Kazuo Ryoyama

Studies on the biological properties of coelomic fluid of sea urchin 1. Naturally occurring hemolysin in sea urchin

Abstract 1. 1. Biological properties, especially hemolytic activity, of coelomic fluid of sea urchin were investigated. 2. 2. Dialyzed coelomic fluid preparation obtained from Anthocidaris crassispina was found to have hemolytic activity against rabbit, mouse and human erythrocytes, but not against erythrocytes of other species of animals so far examined, i.e. rat, guinea pig, goat, sheep, dog, pig, hen, snake and frog. A similar hemolysis spectrum was obtained with a coelomic fluid preparation from Pseudocentrotus depressus, while coelomic fluid preparation from Hemicentrotus pulcherrimus showed a capacity to lyse erythrocytes of rat and guinea pig, besides those of rabbit, mouse and man. 3. 3. The hemolytic activity of coelomic fluid preparation was greatly enhanced by the addition of Ca2+ to the medium, but not by Mg2+. Addition of EDTA, even in the presence of Ca2+, resulted in a loss of potency. 4. 4. The degree of hemolysis induced by coelomic fluid preparation was dependent on concentration and temperature of incubation. The kinetic analysis as a function of concentration indicated that the hemolysis curve resembles those of saponin and streptolysin O. 5. 5. The hemolytic factor in coelomic fluid preparation was so labile that complete inactivation was effected after 30 min of incubation at 56°C, and after storage for 24 h at pH 11.3. 6. 6. The active factor may be not a saponin but a protein or a protein-like substance, since its activity was abolished irreversibly by treatment with trypsin or 2-mercaptoethanol.

Adriamycin induced immunomodulation: Dependence upon time of administration

The immunomodulating capabilities of the anti-neoplastic agent, Adriamycin, were investigated. The day of Adriamycin administration to mice was varied from -15 to -1, day 0 being when mice were either immunized or sacrificed and their spleen cells sensitized in culture. Humoral and cellular immune responses against allogeneic or xenogeneic cellular antigens in mice and in culture, as well as phagocytic and ADCC activities were evaluated using spleen cell populations. The cellular responses and phagocytic activities were affected in a cyclical manner with time after Adriamycins administration. Peaks of increased activity were seen subsequent to day -5 and day -11, administration and low activities following day -1, -3 and day -7, -9 administration. The humoral responses were not affected in a biphasic manner but single peaks of increased activity were seen which corresponded to the times of low cellular cytolytic and phagocytic activities. The ADCC was independent of time of Adriamycin administration. The significance of these findings to the design of therapeutic protocols is discussed.

Inhibition of macrophage nitric oxide production by arachidonate-cascade inhibitors.

We examined whether inhibitors of the arachidonic acid cascade inhibited nitric oxide (NO) production, as measured by nitrite concentration, either in macrophages or by their cytosolic fractions. Nitrite production by peritoneal macrophages from mice receiving OK-432 treatment was significantly inhibited by phospholipase A2 inhibitors [dexamethasone and 4-bromophenacyl bromide (4-BPB)], lipoxygenase inhibitors [nordihydroguaiaretic acid (NDGA) and ketoconazole] and a glutathioneS-transferase (leukotrienes LTA4-LTC4) inhibitor (ethacrynic acid). However, caffeic acid and esculetin, inhibitors of 5- and 12-lipoxygenase respectively, were not inhibitory. On the other hand, indomethacin, a cyclooxygenase inhibitor, slightly inhibited whereas another inhibitor, ibuprofen, did not. Inhibition of the nitrite production by dexamethasone, 4-BPB, NDGA and ethacrynic acid was also demonstrated when the macrophages were restimulated ex vivo with OK-432 or with lipopolysaccharide. The inhibitory activity of dexamethasone, NDGA and ethacrynic acid was significantly reduced by ex vivo restimulation with OK-432, whereas that of 4-BPB was hardly affected. Furthermore, the inhibitory activity of dexamethasone, NDGA and ethacrynic acid was much higher when the macrophages were continuously exposed to the agents than when they were pulsed. Meanwhile, inhibition by 4-BPB was almost the same with either treatment. In addition, the inhibitory activity of these agents was not blocked withl-arginine, a substrate of NO synthases, or with arachidonate metabolites (LTB4, LTC4 and LTE4). Ethacrynic acid and 4-BPB, but not dexamethasone and NDGA, also inhibited nitrite production by the cytosolic fractions from OK-432-restimulated peritoneal macrophages, and the inhibitory activity of 4-BPB was superior to that of ethacrynic acid. These agents, however, did not inhibit nitrite production from sodium nitroprusside, a spontaneous NO-releasing compound. These results indicate that dexamethasone, 4-BPB, NDGA and ethacrynic acid inhibited the production of NO by macrophages through at least two different mechanisms: one was inhibited by dexamethasone, NDGA and ethacrynic acid and the other by 4-BPB. Furthermore, 4-BPB and ethacrynic acid directly inhibited the activity of the NO synthase in macrophages, suggesting that the agents work by binding to the active site(s) of the enzyme.

Effector molecules from antitumor macrophages induced with OK-432 and cyclophosphamide

SummaryThe present study was designed to determine whether antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent on their ability to produce a soluble factor, that is,l-arginine-dependent nitric oxide as measured by nitrite concentration. Nitrite production by peritoneal macrophages from NIH Swiss mice pretreated with OK-432 (125 KE/kg) i.p. twice at 1-week intervals and with cyclophosphamide (200 mg/kg) i.p. 2 days before the second OK-432 treatment, increased with time for 24 h, and proportionally depended on macrophage numbers. Nitrite production was inhibited by actinomycin D and puromycin but not by mitomycin C.NG-Monomethyl-l-arginine, a specific competitive inhibitor ofl-arginine-dependent nitric oxide synthesis, also inhibited production. There was a close correlation between nitrite production and antitumor activity in macrophages from mice pretreated with either OK-432 and cyclophosphamide, OK-432, or thioglycolate broth. OK-432 increased both nitrite production and antitumor activities when added to the macrophage from mice pretreated with OK-432 but not with thioglycolate broth. Both activities of macrophages from mice pretreated with OK-432 and cyclophosphamide were enhanced with increasing concentrations ofl-arginine (0.125–1 mM) in the culture medium.d-Arginine, however, did not substitute forl-arginine. Neither activity was affected by contact between the macrophage and the EL4 cell. The macrophage showed antitumor activity through a membrane filter though the activity was greatly reduced. This antitumor activity of macrophages through a membrane was also inhibited byNG-Monomethyl-l-arginine, and increased by OK-432. However, conditioned media, obtained by culturing macrophages induced with OK-432 and cyclophosphamide, inhibited growth of EL4 cells. This activity was carried out by dialysable and non-dialysable factors. One of the dialysable factors was nitrite, an oxidized product of nitric oxide. The antitumor activity of non-dialysable factors was heat-stable and production of factors was increased byNG-Monomethyl-l-arginine and OK-432. Also, non-dialysable factors increased both antitumor and nitrite production activities of OK-432-elicited macrophages, when incubated with factors. Such activity of factors was also heat-stable. The production of factors increased with incubation time of macrophages, and was not inhibited byNG-Monomethyl-l-arginine. These results indicate that in vitro antitumor activity of macrophages induced with OK-432 and cyclophosphamide was mainly dependent onl-arginine-dependent nitric oxide, and that macrophageassociated soluble factors other than nitric oxide were also needed to inhibit fully tumor growth in vitro.

Studies of the properties of a streptococcal preparation, OK-432 (NSC-B116209), as an immunopotentiator

SummaryThe present study was designed to examine the mechanism by which OK-432 triggers the cytotoxic activity of peritoneal exudate cells (PEC). When OK-432 was incubated with freshly harvested mouse serum, the formation of complexes of OK-432 with the third component of complement (C3) was demonstrated by using 131I-labeled mouse C3. The formation of C3-OK-432 complexes was totally abolished by a chelating compound, EDTA, which had been shown to inhibit the OK-432 induced activation of the alternative complement pathway. The C3-OK-432 complexes thus obtained bound to the resident PEC, which were subsequently shown to be activated. These activated PEC had augmented cytostatic activity against MM2 cells, a mouse mammary carcinoma.Further, the PEC from mice which had received an IP injection of OK-432 4–5 days previously were cytostatic against MM2 cells and also inhibited the growth of MM2 cells in culture. In contrast, resident PEC stimulated rather than inhibited the 3H-thymidine uptake by MM2 cells and the growth of MM2 cells. The mechanism of PEC (presumably macrophages) activation by OK-432 is discussed.

The differential sensitivity of T cell immune functions to vincristine and vinblastine

Normal C57Bl/6 mouse spleen cells cultured alone for 4 days (suppressor generation culture) were shown, in mixing experiments, to suppress the primary cytotoxic T-lymphocyte (CTL) response of freshly explanted C57Bl/6 spleen cells against allogeneic tumor cells. This suppression was not apparent when spleen cells from mice pretreated with 1 mg/kg vinblastine (VLB), were used for the suppressor generation culture. However, suppression was noted with vincristine (VCR) at the same dose. Similarly, CTL generation cultures of spleen cells from mice treated with 12 LD10 VLB (3 mg/kg) were inhibited while those of spleen cells from mice treated with 12 LD10 VCR (1.5 mg/kg) were not. These effects observed following in vivo injection of the vinca alkaloids were also seen following addition of the drugs in vitro. In these experiments various anticancer drugs [arabinofuranosylcytosine (Ara-C), 200–1000 nM; Adriamycin (AM), 10–100 nM; VCR, 1–100 nM; VLB, 1–100 nM] were added to suppressor cell generation cultures. Ara-C (1 μM) and VLB (10 nM) inhibited the development of suppressor activity, whereas AM (100 nM) and VCR (100 nM) had no effect. The data indicate that these effects were not due to differences in the number of viable cells recovered. On the other hand, when these drugs were added to CTL generation culture at a responder to stimulator cell ratio of 50:1 (a ratio which gave somewhat less than optimal responses), Ara-C markedly inhibited the development of CTL, VCR and VLB both caused a dose dependent inhibition of the response and AM had no effect. In other experiments, the development of a primary humoral response to SRBC in culture with spleen cells from drug-treated donor mice was more sensitive to VCR than to VLB. These data show that VCR and VLB have different selective effects on components and/or functions of immune responses.

Application of the polymerase chain reaction (PCR) to quantify micro-metastasis in an experimental animal

In vitro cultured r/mHM-SFME-1 cells were injected into the hind foot pads of Balb/c mice. Metastasis was detected in the lungs of tumor-bearing mice by means of both PCR and histological methods. Primers for the PCR were set to amplify a 128 bp exon-1 sequence of the human c-Ha-ras1 gene which had been introduced into the cells. Resulted PCR bands were densitometorically quantified using a bioimage analyzer, and more than 1 x 10(4) tumor cells were detectable in the mouse lung. The number of tumor cells per lung estimated from the amount of PCR products was 1 x 10(5), 15 x 10(5), 1 x 10(5) and 40 x 10(5) on days 7, 14, 21 and 28 respectively after the tumor injection. No metastases were histologically observed on days 7 and 14. Then, the possibility of using this model system for evaluation of a treatment against micro-metastases is discussed.

Effects of injection routes of growing tumors PSK or OK-432 on antiproliferative activity of mouse serum

SummaryThe present study examined the effects of various treatments on the antiproliferative activity of mouse serum. Its activity was estimated against the growth of EL4 tumor cells and L929 cells and against splenic blastogenesis in culture. The activity varied among mouse strains tested and among individuals in any strain. However, normal outbred NIH Swiss mice showed the highest activity among the strains and the least variation among individuals. The activity of serum from NIH Swiss mice constantly decreased 7 or 14 days after an injection of 106 Ehrlich or sarcoma 180 tumor cells subcutaneously in the right-hind footpad, intradermally in the right side of the chest or into the palm. Other routes, such as intraperitoneal, intravenous in the tail vein, subcutaneous in the right side of the chest and intramuscular in the left thigh, however, hardly affected the activity. The activity also decreased 7 days after an injection into the footpad of a biological response modifier such as PSK or OK-432. The antiproliferative activity of mouse serum seems to depend on macrophages but not natural killer-cell activity, because treatment with silica but not anti-(asialo-GM1) antibody totally reduced the activity. The active fraction was heat-stable (100° C, 30 min) and its molecular mass was 127–140 kDa.

Induction of suppressor T cells in culture—II. Modification by adriamycin

The present study was designed to examine the effects of Adriamycin treatment of spleen donor mice on the subsequent generation of the suppressor T cells induced in culture in the presence of fetal calf serum. When mice treated with Adriamycin (5 mg/kg; i.v.) 5 days before sacrifice were used as donors of the spleen cells, the suppressive activity which developed in culture was somewhat greater than that which developed with spleen cells from untreated mice; particularly in terms of suppression of PFC development. The suppressive activities of the cultured spleen cells from untreated and treated mice were equally sensitive to X-irradiation and anti-Thy 1.2 antibody plus complement treatment. When only those cells which were nonadherent to plastic were used in the suppressor generation culture, suppression did not develop until day 6 of culture. Again, cells from Adriamycin treated mice developed somewhat greater suppressive activity than that which developed with cells from untreated mice. The effects of Adriamycin treatment of the mice on the subsequent development of suppressive activity in culture was also dependent upon the day of drug administration. Finally, spleen cells from Adriamycin treated mice, also, developed somewhat greater suppressive activity in response to alloantigen but when used as the source of progenitor cytotoxic T cells they were less sensitive to suppression; particularly by alloantigen-induced suppressor cells. Possible correlation between Adriamycin induced modifications of both the development and function of suppressor T cells in culture and its reported modifications of CTL and phagocytic activities are discussed.

Cyclophosphamide modifies the induction kinetics but not cell types and cytotoxic mechanisms of antitumor cells elicited with OK-432 plus attenuated tumor cells

SummaryThe present study was designed to examine whether the antitumor cells induced by treatment with mitomycin-C-treated EL4 cells (EL4MMC) plus OK-432 plus cyclophosphamide differed from those induced by treatment with EL4MMC plus OK-432 in terms of their cell types and antitumor mechanisms. Antitumor activity of peritoneal exudate cells (PEC) from mice receiving either treatment was nonspecific, and inhibition of their target cell growth increased for up to 24 h. Macrophage toxin, silica and trypan blue abrogated the activity in vitro and in vivo, respectively. The activity of the PEC was inhibited with inhibitors of the arachidonic acid cascade, such as dexamethasone, 4-bromophenacyl bromide and nordihydroguaiaretic acid but not esculetin, ibuprofen, indomethacin and BW755C.NG-monomethyl-l-arginine, a specific competitive inhibitor of thel-arginine-dependent nitric oxide synthesis, also inhibited the activity. These results and morphological observations indicated that antitumor cells in the PEC from mice receiving either treatment were macrophages, and that their activity was closely related to the arachidonic acid cascade and to nitric oxide. Antitumor activity of the PEC spontaneously decayed in vitro and this decay was inhibited by the addition of OK-432 or lipopolysaccharide. On the other hand, cyclophosphamide sustained the appearance of antitumor cells in mice treated with EL4MMC plus OK-432. Therefore, cyclophosphamide treatment did not modify cell types and cytotoxic mechanisms of antitumor cells elicited with EL4MMC plus OK-432, but did modify the induction kinetics of such antitumor macrophages.