INDUCTION OF APOPTOSIS IN B16 MELANOMA CELLS UNDER THE ACTION OF DRUG CONTAINING TUMOR NECROSIS FACTOR ALPHA AS A PART OF VIRUS-LIKE PARTICLES <em>IN VITRO</em>
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Aim : to evaluate the antitumor activity of the drug containing TNF-alpha and high-polymer double-stranded RNA (dsRNA) in the composition of virus-like particles (VLP-TNF-alpha) on B16-F10 melanoma cells. Material and Methods . Analysis of the anti-proliferative effect of VLP-TNF-alpha as well as its components, TNFalpha and dsRNA, was carried out using the MTT -test. Apoptosis of melanoma cells was assessed by flow cytofluorimetry with FITC-annexin V. Results . It was shown that the cytotoxic effect of the drug containing the combination of TNF-alpha and dsRNA on melanoma cells significantly exceeded the total cytotoxic effect of TNF-alpha or dsRNA alone (LD50 for combination drug was 0.05 μg/ml, TNF-alpha – 9.5 μg/ml, dsRNA>20 μg/ml). Conclusion . The drug containing TNF-alpha and dsRNA molecules may be a promising drug for the treatment of malignant tumors, including melanoma.Abstract KCl extracts of Melanoma 14, a human melanoma cell line grown in chemically defined serum‐free medium, inhibited leukocyte migration in 19/36 (53%) patients with malignant melanoma. Only 4/23 (17%) controls with non‐melanoma malignancies and 4/28 (14%) normal subjects with no history of cancer were similarly inhibited. Only 2/27 melanoma patients tested against KCl extracts of normal muscle tissue excised from the donor of Melanoma 14 were significantly inhibited. Patients with Stage I (localized) melanoma and patients with Stage III (generalized) melanoma reacted with roughly equal frequency but the number of patients in each group was too small for meaningful statistical analysis. Leukocytes from the donor of Melanoma 14 were tested in a completely autologous system against extracts of Melanoma 14 tissue culture cells and extracts of autologous muscle and were specifically inhibited by the Melanoma 14 tissue culture extract (Migration Index = 0.67) but not by the extract of normal muscle (Migration Index = 0.96). Only 7/32 (22%) melanoma patients were significantly inhibited by an extract of non‐melanoma tumor. These results suggest that melanoma‐associated antigens are present in soluble extracts of this tumor line. Such extracts could provide a continuing source of standard melanoma‐associated antigens for purification and chemical characterization and for diagnostic and prognostic tests in patients with malignant melanoma.
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In a mixed leukocyte culture (MLC) reaction of allogenic mouse spleen cells differing for H-2K or H-2D, only a weak cytotoxic response is generated. This cytotoxic response is augmented significantly if bacterial lipopolysaccharide (LPS), 5 microgram/ml, or polyadenylic acid (poly A):polyuridylic acid (poly U), 20 microgram/ml, is present in the culture. The cytotoxic cells generated in the presence of these two agents are specific for sensitizing H-2K or H-2D antigen. Two lines of evidence suggest that these two agents exert their effect at different steps in the development of cytotoxic lymphocytes: (a) the effect of poly A:U depends on the presence of adherent cells, whereas the effect of LPS is independent of the presence of adherent cells and (b) LPS promotes the development of cytotoxic cells when ultraviolet light-treated stimulating cells are used in the MLC whereas poly A:U does not.
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Abstract The main conclusion from these experiments is that the antigen‐specific suppressor T cell of mice which inhibits the induction of cytotoxic T lymphocytes is not itself a cytotoxic T cell. This conclusion is supported by two main observations: first, a certain cell number from first‐step cultures which was suppressive in the presence of a high dose of antigen actually helped the cytotoxic response at a lower antigen dose. This observation is difficult to reconcile with the hypothesis that suppression is due to the killing of the stimulator or the responder cells in the second‐step culture by cytotoxic T cells. Second, cells from first‐step cultures of cortisone‐treated mice displayed cytotoxic activity but had no suppressive effect on the generation of killer cells. It was further demonstrated that these cells failed to influence in any way the suppressive effect, however weak, of cells from first‐step cultures of normal spleen. We therefore favor the view that the suppression observed in this system is due to a regulatory signal which occurs as a result of the ability of both inhibitory cells and responder cells to recognize and respond to allogeneic determinants expressed on the surface of stimulator cells. The suppressor T cells described here act by linked associative recognition of antigen. That is, suppressor T cells only inhibit the induction of a precursor cytotoxic T cell in the presence of an antigen to which both the precursor cell and the suppressor cell can bind. In this sense, suppressors act in a manner analogous to helper T cells in T‐B cell cooperation; carrier‐specific helper T cells only enhance an anti‐hapten B cell response in the presence of hapten‐carrier conjugates. Similarly, alloantigen a (carrier)‐specific suppressor T cells only inhibit alloantigen b (hapten)‐specific cytotoxic responses in the presence of (a × b)F 1 stimulator cells (hapten‐carrier conjugate), not in the presence of a mixture of parental stimulator cells (a + b).
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Sir: A number of investigators have recently reported that cell membranes, or solubilized proteins derived from them, are capable of inducing cytotoxic responses in secondary cultures (1–6). They have implied thereby that they are gaining insight into the specificity of the structures recognized by cytotoxic T cells. An alternative interpretation seems equally plausible: that the membrane preparations act not directly on cytotoxic memory cells, but rather on helper T cells, which, by secreting soluble mediators, “trigger” cytotoxic cell differentiation. This interpretation is supported by several recent reports. Thus, Ryser et al. (7), Wagner and Rollinghoff (8), and this laboratory (Okada et al. , in press) have shown that cell mediators secreted by primed Lyt I+ T cells can cause the direct differentiation of cytotoxic T cells in secondary (memory) cultures. Moreover, the specificity of the cytotoxic cells induced by these mediators is that of the antigen initially used for priming.
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Objective To study the effect of As2S2 on apoptosis and necrosis of human hepatoma cell line in vitro.Methods BEL-7402 cell line was treated with As2S2 at different concentration.The proliferation inhibition was detected by MTT assay.Cytotoxity was observed by cell counting.Necrosis was estimated by Trypan blue staining.The cell apoptosis and necrosis were detected by cytometer with Annexin-V/PI.The expressions of Apo2.7 and Fas protein were also detected by cytometer.Results The results showed that high dose of As2S2 had cytotoxity on BEL-7402.In addition,cell apoptosis was detected by cytometer,and the expressions of Fas and Apo2.7 were up-regulated.Conclusion As2S2 can induce apoptosis and necrosis of hepatoma cells.
Trypan blue
Annexin A5
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Abstract Sir: A number of investigators have recently reported that cell membranes, or solubilized proteins derived from them, are capable of inducing cytotoxic responses in secondary cultures (1–6). They have implied thereby that they are gaining insight into the specificity of the structures recognized by cytotoxic T cells. An alternative interpretation seems equally plausible: that the membrane preparations act not directly on cytotoxic memory cells, but rather on helper T cells, which, by secreting soluble mediators, “trigger” cytotoxic cell differentiation. This interpretation is supported by several recent reports. Thus, Ryser et al. (7), Wagner and Rollinghoff (8), and this laboratory (Okada et al., in press) have shown that cell mediators secreted by primed Lyt I+ T cells can cause the direct differentiation of cytotoxic T cells in secondary (memory) cultures. Moreover, the specificity of the cytotoxic cells induced by these mediators is that of the antigen initially used for priming.
Priming (agriculture)
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Objective: To study the effect of tumor necrosis factor-α (TNF-α) on acute liver necrosis induced in mice. Methods: Male Balb/c mice received D-Galactosamine and lipopolysaccharide to induce acute liver necrosis. Serum levels of alanine transaminase (ALT) and TNFα were determined. The liver tissues were fixed for histopathologic analysis. The difference with pretreatment by using anti-TNF-α antibody was studied. Results: The mortality rate of mice with acute liver necrosis reached 60% 9 hours after injection. The serum levels of ALT began to increase from the 6th hour, and reached maximum at the 9th hour. The concentration of TNF-α reached a maximal value at the 2nd hour.The levels of TNF-α at every time point were significantly increased than that of control group (P0.01). The liver were induced with massive or submassive necrosis. But there were no death in mice with anti-TNF-antibody pretreatment. Serum levels of ALT were significantly reduced (P0.01). The histologic analysis showed only spotty necrosis or focal necrosis. Conclusion: The TNF-α is a key cytokine that induces liver necrosis. The treatment with anti-TNF-antibody prevents the liver necrosis.
Aspartate transaminase
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Natural Production and Release of Tumour Necrosis Factor (G. Gifford, D. Flick). Possible Relationships between in vivo Antitumour Activity and Toxicity of Tumour Necrosis Factor-a (M. Palladino et al.). Human Tumour Necrosis Factors: Structure and Receptor Interactions (B. Aggarwal et al.). Cytocidal Activity of Tumour Necrosis Factor: Protection by Protease Inhibitors (C. Baglioni et al.). Lymphotoxin: Cloning, Regulation, and Mechanism of Killing (N. Ruddle et al.). Physiological Responses to Cachectin (K. Tracey et al.). Structure-function Relationship of Tumour Necrosis Factor and Its Mechanism of Action (W. Fiers et al.). Relationship of Tumour Necrosis Factor and Endotoxin to Macrophage Cytotoxicity, Haemorrhagic Necrosis and Lethal shock (J. Rothstein, H. Schreiber). Antitumour Effects of Necrosis Factor: Cytotoxic or Necrotizing Activity and Its Mechanism (K. Haranaka et al.). Effects of Tumour Necrosis Factor on Human Tumour Xenografts in Nude Mice (F. Balkwill et al.). Effects of Tumour Necrosis Factor and Related Cytokines on Vascular Endothelial Cells (J. Pober). Antiparasitic Effects of Tumour Necrosis Factor in vivo and in vitro (J. Playfair, J. Taverne). Clinical Studies with Tumour Necrosis Factor (D. Spriggs et al.). Summary (L. Old). Index of Contributors. Subject Index.
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