KOBAYASHI, S., INABA, H., OKADA, S. and NAKADA, T. Culture and Changesin Dynamics of Proliferation of Pulmonary Small Cell Carcinoma Cells. Tohoku J. Exp. Med., 1989, 159 (1), 1-14 - We had developed a method for the selective cultivation of cancer cells in short-term which was simple and required minimal technical skills. There have been so far 23 cases of pulmonary small cell carcinoma in which long-term passage of more than one year and establishment of a cell line were successful by the culture method. In particular, from November 1982 to March 1987, there were 29 patients who were diagnosed as having pulmonary small cell carcinoma on preoperative cytodiagnosis and whose cells were primary cultured. In 24 (83%) of these, isolation could be carried out in a short period and stable passage was possible. Long-term passage of more than one year and establishment of a cell line were successful in 21(72%) of the 29 patients. As a result of primary culture of small cell carcinoma cells, a pattern of a suspended colony was observed in 22 (88%) of 25 cases. As passage advanced, the tendency toward attachment to the basal surface increased. The changes in dynamics of proliferation after long-term passage of more than one year were divided into three types; epithelial like cell type was observed in 8 (32%), the neurite like cell type was observed in 4 (16%) and the suspended colony type was observed in 13 (52%) of the 25 cases. Variant clones composed of relatively large cells appeared as a result of long-term passage or the use of carcinostatics in some pulmonary small cell carcinoma cells.
HIGUCHI, M., HIRANO, H. and MAKI, M. Effect of Human Epidermal GrowthFactor on Lung Surfactant Production in Fetal Rabbit. Tohoku J. Exp. Med., 1989, 159 (1), 15-22 - The effect of human epidermal growth factor (hEGF) on the maturation of fetal rabbit lung was studied. On the 25th day of gestation, 0.1 ml of saline solution containing 0.1μg of hEGF was injected intramuscularly through uterine wall into fetuses in one horn of the uterus (hEGF treated group) and the same amount of saline solution injected into fetuses in the other horn of the uterus (saline-control group). Sham operated group was also studied; saline was injected to fetuses in both horns. On the 27th day of gestation, fetuses were delivered by cesarean section. There were no significant differences in fatal body weight and fetal lung wet weight among three groups. Total phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) concentrations/g dry lung weight were high in order of hEGF treated group, saline injected group and sham operated group. There were no significant differences in PC concentrations among three groups. However, DSPC concentrations in both saline-control and hEGF treated group were significantly higher than that in sham operated group (p< 0.01). The % composition of palmitic acid in PC molecule, which is the main component of saturated fatty acid of lung surfactant, was significantly higher in hEGF group than in sham operated group (p<0.05). Interestingly, there were no significant differences in DSPC level and the % composition of palmitic acid between saline-control and hEGF treated group. This phenomenon might be due to so called a “neighbor effect” that hEGF injected to fetuses in one side of the uterine horn influences the fetuses in the other side of the horn through placentas. These results suggest that hEGF accelerates the production of pulmo nary surfactant, but does not inhibit cell growth of fetal lung.
TAKEUCHI, K., SATO, S., ABE, K., KIMURA, M., ABE, T., YOSHINAGA, K. and INABA, H. Intracellular Compartmentalization of Fura-2 Dye Demonstrated byLaser-Excitation Fluorescence Microscopy: A Problem in Measuring Cytosolic FreeCalcium Concentration Using Fura-2 Fluorescence in Vascular Smooth MuscleCells. Tohoku J. Exp. Med., 1989, 159 (1), 23-35 - Recently, we have developed a novel laser-excitation fluorescence microscope system to study intracellular calcium (Ca2+) in individual cultured vascular smooth muscle (VSM) cells using fluorescent indicators for Ca2+ In the cource of our study, it was shown that the subcellular fluorescence distribution of fura-2 was not homogeneous in VSM cells incubated with the acetoxy-methyl ester form of fura-2, fura-2/AM. The fluorescence appeared spotty or filamentous and resembled in shapes the intracellular organelles, suggesting that there was fura-2 dye compartmentalization in the organelles. To clarify the nature of the subcellular fluorescence, the soluble fraction of cells loaded with the dye was analyzed through high performance liquid chromatography (HPLC). We also examined the excitation spectra of fluorescence in the soluble fraction, which was compared with that in the cell suspension. Using HPLC, it has been shown that no other than fura-2 was found in the soluble fraction, whereas analyses of excitation spectra have indicated that the membrane fraction contained fura-2/AM or its lipophilic metabolite. On the other hand, indo-1 dye fluorescence showed a diffuse intracellular distribution, but the nuclear region had higher or sometimes lower fluorescence levels than the cytoplasm. The present results suggest that it may be necessary to assess subcellular fura-2 compartmentalization and possible interference by fura-2/AM or its lipophilic metabolite for the accurate measurement of intracellular Ca2+ concentration in VSM cells. It is also suggested that indo-1 may be more suitable for estimating Ca2+ concentration than fura-2 in individual VSM cells.
AIHARA, M., SAWADA, Y., TAKAMI, H., KARIYA, K., KUDO, I., KIMURA, A. and YOSHIDA, Y. Factor XIII is Not Involved in Human Platelet-Collagen Interaction. Tohoku J. Exp. Med., 1989, 159 (1), 37-44 - A role of factor XIII (FXIII) on the interaction of human platelets with collagen was investigated using either formaldehyde fixed-washed platelets (FWP) or nonfixed platelets. The adhesion of FWP to bovine type I collagen was measured by using either an aggregometer or a collagen immobilized glass beads column. The interaction of non-fixed human platelets with collagen was measured with in vitro bleeding time (Thrombostat-4, 000), which was performed by passing citrated whole blood through the filter covered with rat type I collagen under the constant shear stress. FWP adhesion to the collagen immobilized column (1, 300μg collagen) was not changed by the addition of commercial FXIII preparation (Fibrogammin); the adhesion was 42.7% in the presence of 1% human serum albumin, 42-43% in the presence of 1-2U/ml of FXIII. The addition of rabbit antibody to FXIII to normal FWP did not change the degree of a dhesion; 42.3% (1:100 anti-FXIII) and 46.1% (normal rabbit serum). Furthermore, platelets from the patient with congenital FXIII deficiency normally aggregated by bovine collagen and the adhesion of the patient FWP to the collagen was similar to that of normal FWP. Prolongation of partial thromboplastin time and the changes of thromboelastograph of normal plasma were observed after mixing with the collagen, and factor VIII, FXIII and von Willebrand factor were adsorbed by the collagen. The amount of FXIII in normal human plasma bound to collagen was 17, 23 and 54% at the concentration of the collagen 250, 500 and 1, 000μg/ml, respectively. The binding of plasma ristocetin cofactor was not different between normal control and the patient with FXIII deficiency. These data suggest that FXIII is not involved in human platelet interaction with the type I collagen, while FXIII in normal human plasma binds to the collagen.
OIZUMI, K., ONUMA, K., WATANABE, A. and MOTOMIYA, M. Clinical Study ofDrug Fever Induced by Parenteral Administration of Antibiotics. Tohoku J. Exp. Med., 1989, 159 (1), 45-56 - The incidence and clinical features of drug fever induced by antibiotics were investigated. Of a total of 390 patients analyzed, 193 had malignant diseases (lung cancer in most cases) and the remaining 197 had non-malignant diseases, of which the majority comprised pulmonary infectious diseases such as pneumonia, lung abscess and chronic infections. β-Lactams most frequently induced drug fever. Piperacillin induced drug fever in 18 of 108 (17%), cefotaxime in 11 of 72 (15%), ceftizoxime in 7 of 49 (14%) and cefoperazone in 6 of 74 patients (8%). In contrast, the incidence of drug fever caused by ampicillin and that by cefazolin were in one of 39 (3%) and in none of 44 (0%), respectively. On the other hand, antimicrobial agents other than β-lactams only rarely induced drug fever. The higher incidence of drug fever caused by newer derivatives of β-lactam antibiotic suggests that the side chain attached to their core moiety might be involved in the mechanism of drug fever. In patients with malignancy who were on antibiotics, respiratory infection was the most frequent cause of fever exceeding 38°C. In contrast, in patients with non-malignant diseases, the use of antibiotic per se was the most frequent cause of the fever which reccurred during antibiotic therapy after a previous febrile episode had subsided. The most common feature of drug fever induced by the use of an antibiotic was as follows: A low-grade fever at the time of onset is followed by a high and remittent fever. The highest diurnal body temperature rises gradually, and then the fever subsides promptly after cessation of the causative antibiotic. The fever of this type accounted for 70% of all the drug fever in this study. A transient elevation of serum level of lactic dehydrogenase was associated with drug fever in one half (25/49, 51%) of the patients. A transient and slight decrease from the normal range in counts of neutrophils and platelets were observed in 11 (23%) and in 4 (8%) of 48 patients with drug fever, respectively. These changes in laboratory findings were considered as the possible consequence of allergic processes involved in the development of drug fever and thus seem to be a helpful index for establishing the diagnosis of drug fever,
NISHIMURA, Y., HIDA, W., TAGUCHI, O., SAKURAI, M., ICHINOSE, M., INOUE, H. and TAKISHIMA, T. Respiratory Muscle Strength and Gas Exchange in NeuromuscularDiseases: Comparison with Chronic Pulmonary Emphysema and IdiopathicPulmonary Fibrosis. Tohoku J. Exp. Med., 1989, 159 (1), 57-68-To examine whether or not the respiratory muscle weakness is correlated with decrease in arterial oxygen tension (PaO2), respiratory muscle and pulmonary functions in 14 patients with neuromuscular diseases (NMD) were studied and compared with those of 12 patients with chronic pulmonary emphysema (CPE) and 15 patients with idiopathic pulmonary fibrosis (IPF). Respiratory muscle strength was assessed by maximal static inspiratory and expiratory mouth pressure at three lung volumes (RV, FRC and TLC). Although mean pulmonary functions in NMD showed virtually normal function, respiratory muscle strength was significantly less than the corresponding values in CPE and IPF. In NMD, maximal inspiratory mouth pressure at RV level (PImax) correlated positively with %TLC and %VC (r=0.652 and r=0.536, respectively). Moreover, PImax was significantly correlated with PaO2 (r=0.561), but not with PaCO2. Maximal expiratory mouth pressure at TLC (PEmax) correlated positively with %TLC and %VC. In CPE and IPF, respiratory muscle strength had no correlation with PaO2 and PaCO2. These findings suggest that inspiratory muscle dysfunction in NMD may be one of the factors responsible for determination of the level of hypoxemia and lung volume.
TAMURA, G., SAKAI, K., TANIGUCHI, Y., IIJIMA, H., HONMA, M., KATSUMATA, U., MARUYAMA, N., AIZAWA, T. and TAKISHIMA, T. Neurokinin A-InducedBronchial Hyperresponsiveness to Methacholine in Japanese Monkeys. Tohoku J. Exp. Med., 1989, 159 (1), 69-73-The present study was designed to investigate whether neurokinin A (NKA) and substance P (SP) increase bronchial responsiveness to methacholine (MCh). Before and 1, 2, 3 and 4 weeks after three Japanese monkeys had inhaled 10-8M NKA and 10-8M SP for 2min, bronchial responsiveness to inhaled MCh was evaluated. Pulmonary flow resistance (R1) and dynamic lung compliance (Cdyn) were used as pulmonary function tests. Dose-response curves of R1 to inhaled MCh were shifted to the left 1 to 4 weeks after NKA treatment, compared with that obtained before treatment. However, SP treatment did not change the R1 dose-response curve. Thus, bronchial responsiveness to MCh was increased by inhalation of 10-8M NKA in Japanese monkeys.
NAKAZAWA, I., IWAIZUMI, M. and OHUCHI, K. Some Features of the MetastaticCancer Cells in Prostaglandin Production. Tohoku J. Exp. Med., 1989, 159 (1), 75-78-In order to establish metastatic lesions, 2.5×106 AH100B cells were injected into the left carotid artery of male Donryu rats. Each metastatic nodule in the liver or kidney, 1mm or less in diameter, thus obtained was then injected into the peritoneal cavity in which these metastatic cells come to free. About 3 weeks later, each ascites was collected from the rats, while not bloody. Then, cancer cells obtained from each ascites were suspended in Dulbecco's PBS without Ca2+ and Mg2+ (pH 7.2) after washing. Then, 106 metastasized or control cancer cells were incubated in 0.1ml of PBS mentioned above together with 0.1μCi of (1-14C)-AA at 24°C for 3min, respectively. After the extraction procedure, AA metabolites formed were separated by means of TLC, and each TLC plate was subjected to autoradiography. In the metastasized cells, PG production ability was generally accelerated and especially in that of PGF2α as compared with that of the control.
KOIWA, Y., KIKUCHI, J., TAKAGI, T., HONDA, H., HOSHI, N. and TAKISHIMA, T. Human Left Ventricular Wall Vibration Responded to Precordial Minute Vibration. Tohoku J. Exp. Med., 1989, 159 (1), 79-80-We examined, noninvasively, the response of the left ventricular (LV) wall to precordially applied vibration in seven normal subjects. The LV posterobasal wall vibration was detected by an esophageal vibration sensor. Even though the amplitude of input vibration was maintained at a constant level, a sharp change in amplitude of the LV wall vibration was observed during the cardiac cycle, especially when 1) the subject was lying in a 45° left anterior oblique supine position, 2) the sensor was positioned at approx. 40cm from the incisor teeth and 3) breath was at expiration. The pattern of this amplitude modulation changed with the input frequency. We concluded that the change in amplitude of output signal must have resulted from LV contraction and relaxation.
YAMAUCHI, H., NITTA, A., ICHINOHASAMA, R. and NAMIKI, T. Non-InvasiveAdenomatous Component in Carcinoma of Papilla of Vater: An Immunohisto-chemical Study of Carbohydrate Antigen, CA19-9. Tohoku J. Exp. Med., 1989, 159 (1), 81-82-Eleven patients with carcinoma of papilla of Vater accompanied by non-invasive adenomatous component (nac) had good prognosis after surgical resection. These carcinomas were associated with low serum CA19-9 levels seldom invaded or metastasized to the pancreas, duodenum, lymph node and vein. On the contrary, 12 patients whose carcinomas lacked in `nac' had poor prognosis after resection. The `nac' negative carcinomas invaded or metastasised more frequently to the pancreas, duodenum, lymph node and vein. A half of the `nac' negative carcinoma showed high serum level of CA19-9. A significant different immunohistochemical pattern of CA19-9 was demonstrated between the `nac' positive and negative carcinomas. The former showed partial, while the latter showed diffuse distribution pattern.