Takao Iwaguchi

Inhibition of angiogenesis by vitamin D3 analogues

The effects of vitamin D3 and two analogues on embryonic angiogenesis were studied in 4.5-day-old chick embryo chorioallantoic membranes. The active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3, and a synthetic vitamin D3 analogue, 22-oxa-1 alpha,25-dihydroxyvitamin D3, inhibited angiogenesis in a dose-dependent manner, the inhibition occurring in the picomolar range. In contrast, vitamin D3 was not effective. The results suggest that these two vitamin D3 analogues might be promising anti-angiogenic agents for controlling the angiogenesis which occurs in several pathological conditions, including tumor development.

Novel function of ascorbic acid as an angiostatic factor.

Endothelial permeability is increased by vascular endothelial cell growth factor and decreased by antioxidants. Whether or not l-ascorbic acid (Asc), which decreases endothelial permeability by stimulating the endothelial barrier function, is anti-angiogenic (angiostatic) remains unknown. We examined the role of Asc on angiogenesis using two assay systems. At first, the potential role of Asc on four steps of angiogenesis was investigated in cultured bovine microvascular endothelial cells. Asc inhibited the formation of vessel-like tubular structures of endothelial cells cultured on Matrigel; however, it did not decrease the activity of plasminogen activator (PA), which creates the space into which vascular vessels extend. Furthermore, even at high concentrations, Asc did not inhibit either the proliferation or migration of endothelial cell cultures. Secondly, whether Asc inhibited in vivo angiogenesis or not was studied on chick chorioallantoic membrane (CAM) during the 4–6 days of embryogenesis when neovascularization is rapid. It also revealed that angiogenesis was dose-dependently inhibited by Asc from 0.5 μmol/CAM with half-maximal inhibition at 2.5 μmol/CAM. Because it was previously reported that the endothelial barrier function decreases permeability via the stimulation of collagen synthesis induced by Asc, we treated CAM with the inhibitor of collagen synthesis, l-azetidine 2-carboxylic acid (AzC). This compound partially attenuated the angiostatic function of Asc on CAM. To understand the involvement of an antioxidant activity in the angiostatic function of Asc, we further examined the effect of glutathione (GSH), which is an endogenous antioxidant, on angiogenesis in CAM and endothelial cells. GSH inhibited CAM angiogenesis, as well as the formation of vessel-like tubular structures of endothelial cell cultures on Matrigel. Both Asc and GSH inhibited hydrogen peroxide (H2O2) induced tubular morphogenesis. These findings suggest that Asc affects angiogenesis through both its antioxidant properties and the stimulation of collagen synthesis. As the angiostatic activity of Asc may be one of the many effects involved in host resistance to the growth or invasiveness of solid cancer, it may be useful as a supplementary therapy in various angiogenic diseases.

In vitro and in vivo antitumor activity of the interferon inducer bropirimine

In vivo and in vitro antitumor effects of an interferon (IFN)-alpha inducer, bropirimine (2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone), were examined in the murine tumor system. The antitumor effects were studied in Meth A cells, which were the most sensitive to bropirimine in the murine cell lines tested. The direct inhibitory activity of the drug was not reduced when Meth A cells were incubated with bropirimine and anti-IFNs, indicating that the inhibition is not due to autocrine IFN induction from tumor cells. The drug partially inhibited the uptake of [3H]thymidine, [3H]uridine and [3H]leucine. Cell cycle analysis with flow cytometry showed that the drug decreased G0/G1 phase and increased G2/M phase Meth A cells. The drug administered i.p. exhibited a remarkable antitumor effect against Meth A cells which were implanted i.p. These results suggested that the drug induced the in vivo antitumor effect by its direct antitumor activity.

The Lyt phenotype of the T cells responsible for in vivo tumor rejection in syngeneic mice

SummarySpleen cells of BALB/c mice hyperimmunized with a transplantable methylcholanthrene-induced sarcoma Meth A (Meth A-Im-SPL) inhibited the growth of Meth A tumor in vivo in a tumor neutralizing test. Meth A-Im-SPL did not neutralize another antienically distinct sarcoma, Meth 1, indicating that the antitumor activity is tumor specific. Lyt-1+2− cells of Meth A-Im-SPL (Im-Lyt-1+2−) were the effectors since in vitro treatment of Meth A-Im-SPL with anti-Thy 1.2 or anti-Lyt 1.2 antibody plus complement completely abrogated their neutralizing activity, whereas treatment with anti-Lyt 2.2 plus complement did not. To further confirm the effector activity of Im-Lyt-1+2− cells, T cell subpopulations were separated from Meth A-Im-SPL by the panning method. The purified Im-Lyt-1+2−, but not Im-Lyt-1+2+ cells neutralized the tumor in athymic nu/nu mice as efficiently as in +/+ mice, suggesting that the donor Im-Lyt-1+2− cells but not recipient T cells were primarily responsible for neutralizing the tumor. The present study, however, did not exclude the possible contribution of recipient T cells to the tumor neutralization and this is open to further investigation.

Cyclophosphamide-induced suppressor cells in nude mice.

Lymphocytes regenerated after treatment with a high dose of cyclophosphamide (CY) were characterized in nude mice. Ten days after a single injection of 200 mg/kg CY into nude mice, regenerated spleen cells suppressed in vitro primary and second antibody production against sheep red blood cells. The CY-treated spleen cells exhibited normal natural killer (NK) activity, very low B and T cell content, but increases in cell surface charge [electrophoretic mobility (EPM)] and histamine receptors. The suppressor cells could not be removed by treatment with anti-Thy-1 plus complement (C), or treatment with anti-asialo GM, (aGM,) plus C, which abrogated NK activity. It was concluded that CY-treated spleen cells, which exhibited high EPM and histamine receptors, comprise the natural suppressor cells which are Ig−, Thy-1− and aGM−1.

A reverse effect of in vitro and in vivo concanavalin A-induced spleen cells on anti-sheep red blood cells response detected with a new method based on flow cytometry

A simple flow cytometric method for detecting humoral immunity against sheep red blood cells (SRBC) is described. The SRBC were incubated with the serum from SRBC-immunized mice, monoclonal anti-SRBC, or the supernatant which was obtained from the in vitro primary culture of spleen cells with SRBC. The antibodies which bound to SRBC were estimated by means of an immunofluorescence and a flow cytometry. When the channel number of the peak in the histogram of flow cytometry was measured as an index of fluorescence intensity of SRBC, the number significantly correlated with the concentration of IgM and IgG classes of anti-SRBC. The flow cytometry method and hemagglutination (HA) test, as a classic method, were compared in SRBC-immune sera and monoclonal anti-SRBC antibody. The sensitivity determined with flow cytometry was much higher than that with HA. The minimum detectable concentration of anti-SRBC antibody was found to be 3.4 ng/ml by the flow cytometry. The dose response of SRBC in in vitro primary culture was detected by the flow cytometry, not by HA, and the response increased with the dose of SRBC. Using this method, the effect of in vitro and in vivo concanavalin A (Con A)-induced spleen cells on humoral response against SRBC was examined in an in vitro culture system. Anti-SRBC response (IgM and IgG) was found to be suppressed by in vitro Con A-induced lymphocytes but enhanced by in vivo Con A-induced lymphocytes. Thus, this new approach is found to be a good method for detecting the in vitro primary humoral antibody response, which is known to have a low reactivity.

Analysis of electrophoretic mobility histograms of mouse splenocytes and thymocytes during tumor growth and after combined chemotherapy and immunotherapy.

Changes in electrophoretic mobility histograms of splenocytes and thymocytes were studied in plasmacytoma X5563-bearing mice as an indicator of response to treatment with mitomycin C (MMC) alone or combined with the immunomodulator Krestin (PSK). Tumor growth was inhibited by 80-90% in the MMC-treated and was further inhibited in the MMC and PSK-treated group. Electrophoretic mobility histograms of splenocytes were used to determine the fraction of cells having intermediate mobility between high mobility (T cells) and low mobility (B cells). This fraction of intermediate-mobility cells increased in tumor-bearing mice, but a normal electrophoretic mobility pattern was obtained following successful antitumor treatment. In the electrophoretic mobility histogram of thymocytes, on the other hand, the low-mobility cells (cortical thymocytes) decreased in number during tumor growth and were further reduced in the MMC-treated group. This reduction was less in the MMC and PSK-treated group. These results suggest that combined therapy with MMC and PSK prevents damage of the host defence mechanism and allows more efficient antitumor treatment. Analysis of electrophoretic mobility histograms of splenocytes and thymocytes using a fully automated cell electrophoretic instrument makes possible the rapid evaluation of the immunological effects of drug therapy of tumor-bearing mice.

Immunological characterization and clinical significance of low mobility cells appearing in the peripheral blood mononuclear cells of cancer patients

The characteristics of low mobility cells (LMC) in the peripheral blood mononuclear cells (PBMC) of cancer patients were studied using a Parmoquant-L, a fully automated electrophoresis instrument, and a fluorescence-activated cell sorter (FACS). The L/H ratio (%) of LMC (less than 0.95 micron/s/V/cm) to high mobility cells (HMC) (greater than or equal to 0.95) was used to analyze the electrophoretic mobility (EPM) histogram of PBMC. In the cancer patient, the L/H ratio increased as a result of the appearance of LMC. As the ratio closely correlated with clinical stage of cancer, recurrence of cancer, and performance status in the patients, the method is a useful parameter for prognosis of cancer, practically, for early detection of recurrence in the follow-up study of cancer patients. The EPM of monocytes and subsets of lymphocytes was determined to characterize LMC. Monocytes were in LMC. T-Cells such as helper/inducer (Leu-3+), suppressor (Leu-2+Leu-15+) and cytotoxic (Leu-2+Leu-15-) cells and natural killer (NK) cells (Leu-7+ or Leu-11+) were in HMC. The percentage of monocytes correlated significantly with the mean EPM of PBMC. In cancer patients, the percentage of monocytes increased significantly. On the other hand, suppressor T-cells did not increase enough to account for LMC. The appearance of suppressor T-cells correlated with that of monocytes. These results suggest that LMC in cancer patients consisted of mainly monocytes rather than suppressor T-cells.

Lymphocyte electrophoresis as an indicator of modulation of concanavalin A-induced suppressor T-cells in vivo by anti-cancer drugs

We have previously reported that concanavalin A (Con A)-induced suppressor T-cells in vivo are located in the lower electrophoretic mobility (EPM) zone as judged by the ratio of low mobility T-cells to high mobility T-cells (LMT/HMT ratio) in terms of lymphocyte electrophoresis, and that the administration of 5-fluorouracil (5-FU) eliminates their suppressor activity as assayed by Con A-induced lymphocyte blastogenesis in vitro. The current study was undertaken to clarify the relation between changes in EPM and suppressor activity of Con A-induced suppressor T-cells by the administration of antitumor drugs such as cyclophosphamide (CY) and 5-FU when assayed by production of anti-sheep red blood cell antibody in vitro. The pretreatment with 5-FU, but not CY, abrogated the suppressor activity of Con A-induced T-cells followed by restoration of the LMT/HMT ratio of Con A-induced T-cells to that of normal T-cells. These finding suggest that lymphocyte electrophoresis can be applied to an assay for screening immunomodulating agents in Con A-induced suppressor T-cells.

Effects of CD4+ and CD8+ T cells in tumor-bearing mice on antibody production

The function of T cell subsets in tumor-bearing mice was examined using an in vitro culture system of anti-(sheep red blood cell) antibody production, which is known to be dependent on T cells. The helper function of T cells of fibrosarcoma-MethA-bearing mice in antibody production decreased with the tumor stage of the mice. T cells were separated into CD4+ and CD8+ cells for further analysis of T cell subsets by the panning method using monoclonal antibodies. The helper function of CD4+ T cells in antibody production began to decrease significantly in tumor-bearing mice 1 week after the tumor transplantation. On the other hand, the suppressive function of CD8+ T cells was retained and had not decreased in the mice even 3 weeks after the transplantation. The same changes in function of CD4+ and CD8+ T cells were also observed in Methl-bearing mice. These results suggested that this tumor-associated immunosuppression in antibody production is attributable to the decrease in helper activity of CD4+ T cells and the maintenance of the suppressive activity of CD8+ T cells.