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The effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6, which was inoculated at low density in a serum-free medium was examined. Several supplements were required to enhance cell growth of HuH-6. These included cholera toxin (CT), glucagon (Glu) and selenium (Se). Type IV collagen (C-IV) provided the most conductive environment tested for cell growth. These results suggest that CT, Glu, Se, and C-IV are important stimulators for the continuous growth of HuH-6 in a serum-free medium at low density.

Effects of capsaicin on the circular muscle motility of the isolated guinea-pig ileum were investigated. Capsaicin produced a contraction followed by a relaxation of the circular muscle. Both responses were easily desensitized. As the late relaxation response was not sufficiently intense to be analyzed, the inhibitory effect of capsaicin on substance P-induced contractions was explored. Capsaicin abolished the substance P-induced contractions. This inhibitory effect was not affected by tetrodotoxin, and the effect was desensitized. Therefore, all effects of capsaicin on circular muscle motility seem to be due to the release of sensory neuropeptides, similarly to those elicited in the longitudinal muscle.

Changes in the hemodynamics of six patients having received Fontan-like operations were closely observed during the first 48 h after the operation. Catheterization studies and simultaneous angiocardiography were also performed before and after the operation. Hemodynamic derangement was particularly severe during the first 24 h postoperatively as indicated by a low cardiac output of less than 2.01/min/m2, which persisted in spite of very high central venous pressure. Furthermore, the central venous pressure needed to re-establish the circulation soon after the Fontan procedure significantly correlated with the angiocardiographically assessed preoperative size of distal pulmonary arteries. Accordingly, the preoperative evaluation of the distal pulmonary arterial size is very important, that provides a good guide-line for the degree of circulatory volume expansion necessary to elevate the central venous pressure and to sustain the circulation in the early postoperative period.

The effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the organization of cytoskeleton and growth of normal and established chick embryo cells (CEC) were studied. The cytoskeleton of normal CEC formed stress fibers, while that of the CEC lines established in our laboratory formed no stress fibers. TPA treatment of normal CEC resulted in disorganization of the stress fibers into amorphous structure, while that of the established CEC lines induced no reorganization of the cytoskeleton. TPA had no promotional effect in vitro or in vivo on tumor growth in normal or the established CEC.

Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer. The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E. coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label. Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA. The long tag sequence derived from the M13 vector may increase the sensitivity of the detection. The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates. As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development.

Dynamic ergometer exercise in a supine position was applied to 64 patients more than 1 year after valvular heart surgery, and the left ventricular reserve was evaluated echocardiographically. The left ventricular reserve declined in the mitral stenosis-mitral valve replacement group, while it was better maintained in the mitral stenosis-mitral commissurotomy, aortic regurgitation and aortic stenosis groups. The patients were divided into 3 groups depending on whether the percentage increase during exercise of stroke index, an index of left ventricular pump function, increased, unchanged, or decreased. The percentage increase of mean velocity of circumferential fibre shortening (y) and that of left ventricular end-diastolic diameter (x) during exercise were plotted for each group. The increased group was isolated from the unchanged group by the line of y = -5.02x + 30.1; the unchanged group was isolated from the decreased group by that of y = -5.68x-10.0, and the increased and unchanged groups were clearly isolated from the decreased group by that of y = -6.86x-4.76. We conclude that dynamic ergometer exercise echocardiography is useful for evaluating the left ventricular reserve of postoperative patients with valvular heart disease. It was also thought that the subclinical state of cardiac failure can be effectively detected by the present method.</P>

We studied the in vivo antitumor effects of natural human tumor necrosis factor-alpha (nHuTNF-alpha) and natural human interferon-alpha (nHuIFN-alpha), both of which were produced by HVJ (hemagglutinating virus of Japan)-stimulated acute lymphatic B cell leukemia line, BALL-1 cells. To clarify the interaction between nHuTNF-alpha and nHuIFN-alpha, we used novel experimental models of lung metastasis and intraabdominal carcinomatosis which we developed in nude mice using a human tumor line, RPMI 4788. While the intravenous administration of nHuTNF-alpha or nHuIFN-alpha alone inhibited lung metastasis, the two cytokines given in combination synergistically inhibited lung metastasis. In a comparative study, nHuTNF-alpha and recombinant human interferon-gamma (rHuIFN-gamma) in combination also synergistically inhibited lung metastasis. Treatment with nHuTNF-alpha and nHuIFN-alpha combined significantly prolonged the survival of nude mice with intraabdominal carcinomatosis. Complete regression of five different human tumor xenografts was achieved by the simultaneous intratumoral injection of nHuTNF-alpha and nHuIFN-alpha. Histological examination revealed that tumor cell lysis occurred 24 h after the intratumoral administration of the cytokines. No significant signs of toxicity to nude mice were observed at any dose tested. The synergism of nHuTNF-alpha and nHuIFN-alpha may allow treatment at a relatively low dose range, thus minimizing side effects. The wide range of anticancer activity of these agents may provide better therapeutic efficacy. The in vivo assay systems which we have developed are useful for the analysis of the biological activities and interactions of cytokines and chemotherapeutic drugs.

In the human lymphoreticular system, the alpha and beta subunits of S-100 protein are found in ordinary monocyte-macrophages and non-phagocytic histiocytes such as Langerhans cells and interdigitating reticulum cells, respectively. The beta subunit is also present in some CD8+ T cells. In the present study, we investigated the ontogeny of these histiocytes and lymphocytes in humans. Yolk sacs and 4 to 21-week fetuses were examined immunohistochemically for the presence of S-100 protein subunits using antisera monospecific to each subunit. S-100 alpha + macrophages were present in the yolk sacs and the hepatic sinusoids of the 4th week embryos prior to bone marrow hematopoiesis. These macrophages later appeared in other lymphoid organs when anlagen of these organs were formed. No S-100 beta + cells were found in the yolk sacs. S-100 beta+ histiocytes were first detected in the hepatic sinusoids of the 5th week embryo, and after the 8th week of gestation, they were distributed in other lymphoid organs. S-100 beta+ lymphocytes were not found in the liver. They were first detected in the thymus at the 12th week of gestation, and were subsequently distributed in other lymphoid organs. These results suggest that S-100 beta+ lymphocytes and histiocytes may belong to different cell lineages, and the former may not be the precursor of the latter.

Isozyme patterns of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) in human cell lines derived from primary hepatomas were compared with those in HeLa cells. Some cell lines derived from primary hepatomas having type B G6PD showed one or two isozymes of LDH. On the other hand, HeLa cells having type A G6PD showed four LDH isozymes. These findings suggest that not only G6PD, but also LDH may be useful for the detection of HeLa cell contamination of a culture in some cases.

Markers of hepatitis A and B virus were tested in 88 adult Sudanese subjects in Khartoum, Sudan. The subjects consisted of 25 control hospitalized patients, 21 volunteer blood donors, 23 patients with hepatosplenic schistosomiasis, 13 patients with liver cirrhosis and 6 patients with hepatocellular carcinoma (HCC). Antibody to hepatitis A virus was detected in 96% of the total. Hepatitis B surface antigen (HBsAg) was positive in 4, 24, 22, 31, and 67% of the subject groups, respectively. Antibody against hepatitis B core antigen (HBcAb) of undiluted serum was positive in 60, 57, 65, 77 and 83%, and there was no difference in incidence among the groups. It was positive in 200X diluted serum in 4, 24, 17, 23 and 60%. HBsAg and HBcAb (200X) were detected more often in HCC patients than in the control subjects (p less than 0.01). Hepatitis B virus is an important factor in the etiology of HCC in the Sudan.

An attempt was made to isolate the cell proliferation stimulation factors in the supernatant of embryo carcases and adult muscles of chickens. Evidence was obtained for the presence of at least two or more stimulating factors in both the embryonic and adult muscular supernatants. These factors did not require a supplement of sera or other supporting agents. Furthermore, the use of the salting-out method with ammonium sulfate revealed two or more growth stimulants in the supernatant of chick cells.

The effect of an intravenous injection of squid-ink (sepia-melanin) solution on adult mouse spheroid alveolar epithelial cells was observed by the electron microscope. Sepia-melanin particles were seen in all alveolar wall cells examined that seems to suggest the entrance of sepia-melanin particles into the spheroid alveolar epithlial cells from the alveolar blood capillary. In cases of large penetrations of sepia-melanin particles into spheroid alveolar epithelial cells, a greater increase was found in the intramitochondrial granules. In addition, the so-called inclusion body believed to be formed by the degeneration of mitochondria had very high electron density and its quantity was abundant. On the contrary, in cases where the quantity of sepia-melanin entrance into the spheroid alveolar epithelial cell was small, neither an increase of intramitochondrial granules, an increase of the electron density nor an increase in the quantity of specific inclusion body was found.

Forty-five patients with acute leukemia were compared on cellular immunity measures versus prognosis. The patients were treated according a multicombination therapy protocol. The purified protein derivative (PPD) test and dinitrochlorobenzene (DNCB) test on admission indicated low positive percentages. In acute non-lymphocytic leukemia (ANLL) patients, the 50% survival durations were 11 months in the PPD positive group and 6 months in the PPD negative group. In acute lymphocytic leukemia (ALL) patients, the 50% survival durations were 21 months in the PPD positive group and 13 months in the PPD negative group. Peripheral lymphocyte blastogenesis by phytohemagglutinin (PHA) stimulation was examined at various clinical stages. The stimulation indices were generally low, and no correlation was found between the PHA test and clinical stages. These cellular immunity measures appeared to reflect one aspect of the clinical condition in acute leukemia patients, and further studies are needed for predicting prognosis.

Electron microscope observations were conducted on the relationship between mitochondria and inclusion body in mice spheroid alveolar epithelial cells after injection of trypan blue, an acidic dye and Alcian blue 8GS, a basic dye, by vital staining procedures. When both dyes were injected, the mitochondria of the spheroid alveolar epithelial cell became degenerated; however, in injection of only trypan blue, the cristae showed an increase in electron density. In injection on only Alcian blue 8GS, the cristae showed negative contrast. In most cases the trypan blue particles did not enter into mitochondria, whereas particles of Alcian blue 8GS sometimes entered into the mitochondria. When trypan blue particles entered mitochondria, deposits were not evident in the inclusion body, whereas when Alcian blue particles entered mitochondria deposits were seen in the inclusion body. In both of these cases only a few inclusion bodies were formed so that only traces or no inclusion bodies with vacuolar appearance were observed. From these findings it is suggested that mitochondria maybe convert to inclusion bodies.

The relationship between alveolar macrophages and spheroid alveolar epithelial cells was studied with the electron microscope after injection of squid-ink solution into the trachea of the mouse. At 20 hours after injection of squid-ink solution slight degeneration was evident in alveolar macrophages with sepia-melanin particles being phagocytized with partial digestion by lysosmes. Furthermore, hardly any changes were seen in mitochondria and inclusion bodies of the spheroid alveolar epithelial cells. In contrast, at one week after injection of squid-ink solution, almost all alveolar macrophages were degenerated with destruction of the ectoplasm in which the ingested sepia-melanin particles were digested by lysosomes into fine particles, and the mitochondria of spheroid alveolar epithelial cells were degenerated and the inclusion bodies were hardly formed. At three weeks after injection of squid-ink solution, alveolar macrophages as well as speroid alveolar epithelial cells showed almost complete recovery of functional structure. As the phagocyte in the alveolar space, neutrophile leucocytes were also observed in addition to the so-called alveolar macrophage.

Tissue localization of a subcomponent of the first component of complement (CLq) was examined in one postmortem case of HBs antigen (HBs Ag) positive hepatocellular carcinoma and in six cases of chronic hepatitis from liver biopsy specimens. The direct immunofluorescent method was used after fixation with 2% para-formaldehyde in concentrated ammonium sulfate. CLq localization was found in collagen fibers and the cytoplasm of fibroblasts in the connective tissues of specimens examined. The localization was particularly marked in the region of the fundal glands of the gastric wall. Apart from collagen fibers, other sites of localization included the surface membrane of lymphocytes, especially those cells of the mesenteric lymph nodes. In HBs Ag positive specimens, immune deposit-like substances appeared localized intra-hepatically and in the renal glomeruli. Since C3 and C4 were identified concomitantly, it indicates that these substances were indeed immune diposits. Despite the finding that C3 and C4 were identified together in the hepatic cell cytoplasm, C1q itself was not demonstrated in all hepatic cell cytoplasms.

Today Vitallium is used for surgical implants. It is a casting alloy which, with advances in casting technology, is also used commercially for making instruments of fairly complex shape. Because of its expense, however, it is not widely used in Japan. Instead, a series of 18-8 Mo alloys are used in Japan even though of insufficient strength. Used over a long period of time in the body, especially for the purpose of preserving structual functions as part of the human skeleton, it often corrodes, resulting in either abnormalities in tissue cells or, because of its insufficient strength, danger of bending and breaking with aging. In spite of a marked advance in fracture treatment, we have hardly any suitable materials for making instruments appropriate to the internal fixation of fractures in Japan. We, therefore, conducted various experiments to develop an alloy with sufficient corrosive resistance and strength that could be formed into a complex shape to take the place of Vitallium alloy, finally succeeding in developing an alloy we call "COP". The characteristic properties of COP may be summarized as follows: 1. The main components are 20% Cr, 20% Ni, 20% Co and 4% Mo aside from 0.2% P. 2. As it contains "P", it shows a marked age-hardening. In its molten state its machinability is excellent, and later it can readily be hardened by heat-treatment. 3. It has not only a marked yield point and tensile strength but also has toughness in elongation and reduction of area, showing a strength which surpasses Vitallium. 4. Its corrosive resistance is great. 5. Its cost is far cheaper than Vitallium.

Further purification and characterization are reported on rat cytosol cornin (RLCC), an antimitotic substance. Fraction I (purified RLCC) was purified more than 10-fold from crude RLCC with Sephadex G-50 column chromatography and showed a remarkable inhibitory effect on division of inseminated sea urchin eggs and mouse fibroblast cells. Fraction I was observed as one spot, and the molecular weight was estimated to be about 25,000 by thin layer gel filtration. Fraction I contained protein (92%) and RNA (8%), but the antimitotic activity was scarcely affected by treatment by pancreatic RNase. The protein of Fraction I was separated into two bands by SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated as 10,000 and 15,000, respectively. The 50% inhibition dose of Fraction I on the first division of inseminated sea urchin eggs and on proliferation of mouse L cells was about 2.5 X 10(-5) g/ml and 5 X 10(-4) g/ml, respectively. The yield of fraction I was about 35 mg from 100 g rat liver.

The present study was conducted to investigate the usefulness of the direct leucocyte migration agarose method for studying cell-mediated immunity in vitro. Comparative studies of the purified protein derivative (PPD) skin test and the leucocyte migration inhibition test (LMIT) in which PPD was used as test antigen indicated a significant qualitative and a weak quantitative correlation between these two tests. Furthermore a positive correlation was found between the LMIT and the macrophage migration inhibition test (MIT) using ultrasonicated authochthonous tumor antigen. Comparative studies of the LMIT, MIT, PPD skin and DNCB tests on the same patients showed that cases responding positively to the the PPD skin and DNCB tests tended to respond positively to the LMIT and MIT. Patients with digestive organ cancers were examined by the LMIT. With the advance of cancer, decreased positive test test rates were found. After gastric cancer operations the LMIT findings were divided into two groups: one type changed from positive to negative, and the other type changed from negative to positive. The former response was suggestive of a successful operation, and the latter response was suggestive of a non-curative operation. These results indicated that the direct leucocyte migration inhibition agarose test was useful investigating cell-mediated immunity.