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Biomedical subjects

D Mizuno

Publications and source records attributed to D Mizuno.

At least 55 records · Page 3Linked to original sources
PubMed1989

Immunohistochemical evidence for the production of tumor necrosis factor by murine MH134 tumor cells as well as monocytes in tumor lesions after systemic administration of a polyalcoholized mannoglucan from Microellobosporia grisea.

The endogenous production of tumor necrosis factor (TNF) by tumor tissues was examined. MH134 tumor cells as well as monocytes in MH134 tumor tissues produced TNF after systemic administration of a polyalcoholized mannoglucan (MGA) from Microellobrosporia grisea as shown by the indirect immunofluorescence technique. MH134 tumor cells also produced TNF when stimulated with lipopolysaccharide in vitro. These results suggest that regression of MH134 hepatoma can be ascribed to TNF produced not only by monocytes but also by tumor cells themselves in the tumor tissue.

Animals↗
PubMed1989

Secretion of tumor necrosis factor during fetal and neonatal development of the mouse: ontogenic inflammation.

Inflammation plays an important role in homeostasis of the body. We therefore can assume that an inflammatory state occurs during ontogenesis of animals. To address this problem, we examined the ability of tumor necrosis factor (TNF), one of the inflammatory mediators, to be secreted by mouse cells during development. We cultured cells prepared from various parts of fetuses (10-19 days of gestation) and postnatal brains by collagenase digestion and assayed the secreted TNF activity by the L-929 cytotoxicity test. We found TNF activity by fetal cells without any stimulation. The spontaneous secretion of TNF was relatively high at around 13-15 days of gestation. The secretion was enhanced by lipopolysaccharide (LPS), showing that fetal cells are in an activated state for TNF secretion. These TNF activities were neutralized completely by rabbit anti-murine TNF antibody. Spontaneous and LPS-enhanced secretion by postnatal brain cells reached a peak around 7 days after birth, and thereafter declined rapidly. This time course was well correlated to the increase in the weight of brain. The producing cells were negative in macrophage marker surface antigen, and heterogeneous in relation to adherence and phagocytic activity, showing that TNF is secreted by various types of cells in the fetal body. These results suggest the presence of an inflammation-like state during ontogenesis. We consider that this "ontogenic inflammation" may be the prototype of inflammation, which can regulate homeostasis of the adult body.

Animals↗
PubMed1989

Production in Escherichia coli of human thymosin beta 4 as chimeric protein with human tumor necrosis factor.

We have produced thymosin beta 4 protein in Escherichia coli as a chimeric protein with tumor necrosis factor (TNF). The protein was abundantly expressed, was immunoreactive against both anti-thymosin beta 4 and anti-TNF antibodies, and retained cytotoxicity in a TNF assay using mouse L929 fibroblasts. This latter characteristic enabled the easy and simple purification of thymosin beta 4 merely by following the TNF activity. The chimeric protein was designed to have an Asp-Pro bridge between thymosin beta 4 and TNF which could be specifically cleaved under suitable acidic conditions to release the thymosin beta 4 from the chimeric protein. These results indicate that the expression system in E.coli of a chimeric protein composed of thymosin beta 4 and TNF is appropriate for obtaining an abundant amount of the beta 4 peptide, especially since its purification from tissues is usually difficult because of limited yield and obscurity of its biological activity.

Amino Acid Sequence↗
PubMed1989

Exogenous and endogenous tumour necrosis factor therapy.

Although tumour necrosis factor (TNF) is known as a tumoricidal cytokine, it is also important in maintaining homoeostasis in living organisms by inducing an inflammation-like state. We consider that this monokine may also regulate ontogenesis in animals, because we found that a considerable amount of TNF is secreted by mouse embryonal cells at various stages of development. We named the inflammation-like state in which the TNF concentration was high 'ontogenic inflammation'. Induction of endogenous TNF in the adult body as in ontogenic inflammation should restore homoeostasis in patients suffering from chronic diseases including tumours. We have established several methods for inducing sufficient endogenous TNF to cause tumour regression in patients. For more efficient tumour therapy by ontogenic inflammation, the localization of TNF around tumour lesions was found to be critical. By using a newly constructed rTNF-S, which, like endogenous TNF, has a broader cytotoxic spectrum and fewer side effects than conventional rTNF, we developed a new therapy--exogenous/endogenous TNF therapy (EET therapy). With this therapy, exogenous and endogenous TNF can be targeted to and localized in tumours where they act synergistically. Our results suggest that ontogenic inflammation may be important, even in adults, for maintaining homoeostasis. The appropriate application of ontogenic inflammation should also be effective in treating other diseases, such as rheumatism.

Animals↗
PubMed1989

Antitumor effect of systemic administration of novel recombinant tumor necrosis factor (rTNF-S) with less toxicity than conventional rTNF-alpha in vivo.

This study reported that acute toxicity (expressed as the LD50 value) of rTNF-SAM1 and rTNF-SAM2 which we constructed was considerably lower than that of rTNF-alpha (1). Following this finding, the antitumor effects of systemic administration of these novel recombinant tumor necrosis factors (TNF-S) (named rTNF-SAM1 and rTNF-SAM2) were examined in vivo on murine tumors (Meth A fibrosarcoma, MH134 hepatoma, and B16 melanoma). Both rTNF-SAM1 and rTNF-SAM2 could be administered systemically to tumor-bearing mice at doses up to 3 X 10(4) to 10(5) U. Growth of all tumors was significantly inhibited by systemic administration of rTNF-SAM1 or rTNF-SAM2 at a dose of greater than 3 X 10(4) U; at this dosage conventional rTNF-alpha could not be administered systemically to any of the mice strains due to its toxicity. These results confirm our previous finding that the rTNF-SAM group can be administered with safety at higher doses than rTNF-alpha, and revealed that these novel rTNF-S could be more promising antitumor drugs than rTNF-alpha in view of their lower toxicity compared with antitumor effect.

Animals↗
PubMed1988

A possible role of glucocorticoids: an intrinsic inhibitor of the cytotoxic activity of tumor necrosis factor.

The cytotoxic activity of tumor necrosis factor (TNF) against L929 fibroblasts in vivo was noncompetitively inhibited by physiological concentrations of glucocorticoids such as hydrocortisone (10(-7) M), corticosterone (5 X 10(-8) M) and dexamethasone (5 X 10(-9) M). The inhibition was abolished by the addition of actinomycin D (0.5 microgram/ml) or cycloheximide (4 microM). A phospholipase A2 inhibitor, quinacrine (2 X 10(-6) M), also inhibited the TNF cytotoxicity. These findings suggest that the antitumor cytotoxic reaction by TNF is regulated by glucocorticoid through some mechanism involving de novo transcription and translation and that this regulatory mechanism may involve inhibition of phospholipase A2 activity.

Animals↗
PubMed1988

Endogenous tumor necrosis factor induction with Bordetella pertussis vaccine as a triggering agent and its therapeutic effect on MM46 carcinoma-bearing mice.

Induction of endogenous tumor necrosis factor (TNF) by administration of Bordetella pertussis vaccine (BPV) as a triggering agent and its therapeutic effect against MM46 carcinoma were investigated in C3H/He mice. Test triggering agents were injected intravenously into mice after intravenous injection of 4-fold dilution of macrophage activating factor (MAF) or 10(4) units of murine interferon-gamma (Mu-IFN-gamma). Then sera were obtained from the mice, and their TNF activities were assayed on L-929 cells by the method of Ruff and Gifford. The triggering activity of BPV was the highest among those of conventional triggers, such as lipopolysaccharide (LPS) of Escherichia coli, and OK-432. The levels of serum TNF activity triggered by BPV (4 X 10(9) cells), LPS of E. coli (3 micrograms) and OK-432 (3 KE) were 5350, 85 and 102 units/ml, respectively. Growth of MM46, a spontaneous mammary carcinoma cell line of C3H/He was observed for 35 days after tumor inoculation and was suppressed significantly by intravenous injection of MAF and BPV (4 X 10(9) cells). On local injection of BPV (2 X 10(9) cells) into murine tumors, complete regression was observed in 67% of the mice tested with or without MAF priming on day 25 after tumor inoculation, and intratumoral TNF activity was observed even in the case of the single injection of BPV.

Animals↗
PubMed1988

Biological activities of novel recombinant tumor necrosis factor having N-terminal amino acid sequences derived from cytotoxic factors produced by THP-1 cells.

Eight species of novel recombinant tumor necrosis factor-S (rTNF-SAM group) were constructed in which N-terminal amino acid sequences were based on that of TNF-S from THP-1 cells with higher basicity than conventional rTNF-alpha. Two of this rTNF-SAM group, denoted as rTNF-SAM1 and rTNF-SAM2, showed more cytocidal activity on A549 lung carcinoma cells and G401 Wilm's tumor cells than did rTNF-alpha. In addition to these cell lines, rTNF-SAM1 revealed strong cytocidal activity on T24 bladder carcinoma cells, which are resistant to rTNF-alpha. Moreover, possible cachectin activity of rTNF-SAM2 seemed to be lower than that of conventional rTNF-alpha, suggesting that rTNF-SAM2 has less side effects. Actually, toxicity as expressed by LD50 value of rTNF-SAM2 as well as others of the rTNF-SAM group was significantly lower than that of conventional rTNF-alpha. Thus, newly constructed rTNF-SAM1 and rTNF-SAM2 should be more promising antitumor reagents for clinical use, since they were shown to be superior to conventional rTNF-alpha both in antitumor effect and in less side effects.

Amino Acid Sequence↗
PubMed1988

TNF induces endogenous TNF in vivo: the basis of EET therapy as a combination of rTNF together with endogenous TNF.

Enough amounts of tumor necrosis factor (TNF) in mice serum for the therapy were observed by treatment with 100 units of recombinant human TNF-alpha (rHuTNF-alpha) followed by administration of OK-432 (a streptococcal preparation). Optimal time interval between rTNF and OK-432 to produce endogenous TNF was 3 h, and priming activity of rTNF persisted for at least 10 h. The same effect was observed using novel human recombinant TNF-SAM2 (rHuTNF-SAM2) developed by our group. Production of endogenous TNF using rTNF-alpha or rTNF-SAM2 as a priming reagent was almost equal among various mice strains. Induced TNF in mice serum was completely neutralized by anti-MuTNF antiserum, but not by anti-HuTNF monoclonal antibody. rMuTNF could also induce the priming state; however, the dose-response kinetics of the priming effect to produce endogenous TNF was different between rHuTNFs and rMuTNF-alpha, suggesting species specificity among rTNFs used. The therapeutic effect against Meth A and MH134 tumors in mice treated by rHuTNFs in combination with OK-432 was superior to that by single administration of either OK-432 or rHuTNFs or by successive administrations of OK-432. Especially, the antitumor effect against MH134 hepatoma was superior to that of any other treatment using known biological response modifiers so far experienced. These results suggest that such combination antitumor therapy as rTNF together with OK-432 should be applicable to cancer patients.

Animals↗
PubMed1987

Improvement of cytotoxicity of tumor necrosis factor (TNF) by increase in basicity of its N-terminal region.

Two new recombinant TNFs (named rTNF-Scw1 and -Scw2) with higher basicity than conventional recombinant human TNF-alpha (rTNF-alpha) in the N-terminal region were constructed. Their sequences were constructed based on those of partially purified cytotoxic factors from the culture supernatant of acute monocytic leukemia cells THP-1, which unlike rTNF-alpha are cytotoxic to T24 bladder carcinoma cells in vitro. These new rTNF-Ss showed a broader cytotoxicity to tumor cells than rTNF-alpha. This increase in the basicity of the N-terminal region over that of conventional TNF significantly increased the cytotoxicity on tumor cells in vivo as well as in vitro.

Amino Acid Sequence↗
PubMed1987

Endogenous production of tumor necrosis factor in normal mice and human cancer patients by interferons and other cytokines combined with biological response modifiers of bacterial origin.

The priming effect of endogenous biological response modifiers (BRMs), interferons (IFNs), and interleukin-2 (IL2), and the triggering effect of BRMs of bacterial origin, OK-432 and Corynebacterium parvum, on endogenous production of the tumor necrosis factor (TNF) were investigated in mice. TNF activity in serum was measured by in vitro cytotoxicity assay with L-929 cells as a target. The i.v. injection of OK-432 (3KE per mouse) triggered TNF maximally (mean value: 30 U/ml) after 2 h, with a similar time course to that of triggering by lipopolysaccharide. The priming activities of IFNs and IL2 were examined in the system of TNF-triggering by OK-432. The i.v. injection of recombinant IFN-gamma (rIFN-gamma, 10(4) U per mouse) increased TNF production to 790 U/ml; this priming effect was observed just after its injection, was maximal after 2 to 6 h, and disappeared after 24 h. Other types of interferon, rIFN-alpha A/D(Bgl) (2 X 10(5) U per mouse), rIFN-beta (10(6) U per mouse), and natural IFN-alpha/beta (10(6) U per mouse) showed maximal priming activity 6 h after their injection (200 to 800 U/ml) but no effect just after their injection. Recombinant IL2 (10(6) U per mouse) had priming activity that showed a similar time course to that of interferons other than IFN-gamma (a maximal TNF production: 100 U/ml). The i.v. injection of C. parvum, like OK-432, triggered TNF production at doses of 0.06 and 0.3 mg per mouse 2 h after its injection and the triggered TNF activity was enhanced by rIFN-gamma. These findings suggest that combinations of the above endogenous BRMs as priming agents and OK-432 or C. parvum as a triggering agent could induce endogenous production of TNF even in human cancer patients. In fact, combined administration of rIFN-gamma and OK-432 produced TNF in human cancer patients. The advantage of this method for treatment of human cancer patients is discussed.

Animals↗
PubMed1987

Endogenous production of cytotoxic factor in mice induced by a combination of interferon-gamma and heterologous fibrinogen.

The ability of heterologous fibrinogen in combination with interferon (IFN)-gamma to induce endogenous production of cytotoxic factor was examined. Heterologous but not homologous fibrinogen induced high production of cytotoxic factor in IFN-gamma-primed mice. The cytotoxic activity was maximal 1 h after this triggering. The LD50 value of heterologous fibrinogen in mice was greater than 250 mg/kg i.v. But heterologous fibrinogen induced antibody, causing anaphylaxis. Therefore, the effect of successive injections of fibrinogens from a different species was tested. Cytotoxic factor could be produced repeatedly by successive treatments with a combination of IFN-gamma and heterologous fibrinogen from one species for 1 week, although the cytotoxic activity induced by successive injections gradually decreased. After the decrease of the triggering effect of heterologous fibrinogen of one species, heterologous fibrinogen from a different species could induce cytotoxic activity at the same level as that after the first triggering. Thus, a combination of IFN-gamma and heterologous fibrinogen is effective for cytotoxic factor production, provided different heterologous fibrinogens are used successively. This combination should be useful for endogenous cytotoxic factor production in clinical trials.

Animals↗
PubMed1987

Endogenous production of TNF-like cytotoxic factor in BCG-primed mice by heterologous fibrinogen.

The triggering activities of heterologous fibrinogen and fibrin on endogenous production of tumor necrosis factor (TNF)-like cytotoxic factor in vivo were examined. The triggering activities of fibrinogen or fibrin from four species injected into the peritoneal cavity of C3H/He mice infected i.p. with bacillus Calmette-Guérin (BCG) were tested. Heterologous, but not homologous, fibrinogen and fibrin showed triggering activity. The route of triggering by heterologous fibrinogen to elicit TNF-like activity systemically was studied. Injection of heterologous fibrinogen i.v. into mice infected i.v. with BCG resulted in a 40-fold higher serum TNF-like activity level than after its i.p. injection. The serum TNF-like activity level was maximal 1 h after i.v. injection of heterologous fibrinogen. When heterologous fibrinogen was injected several times i.v. into mice bearing solid-tumors, TNF-like activity was also released into the serum after every injection, although the activity decreased progressively on second and third injections to 10 and 1%, respectively, of that after the first injection. We used heterologous fibrinogens derived from different species for triggering every week to avoid this gradual decrease of TNF-like activity. In this way TNF-like activity was induced as highly as the primary induction. These results showed that TNF-like cytotoxic factor could be produced in vivo locally or systemically by heterologous fibrinogen or fibrin. Thus both agents should be useful as nontoxic triggering agents.

Animals↗
PubMed1987

[Partial response of lung metastases from a renal cancer treated with endogenous tumor necrosis factor].

The strong tumor-selective cytocidal action of tumor necrosis factor (TNF) has been observed in vitro and in vivo. Sato et al. have reported that it was possible to induce a primed state of TNF triggering by injection of purified protein derivative (PPD) even a long time after BCG sensitization, suggesting that this treatment could be applied to human patients. In the present study, we achieved a partial response of a metastatic lesion in a patient with renal cancer by the induction of endogenous TNF by PPD and OK-432 (a streptococcal preparation). This study suggested the possible application of this therapy to also patients with malignant tumor which are highly resistant to any conventional antitumor therapy.

Biological Products↗
PubMed1987

Role of in vivo scavenger function of macrophages in priming for endogenous production of tumor necrosis factor.

The effects of systemic administrations of immune complex, complement activators, and insoluble particles on endogenous production of tumor necrosis factor (TNF) were investigated in mice. Production of serum TNF was triggered by i.v. injection of OK-432, a streptococcal preparation, and measured by in vitro L-929 cytotoxicity assay. Intravenous injection of IgG-opsonized sheep red blood cells (10(8)/mouse) enhanced OK-432-triggered TNF production significantly. This effect was maximal (about 30-fold enhancement) 1.5 to 3 h after the injection and disappeared within 10 h. Complement activators other than immune complex also possessed this activity. Zymosan (0.1 mg/mouse) enhanced OK-432-triggered TNF production maximally (about 25-fold) 3 to 6 h after its i.v. injection, its effect lasting for 10 h, and disappearing within 24 h. Heat-aggregated IgG and cobra venom factor also had similar enhancing effects. In addition, systemic pretreatment with insoluble particles enhanced OK-432-triggered TNF production. The enhancement by latex beads (2 microliters volume of solid/mouse) was maximal (about 60-fold) 3 to 6 h after their i.v. injection, was sustained for at least 20 h, and disappeared within 48 h. Glass beads, dextran beads, alum, silica, and carbon particles all had similar enhancing effects. Based on these results, the in vivo scavenger function of macrophages, as well as direct activation with cytokines, may participate in priming for endogenous production of TNF; alternatively, particles or macromolecules which can be scavenged by macrophages may activate macrophages and prime for TNF production.

Animals↗
PubMed1986

Priming effect of interferons and interleukin 2 on endogenous production of tumor necrosis factor in mice.

The effects of interferons (IFNs) and interleukin 2 (IL 2) on endogenous production of tumor necrosis factor (TNF) were investigated in mice. Production of serum TNF was triggered by iv injection of OK-432 and tested by in vitro cytotoxicity assay. Injection of recombinant IFN-gamma with OK-432 and tested by in vitro cytotoxicity assay. Injection of recombinant IFN-gamma with OK-432 or of IFN-alpha/beta, recombinant IFN-beta, recombinant IFN-alpha A/D or recombinant IL 2 six hours before OK-432 enhanced TNF production about 10-fold, which indicated priming actions of these compounds in TNF production. These findings suggest that these compounds could also be used as priming agents for endogenous production of TNF in cancer patients.

Animals↗
PubMed1986

Calcium-dependent and -independent tumoricidal activities of polymorphonuclear leukocytes induced by a linear beta-1,3-D-glucan and phorbol myristate acetate in mice.

Some antitumor immunomodulators, such as a linear beta-1,3-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140 (TAK), induce potent tumoricidal activity of polymorphonuclear leukocytes (PMNs). In the present study we investigated the role of calcium on the tumoricidal activity of PMNs induced by immunomodulators, especially TAK. The calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) almost completely inhibited TAK-induced PMN cytotoxicity and this inhibition was restored by Ca2+ but not by Mg2+. In Ca2+- and Mg2+-free medium, PMN cytotoxicity induced by TAK was recovered by the addition of Ca2+ provided that Mg2+ was also present. By scopoletin assay, hydrogen peroxide released from PMNs by TAK was also observed in the presence of Ca2+ but not in its absence. The PMN cytotoxicities induced by the other immunomodulators, Propionibacterium acnes, Bacillus Calmette-Guérin, zymosan A, and Nocardia cell wall skeletons were also Ca2+ dependent, judging from studies with EGTA and measurement of hydrogen peroxide release in the presence and absence of Ca2+. The Ca2+ dependency of these PMN cytotoxicities suggests that Ca2+ influx is involved in the cytolytic process, but PMN cytotoxicity was not induced by simple addition of the calcium ionophore A23187. Like TAK, phorbol myristate acetate induced PMN cytotoxicity but this cytotoxicity was not Ca2+ dependent. The present report demonstrates the difference in Ca2+ dependency of these PMN cytotoxicities; i.e., extracellular calcium was required for immunomodulator-induced PMN cytotoxicity, but not for phorbol myristate acetate-induced PMN cytotoxicity. This suggests that the processes of induction of PMN cytotoxicity by the two types of activators are not identical.

Adjuvants, Immunologic↗