phenylhydrazine anemia of rabbits by introducing a mass of red cells into vein at one time and reached the following conclusions. 1. After red cell transfusion in a mass to animal showing a marked hematopoietic activity, anisocytosis or macrocytosis becomes distinct with the appearance of big reticulocytes and red cells as large as four times the normal in volume. This suggests, judging from their volume, the accelerated denucleation of erythroblast as early as at the late basophilic stage. 2. Observations on bone marrow at this stage revealed the reduction in the number of erythroblasts of undifferentiated type with the increase of rather differentiated ones. In erythroid islet, undifferentiated cells are found surrounding a reticulum cell located in the center, while well differentiated ones in the outskirt area are situated near the sinusoid. Such a cell arrangement suggests that the erythroid cell requires a high oxygen tension for its differentiation. 3. From these observations and other results obtained from the studies on reticulocyte maturation and RNA synthesis of erythroblast, the author stresses that erythroid cells can differentiate as long as it is provided with a certain level of oxygen, even though it may develop m-RNA for differentiation. In other words, there should be two steps in the differentiation of erythroblast, the first is m-RNA synthesis induced by the information and the second is the somatic protein synthesis with oxygen supply. This seems to be directly connected to the control mechanism of hematopoiesis by oxygen.

1. For the elucidation of the mechanism of isovalthinuria induction, some precursors of cholesterol biosynthesis and some other compounds were tested in guinea pigs to know whether they induced isovalthinuria or not. 2. Among the cholesterol precursors tested, isovaleric, β-methylcrotonic, and β-methylglutaconic acids were found to be strong inducers of isovalthinuria, but acetic acid, acetoacetic acid, β-hydroxy-β-methylglutaric acid, squalene, α-ketoisocaproic acid, and leucine were not. 3. Among the other compounds tested, palmitic acid and L-cysteine were found to be weak inducers of isovalthinuria. 4. Some antagonistic phenomena were found to occur. The inducing effect of isovaleric acid was inhibited strongly by dexamethasone or dehydrocholic acid, and weakly by insulin. The inducing effect of dexamethasone was inhibited by dehydrocholic acid or thyroxine. The inducing effect of dehydrocholic acid was inhibited by insulin or dexamethasone. 5. Although ethionine was not an inducer in itself, it acted as a substitute for methionine or cysteine in the case of the sulfur deficiency produced by prolonged feeding on bile acid. 6. Isovalthinuria was observed by the administration of bromural, but this effect may be a detoxication but not induction,