Nobuko Satomi

Purification and partial amino acid sequence of rabbit tumor necrosis factor

Good production of tumor necrosis factor (TNF) in the rabbit was obtained using Propionibacterium acnes IID 912 as a priming agent and subsequent administration of lipopolysaccharide. The physicochemical characteristics of rabbit TNF were very similar to those of murine TNF. The molecular weight of rabbit TNF was 39,000 as estimated by gel filtration, and 18,000 by SDS‐PAGE. The isoelectric point was determined as ph 4.0 by isoelectric focusing. Rabbit TNF was stable within the ph range of 5.5 to 11.0, and was stable at 56°C for 8 hr. It was digested by trypsin, pancreatic protease and elastase, but was resistant to neuraminidase . The amino acid sequence of rabbit TNF was determined as Ser‐Ala‐Ser‐Arg‐Ala‐Leu‐ …. Monoclonal antibody against rabbit TNF completely inhibited both the in vivo and in vitro activity of rabbit TNF. However, this antibody could not inhibit the action of murine TNF. Antitumor activity of rabbit TNF was shown against murine and human cancer cells in vivo and in vitro, and rabbit TNF was also capable of distinguishing malignant cells from normal cells.

Effects of tumor necrosis factor (TNF) on transplanted tumors induced by methylcholanthrene in mice. A histopathologic study.

SummaryThe effects of a highly purified tumor necrosis factor (TNF) on transplanted methylcholanthrene (Meth A)-induced murine tumors were compared with those of lipopolysaccharide (LPS). TNF caused immediate subepidermal edema and hyperemia followed 2 h later by fibrin thrombi in tumor blood vessels. Finally hemorrhagic necrosis with dispersal of tumor cells occurred. LPS produced similar hemorrhagic necrotizing changes. However, the necrotic action of LPS was delayed and complete tumor regression was not achieved with LPS. These findings suggest that tumor necrosis induced by TNF is due to circulatory disturbance associated with a microvascular injury in the tumor manifested by hyperemia and multiple fibrin thrombi.

Role of first stimulating agents in the production of tumor necrosis factor.

SummaryThe conditions and kinetics of tumor necrosis factor (TNF) production were examined. For TNF production, dual stimulation is necessary. Priming agents such as BCG, Corynebacterium parvum, and zymosan, which can stimulate the reticuloendothelial system (RES), are good substances for TNF production with the aid of lipopolysaccharide. Wide differences are observed in TNF producibility among different priming agents. The producibility of TNF depends on the degree of stimulation of the RES by the priming agents. Those priming agents, e.g., Propionibacterium acnes and Corynebacterium anaerobium, that are able to induce substantial RES hyperplasia are also able to induce high levels of TNF activity. Following administration of large doses of BCG or zymosan, mice were found to produce TNF activity. However, PPD, OK 432, PSK, and Choreito were unable to induce TNF activity.

Necrotizing activity of tumor necrosis factor: histopathological investigation using Meth A sarcoma and granulation tissue

SummaryThe action of tumor necrosis factor (TNF) was investigated histopathologically in mice using methylcholanthrene A (Meth A)-induced sarcomas and granulation tissue induced by autotransplantation of fragments of liver and spleen. Highly purified murine TNF caused hemorrhagic necrosis of both the tumors and the granulation tissue. Proliferation of tumor capillaries, demonstrated microangiographically, occurred 2 h after TNF administration and hyperemia of tumor vessels was obvious after 3–6 h. Hyperemia and capillary leakage were also observed in the granulation tissue 6 h after TNF injection and hemorrhage was noted in the epidermis after 12 h. These results strongly suggest that the in vivo necrotizing action of TNF is mainly related to capillary injury.

Tumour necrosis factor and the lysosomal enzymes of macrophages or macrophage-like cell line

SummaryThe relationship between tumour necrosis factor (TNF) and macrophages or macrophage-like cell line, especially the lysosomal enzymes was investigated. The serum lysosomal enzymes and LDH activities were increased in proportion to the TNF production even in different strains of mice. Lysosomal enzymes and TNF activity were released into the supernatant of the culture medium of macrophage-enriched peritoneal exudate cells (PEC) or spleen cells derived from Propionibacterium acnes-primed mice after addition of lipopolysaccharide (LPS). After passage through a Sephadex G-10 column, TNF activity could not be detected in the supernatant of these spleen cells after addition of LPS. Also TNF activity could not be detected in the supernatant following destruction of PEC. These results suggest that TNF producibility is strongly related to the degree of activation of macrophages, especially the lysosomal enzymes. The murine macrophage-like cell line, J 774, also released TNF activity and lysosomal enzymes after addition of LPS.

Traditional Chinese Medicines and Drugs in Relation to the Host-Defense Mechanism

Pseudomonas aeruginosa infection is observed mostly under immunosuppressive conditions. We have clarified that the combined use of antibiotics and specific antibodies is effective against mouse P. aeruginosa infection in vivo [1–5]. From our previous experiments, traditional Chinese medicines and crude drugs are known to cause stimulation of the reticuloendothelial system [6]. Kiger et al. [7] found that P. aeruginosa treatment (108 killed organisms) had a capacity to induce release of tumor-necrotizing factor in the serum of bacille Calmette-Guerin (BCG)-pretreated mice to the same extent as lipopolysaccharide (LPS) treatment. Our experiments on tumor necrosis factor (TNF) have demonstrated the production of TNF as well as its associated severe toxicity [8–10]. Recently, recombinant human TNF has been mass produced and many actions of this factor have become clear. We have found that by employing traditional Chinese medicines as priming agents, the toxic symptoms after administration of LPS could be reduced.

The Combined use of Antibiotics and Specific Antibodies against Mouse Pseudomonas aeruginosa Infection In Vivo and the Phagocytosis of Peritoneal Exudate Cells In Vitro

Pseudomonas aeruginosa infection is one of the most difficult diseases to cure because it has a strong natural resistance to many antibiotics, and there are too many different serotype strains to make active or passive immunotherapy feasible. This infection can also be observed in immunosuppressive conditions such as so-called superinfections.

Preventive effect of several drugs against Pseudomonas aeruginosa infection and the toxicity of combined tumor necrosis factor with lipopolysaccharide: relationship between lethality and the arachidonic cascade.

The participation of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) inPseudomonas aeruginosa (Pa) infection was examined. The lethal challenge of Pa or TNF and LPS injection could be prevented by pretreatment with anti-TNF antibody, polymyxin B, ONO 1078, or Shosaiko-to. The combined effects of TNF and LPS may be deeply related to the lethality of Pa infection. The activities of leukotriene(LT) C4/D4/E4 or platelet activating factor (PAF) were also related to the lethality of Pa infection, probably due to the subsequently produced TNF which acts in combination with LPS. Activating the host defence mechanism with biological response modifiers like Chinese medicines was effective against Pa infection. One mechanism could involve an activity as an LT inhibitor or PAF antagonist. Following the administration of TNF and/or LPS, the serum levels of arachidonic cascade products underwent various changes. With a combination of TNF and LPS, there was a synergistic increment of prostaglandins, thromboxane, and LT. Following pretreatment with Shosaiko-to, suppression of LTs was dominant even with the combination of TNF and LPS, which might be related to the lethality of the infection or combined TNF with LPS.

Establishment and characterization of human myelomonocytic (TYS) and histiocytic (TYH) cell lines.

Two human hematopoietic cell lines (TYS and TYH) with monocytic characteristics were derived from the peripheral blood of a patient with acute myelomonocytic leukemia and of another with a follicular large‐cell type of malignant lymphoma. The TYS cells, derived from the leukemia patient, revealed a monocytic appearance with microvilli at one side and had many granules and vacuoles. They showed strongly positive reactions with α‐NBE, NASDAE, and AcP, and were reactive with monoclonal antibodies such as Oklal, 12 and B1. The TYS cells, which phagocytized carbon particles and antibody‐coated SRBC but not latex particles, released lysosomal enzymes and tu‐ moricidal factor into the supernatant. The TYH cells, derived from the malignant lymphoma patient, had abundant cytoplasm and pseudopods detectable by electron microscopy with a monocytoid appearance and virus‐like particles in the cytoplasm. They showed strongly positive reactions with α‐NBE, NASDAE and β‐Gase, but no reactivity with monoclonal antibodies or with surface markers except Fc‐γ‐R. TYH cells phagocytized latex particles very well. Two different human monocyte‐histiocyte lineages were thus established. During culture, the TYS and TYH cells maintained their characteristics over 28 and 16 months of Passage, respectively.

Purification characterization and anti‐tumor activity of a new cytokine, histiocyte‐secreted‐factor (HSF)

Purification of cytokines was carried out while monitoring their in vivo anti‐tumor activity and in vitro cytotoxic activities. As a result, purified new cytokines were obtained from culture supernatant of a histiocytic cell line and from rabbit serum. Briefly, a new cytokine (HSF, histiocyte‐secreted‐factor) was purified from the culture supernatant of the histiocytic cell line (TYH) which we established from the peripheral blood of a malignant‐lymphoma patient. The purified samples exhibited suppressive effects on tumor growth but no necrotizing activity towards transplanted murine tumors. The substance displayed no cytotoxic activity against L cells (mouse fibroblast cells). The molecular weight of human HSF was about 42 kDa as estimated by SDS‐PAGE. Amino‐acid sequencing of the purified HSF from the culture supernatant was performed, but the N‐terminal was blocked. Next, a new cytokine was purified from rabbit serum stimulated with Propionibacterium acnes and elicited with lipopolysaccharide. The rabbit HSF was isolated by the same procedures as those used for the human HSF purification steps. Amino‐acid sequencing was carried out after enzyme digestion. Three parts of the amino‐acid sequence of the rabbit HSF were determined as LPPGLLAPMRQLRS‐, NLEXFTNGMEQHYAQL‐, and NPAENQAHELPNQLN‐. A computer‐based homology search demonstrated that these sequences were novel. The molecular weight of HSF as determined using anti‐peptide antibodies revealed the following values: human HSF, 41 and 46 kDa; rabbit HSF, 35, 42 and 55 kDa.