Okayama University Medical School

A factor, cornin, inhibiting the growth of L cells cultured in monolayer was extracted from bovine liver with boiling water and was partially purified by gel filtration with Sephadex G-200. The factor was (1) precipitable with ethanol at the concentration between 70% and 90%, (2) impermeable through dializing memo brane, (3) eluted as the last peak at the gel filtration and (4) containing protein and RNA but no DNA.

Oncogenesis of human adenovirus type 12 in the brain of rats was examined. Newborn rats of Sprague-Dawley and Donryu　strains were injected intracranially with human adenovirus type 12.　The incidence of intracranial tumors was 91% (30/33) in SpragueDawley　and 56% (14/25) in Donryu rats. Except for one tumor nodule　located in the parietal cortex of a Sprague.Dawley rat, all tumors　developed in the paraventricular areas or in the meninges. Tumors were quite similar histologically to those induced in hamsters and　mice resembling the undifferentiated human brain tumors such as medulloblastoma,　ependymoblastoma and embryonic gliomas. From　the histological features and primary sites of tumor development, it is　suggested that the tumors in the brain of rats induced by adenovirus type 12 originate from the embryonic cells in the　paraventricular area　and also from the undifferentiated supporting cells of the peripheral　nerves in the　leptomeninges.

Detailed morphologic characteristics of type C virus particles observed in an X-ray-induced C58 mammary tumor and its transplants have been described. The particles are round and 75 to 100 mμ in diameter, containing an electrondense nucleoid 60 to 70 mμ in diameter. By the negative staining, they do not show obvious spines. Two abnormal types of particles, i. e. cylindrical and aberrant forms have been observed.

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.

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 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.

In the experiments with cultured liver cells it is very important to know whether or not the cells in vitro have the same properties and functions as in vivo. The purposes of this work were to investigate the functions of the cultured liver cells and to identify functionally the liver cells cultured by our present method with the parenchymal liver cells. At first, the albumin production of the cultured liver cells, one of the well known functions of the liver cells, was examined by the immunological methods, especially, the fluorescent antibody technique and the complement fixation test. Culture methods which could display the functions of the liver cells as much as possible were explored simultaneously. The results were as follows: 1. Albumin production was detected in the strain RLN·10 liver cells established from the liver tissues of a Donryu rat with immunofluorescent method and complement fixation test. This confirms that the cultured liver cells maintain the function to produce albumin and these cells have originated from the parenchymal liver cells. 2. Hepatoma strains (AH 66-TC-l, AH 7974-TC-l) also showed the albumin production but the extent of its production was less than that of the strain RLN-10. 3. In the short-term cultured liver cells, the albumin production was testified only slight in one month and was exhibited in a small amount in three months. 4. Every culture method examined exhibited no appreciable difference in the albumin production in the cultured liver cells.

As a link in the series of studies on tumor-specific immunity, in vitro inhibitory effect of sensitized isologous lymph-node cells on the proliferation of C3H mammary cancer was studied. For this purpose tissue culture was conducted with regional lymph-node cells obtained from truly isologous C3H mouse inoculated with A strain cells derived from C3H mouse mammary cancer along with A cells, and the following results were obtained. In the case of tissue culture with those lymph-node cells obtained from the groups of mice 10 days after the inoculation of 5 X 106 A cells, the inhibitory effect on the proliferation of A cells was most marked, followed by that of those taken on day 14, 7, and 5 decreasing in the order mentioned. In the case with those regional lymph-node cells obtained from mice which did not have recurrence of tumor 1 week after extirpation of 2-week old tumor, the inhibitory effect on proliferation of A cells was marked, with the regional lymph-node cells obtained two weeks after transplantation of 1 × 108 A cells there could be observed no inhibitory effect at all. This suggests that at a certain stage after implantation of such regional lymph- node cells there develops a specific anti-tumor activity in the host.

For purpose to study specificity in the growth inhibition effect of sensitized lymph-node cells on target cells, the regional lymph-node cells obtained from the truly isologous mouse previously inoculated with A strain cells (derived from C3H mouse mammary cancer) were cultured with A cells in various ways, and obtained the following results. 1. Those regional lymph-node cells from the isologous mice transplanted with the skin of C3H mouse or MC-induced sarcoma do not inhibit the growth of A cells in tissue culture. 2. The regional lymph-node cells from the mice positive to tuberculin test also do not inhibit the growth of A cells in tissue culture. 3. The regional axillary lymph-node cells (C3H anti-A strain cells) inhibit the proliferation of M cells from Cb mouse mammasy cancer and JTC-ll cells from Ehrlich ascites tumor as well as A cells. However, these axillary lymphnode cells do not inhibit the growth of AH-66F cells from rat DAB hepatoma, Hela-S3 cells from human uterine cancer and L cells from subcutaneous connective tissue of C3H mouse. From these results it is assumed that the sensitized regional lymph- node cells act specifically on cancer antigen.

For the purpose to clarify the relationship between production of humoral antibodies and cellular reactions of the lymphoid system to allogeneic-transplanted cells in mice, a study on cross sensitization was carried out between inbred A(H-2a) and C3H(H-2k) strain mice. The median survival time of skin of C3H transplanted to A (C3H-to-A) was 14.1 ± 1. 4 days, and of A transplanted to C3H(A-to-C3H) was 11.8± 1. 6 days. Repeated A cell injections to C3H induced the formation of humoral antibodies, whereas the C3H cell injections into A did not. In A-to-C3H and C3H-to-A combinations, the immunization induced an increase in white blood cell number in circulating blood successively with the repetition of the antigen injection, and organ weights increased in thymus, spleen, and liver but not in kidney. Weight increases in the organs of A treated with C3H cell injection were less in extent, comparing to those of C3H treated with A cells. Histologic observations revealed that the cellular proliferation in the lymphoid system including plasmocytic responses were obviously predominant in the C3H treated with A cells comparing to those in the A treated with C3H cells. Hemocytoblasts also increased during the immunization in both cases showing no significant differences between the two series of experiments. These cellular reactions were observed not only in the draining lymph nodes but also in the generalized lymphoid tissues. The results of the present study suggest that the definitive factor for producing humoral antibodies is in the differences of the homologous antigenicity between the donor and the recipient but not in the degree of sensitization, and the Dk in H-2 loci is not so strong in antigenicity as to elicit sufficient plasmocytic responses for the formation of humoral antibodies in C3H strain mouse.

Recently, by histochemical observations the changed activities of the enzymes of heartr,nuscle in experimentally induced ischemia have been reported by several investigatorsl~4. SHNITKA and NACHLAS4 demonstrated that the ligation of coronal artery of dog heart induced an increase in the activities of succinic dehydrogenase and NAD-diaphorase four to six hours after the ligation. However, extracorporeal circulation induced no distinct changes in the activities of succinic dehydrogenase and cytochrome oxidase as has been revealed quite recently by BJORK and associate5 from their histochemical studies of the specimen from left ventricular myocardium by a method of drill biopsy, but only the myocardial edema and fibrosis ocurred. This report deals with the distribution and activities of oxidative enzymes of human myocardium of fortyone cases of congenital heart disease and four cases of mitral stenosis and two controls, the specimen of which were obtained at the surgical operation. The purpose is to confirm the damaging effect of occlusion of blood flow in surgical operation on muscle fiber.

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.

For the purpose to clarify whether minimal catalatic activity exists in Japanese acatalasemic cells or not and the manner how extrinsic hydrogen peroxide affects the acatalasemic cells, the author performed tissue cultures using the skin specimens from four acatalasemic persons affected with Takahara's disease and studied the nature of these cultured cells. The results are summarized as follows. 1. Between normal and acatalasemic cultured cells, no morphological differences could be seen and the growth rate of these cell-lines was similar to one another. 2. On the activity of succinoxidase and cytochrome oxidase there could be observed no difference between normal and acatalasemic cells. 3. In each acatalasemic cell line the minimal catalatic activity was observed and it seemed that this activity has an important role in decomposing hydrogen peroxide under normal metabolic pathway. 4. After treating with 10-4M hydrogen peroxide, respiratory enzyme activities and the growth rate in the acatalasemic cells were markedly disturbed, while in normal cells these remained almost intact. 5. There could be observed no differences between normal and acatalasemic cultured cells after X-ray irradiation (200 to 600 r) on the succinoxidase activity, catalatic activity and growth rate.

Chromatography on Sephadex G-200 was performed with the soluble fraction of homogenated rabbit liver, which was extracted with 0.1 M phosphate buffer containing 0.1 M NaCl. and the influences of autolysis on the soluble fraction of liver were also examined. The soluble fraction of liver was different from serum in molecular weight, in electrophoretic character and in components with sedimentation coefficients. The soluble fraction of liver was stable under the influence of Mg and K ions, and rather unstable in the presence of Na ions. Serum was fractionated in three main peaks. The soluble fraction of liver was fractionated in a similar pattern as of serum, but the first peak contained nucleic acid and lipoprotein. The second contained albumin. 32p radioactivity peaks of the stored sample appeared with change in patterns by autolysis from the original, and were observed wide based and continuous figures in retarded peaks. The correlations with the first peak and retarded peaks were represented by the analysis of phosphorus compounds and electrophoresis. In lipid analysis, both diglyceride and monoglyceride gradually decreased, and phospholipid pattern was observed to increase in retarded peaks by autolysis. Lipoprotein or lipid-albumin complex was gradually converted to smaller molecular weight compounds, and appeared in retarded peaks.

In the present experiment, it has been noted that clonizing epithelial-like cells of the intestine 407 were more susceptible to SV-40 virus than normal fibroblasts in primary human cell cultures. In the early stage of the infection the cell growth was enhanced by the inoculation of DNA virus but many cells died, showing lysis characterized by CPE, clumping of chromatin and formation of inclusion bodies. On the other hand, the cells surviving infection have given rise to virus-free long term cultures and cellular responses to the virus characterized by cell proliferation which is. classified in four phases. (Phase. I: infection and cell alteration. Phase. II: crisis. Phase. III: fibro-reticulum cell formation. Phase. IV: recovery and proliferation). The most remarkable morphological characteristic was fibroblastic cell alteration from epithelial cells at 5 weeks of virus inoculation. By this study an interesting generalization of human epithelial-like cells can be made about the differentiation of the transformed cells in relation to SV-40 virus and it has been shown that an established human cell line is still susceptible to the reverting action of the SV-40 virus.

,The effect of infection of human embryonic skin-muscle cell cultures with adenovirus type 12 has been studied. When maintained in YLE containing 20 per cent bovine serum, human embryonic skin-muscle tissue culture cells developed little or no cytopathogenic effect for about 50 days after inoculation of adenovirus type 12, though a small amount of virus was always detected in the overlying medium. From day 50∼60, CPE started appearing and spread over 90 per cent of cells accompanied with the increase of virus in the overlying medium. The addition of human serum to the maintenance medium inhibited the virus release. After removal of human serum about 16∼37 days after its addition, virus-and, later, CPE also-again started appearing. The second virus release-and CPE also-was inhibited by addition of human serum to the medium. When maintained in the medium with human serum for about 200 days, the removal of human serum did not result in the appearance of virus or CPE. The virus isolated from the overlying medium of these cells during the whole process of the experiment was always highly oncogenic to newborn hamsters. Diluted adenovirus-12-immune rabbit serum also showed the effect similar to that of human serum. But, regardless of its much higher antibody titer, the effect of this diluted adenovirus-12-immune rabbit serum was weaker than that of human serum. In one of cell cultures, rapidly growing cells appeared 212 days after virus inoculation. But the available data suggest that these are the cells transformed rather spontaneously in tissue culture than by adenovirus type 12.

Les auteurs out effectue après une irradiation totale de 1200r de rats blancs des deux sexes des examens hématologiques à la suite d'irradiations ainsi que des examens physiologiques et des contrôles. Ils n'ont observe de modification importante des facteurs coagulants qu'au troisieme jour; cette modification était maximum avant la mort, c'est-à-dire au stade terminal. Les temps de coagulation naturelle ont beaucoup diminué, de même que ceux de la thrombine et ceux de la thrombine avec le bleu de toluidine, c'est-à-dire que l'héparine libérée ( = antithrombine semblable à l'héparine) a diminue. Pour les facteurs V et VII et en particulier pour la prothrombine on a observe un fort accroissement de la concentration. Les auteurs pensent que ceci est explicable par le fait que la décomposition des tissus pendant l'irradiation entraine la libération de kinase et d'autres activateurs dans la circulation sanguine, ce qui provoque une anoxemie des tissus. D'autres expériences sont en cours en collaboration avec de nombreux spécialistes et instituts.

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).