Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic effect of this cytokine: relationship with c-myc and Rb.

Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. We have previously shown that the MCF7 resistance to the cytotoxic action of TNF correlates with p53 mutations. In the present study, we used a recombinant adenovirus carrying a wild-type p53 gene (Adwtp53) in order to investigate the effect of wt p53 transfer on modulation of cell resistance to the cytotoxic action of TNF. Our data indicate that infection of TNF resistant MCF7 cells (1001 and MCF7/Adr) with Adwtp53 resulted in the restoration of wt p53 expression and function as respectively revealed by the yeast assay and the induction of p53 inducible genes MDM2 and p21. Furthermore, the restoration of p53 function significantly sensitized TNF resistant cells to TNF cytotoxic action. This correlated with a significant down-regulation of c-myc in both TNF-resistant cell lines and a decrease of Retinoblastoma protein (Rb) in 1001 clone. In contrast, the effect of p53 seems to be independent from Bcl-2 and Bax protein level regulation. The present study suggests that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine.

HIV type 1 glycoprotein 120 amplifies tumor necrosis factor-induced NF-kappa B activation in Jurkat cells.

This article demonstrates that human immunodeficiency virus type 1 (HIV-1) gp120 amplifies the activity of tumor necrosis factor alpha (TNF-alpha), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In CD4-positive Jurkat cells, gp120 potentiates TNF-induced NF-kappa B activation. TNF-mediated activation of NF-kappa B is known to involve the intracellular formation of reactive oxygen intermediates (ROIs). Accordingly, we examined the influence of gp120 on the cellular redox state. We found that gp 120-modulated TNF-induced NK-kappa B activation was inhibited by the antioxidant butylated hydroxyanisole, indicating the involvement of redox-dependent mechanisms. In addition, we showed that gp120 induces intracellular formation of hydrogen peroxide, which is accompanied by a decrease in the ratio of glutathione to glutathione disulfide. In contrast, in the p56lck-deficient J.CaM1.6 T cell line, a derivative of the Jurkat cell line, gp120 was unable to stimulate hydrogen peroxide, to decrease the ratio of GSH to GSSG, and has no effect on TNF-induced NF-kappa B activation. This demonstrated that p56lck protein tyrosine kinase plays an active role in transmitting a signal that increases the oxidative state of the cell and as a consequence amplifies TNF-mediated NF-kappa B DNA binding. We have demonstrated that Tat protein decreased both the Mn-dependent superoxide dismutase (MnSOD) and the cellular glutathione content (GSH). Here we show that, in contrast to Tat, gp120 is unable to inhibit activity and expression of MnSOD and to decrease GSH content. Taken together, our data suggest that gp120 potentiates TNF-induced NF-kappa B activation by stimulating a signal pathway that involves p56lck and the increased formation of reactive oxygen intermediates such as H2O2. These findings may be relevant for the regulation of HIV-1 replication in T cells.

Methyltransferase inhibitor S-adenosyl-L-homocysteine sensitizes human breast carcinoma MCF7 cells and related TNF-resistant derivatives to TNF-mediated cytotoxicity via the ceramide-independent pathway.

In this study we investigated the signalling requirements for TNF-induced cytotoxicity modulated by the methyltransferase inhibitor S-adenosyl-L-homocysteine (AdoHcy) using the TNF-sensitive human breast carcinoma MCF7 cells and its established TNF-resistant clones (R-A1 and clone 1001). Our data indicate that inhibition of methylation reactions by adenosine plus homocysteine, which are known to condense within cells to AdoHcy, markedly potentiated TNF-induced cytotoxicity in MCF7 cells and rendered related TNF-resistant variants, TNF-sensitive by a mechanism independent from the ceramide pathway. We demonstrated that the dominant-negative derivative of FADD (FADD-DN) blocked methylation inhibition/TNF-induced cell death. Moreover, TNF-mediated cytotoxicity modulated by AdoHcy was blocked by the ICE-inhibiting peptide z-VAD-fmk, suggesting that an ICE-like protease is required for the methylation inhibition/TNF-inducible death pathway. In conclusion, these results suggest that the methyltransferase inhibitor AdoHcy potentiates TNF-induced cytotoxicity in MCF7 cells and renders TNF-resistant MCF7 clones, TNF-sensitive via the ceramide independent pathway and that FADD and the ICE-like protease are likely necessary components in transducing methylation inhibition/TNF signals for cell death.

Iron chelation decreases human immunodeficiency virus-1 Tat potentiated tumor necrosis factor-induced NF-kappa B activation in Jurkat cells.

TNF-alpha stimulates HIV-1 replication via activation of the transcription factor NF-kappa B. TNF-mediated activation of NF-kappa B is known to involve the intracellular formation of reactive oxygen intermediates (ROIs). We recently demonstrated that HIV-1 Tat protein potentiates TNF-induced NF-kappa B activation by downregulation of manganese-dependent superoxide dismutase (MnSOD), shifting the cellular redox state towards pro-oxidative conditions. This study shows that treatment of Jurkat cells with iron chelator deferoxamine (DFO) strongly decreases HIV-1 Tat-potentiated TNF-induced NF-kappa B activation but does not modify NF-kappa B activation by TNF-alpha. The ability of iron chelators to reduce Tat-potentiated TNF-induced NF-kappa B binding activity suggests that iron and intracellular hydroxyl radicals (OH.) are required for Tat effect. Moreover, we have shown that exogenously generated OH. markedly enhanced TNF-induced NF-kappa B activation in a dose-dependent manner while was not sufficient to trigger activation of NF-kappa B by itself. In addition, iron chelators had no effect either on MnSOD activity or on the decrease of this activity by Tat. Iron chelators had also no effect on the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), but could elevate the GSH:GSSG ratio decreased by Tat protein. These observations suggest that the formation of intracellular OH. in the presence of iron ions play a major role in HIV-1 Tat enhancement of TNF-induced NF-kappa B activation and that iron chelation may protect Jurkat T cells, at least in part, against oxidative stress induced by Tat.

[Assessment of peripheral blood monocyte function in pulmonary tuberculosis patients]. / Otsenka funktsional'nogo sostoianiia monotsitov perifericheskoi krovi u bol'nykh tuberkulezom legkikh.

As the result of the study of the peripheral blood monocyte function in patients with pulmonary tuberculosis, the ingestive capacity of monocytes has been found to be suppressed, which indicates the pathological state of oxygen-dependent mechanisms governing the bactericidal activity of cells, the most pronounced disturbances of monocyte functions being observed in patients with fibrous-cavernous and disseminated tuberculosis.

[The status of the immunity system in diphtheria]. / Sostoianie sistemy immuniteta pri difterii.

The complex study of cell-mediated and humoral immunity characteristics, as well as nonspecific protective factors, in 30 diphtheria patients, 9 clinically healthy carriers and 54 healthy subjects was carried out. In healthy immunized subjects normal characteristics of all elements of immunity were observed in combination with high titers of antitoxins and sensitization of lymphocytes to diphtheria toxoid. In healthy carriers the presence of cell-mediated and humoral immunity to diphtheria was associated with disturbances of the metabolic activity of phagocytes and a decrease in the proliferation of lymphocytes in response to the mitogen. Diphtheria patients were found to have changes in all elements of immunity, especially pronounced in severe forms of the disease.

[Effect of antimicrobial surface-active agents on the immune response to thymus-dependent antigen in mice]. / Vliianie antimikrobnykh poverkhnostno-aktivnykh veshchestv na immunnyi otvet k timuszavisimomu antigenu u myshei.

The study showed that miramystin, a cationi surfactant, was an immunostimulant inducing an increase in humoral and cellular immunity in response to sheep red blood cells. The observed dose-dependent stimulating effect of miramystin on antibody production and development of DTH recommends its use as an immunologic adjuvant in the laboratory practice. It also indicated to the prospects in investigation of immunologic adjuvant properties of other preparations belonging to surface-active substances.

[Effect of leukocytic interferon on the functional state of bronchoalveolar phagocytes in lung diseases]. / Vliianie leikotsitarnogo interferona na funktsional'noe sostoianie bronkhoal'veoliarnykh fagotsitov pri zabolevaniiakh legkikh.

A study was made of the effect of leukocytic interferon on the phagocytic activity of alveolar macrophages and neutrophils in patients with chronic bronchitis, tuberculosis and sarcoidosis of the lungs. Cells isolated from the patients' bronchoalveolar washing off were used. It was concluded that short-term incubation of macrophages and neutrophils with interferon resulted in phagocytosis enhancement and increased intensity of metabolic processes in them. Interferon produced the most noticeable effect on cells with an initially reduced absorption capacity.

[Immune system in residents of territories contaminated with radionuclides after Chernobyl accident]. / Sistema immuniteta u lits, prozhivaiushchikh na territorii, zagriaznennoi radionuklidami vsledstvie avarii na Chernobyl'skoi AES.

AIM: Study of the immunity and nonspecific defense factors in subjects living at a territory contaminated with radionuclides at a density of 1-5 Ci/km2 after the Chernobyl accident. MATERIALS AND METHODS: A total of 144 subjects aged 18 to 82 years living in the Sasovo region of the Ryazan district were examined. Three groups were distinguished with different density of contamination: 1) n = 54, 1-5 Ci/km2; 2) n = 36, conditionally pure territory; and 3) n = 54, living at the interface of the two territories. Blood analysis was carried out, nonspecific defense factors studied in the NBT test, and cellular and humoral immunity parameters investigated. RESULTS: Values of the NBT test, levels of the natural inhibitory factor and IgA, counts and functional activities of T lymphocytes and their subpopulations differed but negligibly from those in subjects living at pure territories. On the other hand, the counts of large granular lymphocytes were decreased and the incidence of autoimmune reactions to thyroid hormone antigens increased in the population exposed to low-dose ionizing radiation, which might be due to incorporation of radioactive iodine. CONCLUSION: The detected changes in the population exposed to low-dose radiation indicate that the history of exposure cannot be neglected, for such an exposure causes development of some diseases or alters their course.

[Use of plasmapheresis in surgical hematology]. / Ispol'zovanie plazmatsitafereza v khirurgicheskoi gematologii.

Results of 270 plasmacytophereses performed in 64 patients with different forms of hemoblastoses are presented. Indications for using the method are determined. It is shown that plasmacytopheresis included in the complex therapy of hemoblastoses increases the incidence of remissions, shortens the time of treatment, allows to increase single doses and decrease general dose of the cytostatic preparations used.

[The rheologic aspects of washed donor erythrocytes]. / Reologicheskie aspekty otmytykh donorskikh éritrotsitov.

Washing off donors' red blood cell pack with NaCl isotonic solution results in improvement of red blood cell rheologic properties: their aggregation reduces, deformability increases, osmotic resistance rises. Heparin addition to the washing off solution in a dose of 5 IU/ml prevents development of antiheparin activity in red blood cells after washing off. Application of washed-off red blood cells of donors has advantages as compared to transfusion of the whole blood.

[Lymphocytic sensitization to antigens of viruses of the family Paramyxoviridae in diabetes mellitus].

The purpose of this investigation was to study cellular immunity to parotiditis and measles viruses in patients with diabetes mellitus (DM). Lymphocytic sensitization to parotiditis and measles viral antigens and insulin was determined by peripheral blood lymphocytic blast-transformation reaction (a morphological method for reaction assessment) In 10 patients with measles, 18 patients with epidemic parotiditis, 52 patients with DM (23 and 29 patients types 1 and 2 DM, respectively), and 46 apparently healthy individuals. The studies revealed lymphocytic sensitization to parotiditis and measles viral antigens and insulin in most patients in the acute phase of infection with a subsequent reduction in the intensity of proliferation until the point of complete cessation during 12 months. Unlike the controls in whom lymphocytic sensitization to the viruses of the family Paramyxoviridae was detected in 3 persons, prolonged antiviral cellular immunity was found in 17 of the 23 patients with type 1 DM and in 25 of the 29 patients with type 2 DM. Thus, prolonged cellular immunity to viral antigens (measles and/or parotiditis) Is associated with diabetes melhtus. The concordance of a lymphocytic response to viral antigens and insulin suggests that the viruses of the family Paramyxoviridae in some persons initiate a cascade of immune reactions leading to the development of diabetes, the tatter's type is determined by the nature of an immune system response to viral antigens and insulin.

Modulation of tumor necrosis factor-alpha-mediated cytotoxicity by changes of the cellular methylation state: mechanism and in vivo relevance.

A combination of adenosine (Ado) and homocysteine (Homo) enhances tumor necrosis factor (TNF)-alpha cytotoxicity in vitro and in vivo in several tumor cells. Ado and Homo at concentrations that enhanced TNF-alpha-mediated cytotoxicity accumulated S-adenosylhomocysteine (AdoHcy) and as consequence decreased the cellular methylation state, i.e. the ratio of S-adenosylmethionine to AdoHcy. This decrease led to inhibition of the isoprenylcysteine carboxyl methyltransferase (MTase), an enzyme that catalyzes carboxyl methylation of C-terminal cysteine residues on isoprenylated proteins. The effect of Ado and Homo on TNF-alpha cytotoxicity was at least partly mimicked by S-farnesylthioacetic acid, a selective inhibitor of the isoprenylcysteine carboxyl MTase, suggesting involvement of methylations of prenylated proteins in TNF-alpha-mediated cytotoxicity. Blockage of methylation reactions was associated with an enhancement of the TNF-alpha-induced disruption of the mitochondrial membrane potential (delta psi(m)). In nude mice, a combination of Ado, Homo and TNF-alpha led to TNF-alpha-induced hemorrhagic necrosis and growth inhibition of TNF-sensitive L929 tumors, whereas little effect was observed with TNF-alpha alone. Even more important, the TNF-resistant L929 M1 tumors were rendered TNF-sensitive by the combined action of Ado and Homo. We conclude that Ado and Homo together enhance the effectiveness of TNF-alpha in vitro and in vivo, results that may have therapeutic implications.

[The effect of miramistin on the phagocytic activity of urethral neutrophil granulocytes]. / Vliianie miramistina na fagotsitarnuiu aktivnost' uretral'nykh neitrofil'nykh granulotsitov.

Secondary immunodeficiency, one of whose manifestations is depressed phagocytic activity of urethral neutrophilic granulocytes, develops in patients with chronic urethroprostatitis. Miramistin exerts a dose-dependent stimulating effect on the urethral neutrophilic granulocytes; the maximum stimulating effect is observed when phagocytes are treated with 0.001% solution of the drug.

Induction of NO synthesis in macrophages by Newcastle disease virus is associated with activation of nuclear factor-kappa B.

Newcastle disease virus (NDV) has received much attention recently because of its non-specific immune stimulating potential and its various anti-tumor activities. Here we describe that NDV induces synthesis of NO and causes an activation of nuclear factor-kappa B (NF-kappa B) in murine macrophages. These reactions were part of an activation process which included also stimulation of adenosine deaminase and inhibition of 5'-nucleotidase. NDV-mediated NO synthesis and NF-kappa B activation were blocked by an antioxidant (butylated hydroxyanisole), by an inhibitor of protein tyrosine kinase (genistein) and of protein kinase A (H-89), but not by an inhibitor of protein kinase C (staurosporin). These data suggest that signalling requirements of NF-kappa B activation and NO production in NDV-treated macrophages are similar.

Sphingosine-1-phosphate mobilizes intracellular calcium and activates transcription factor NF-kappa B in U937 cells.

Sphingosine-1-phosphate (SPP), a metabolite of sphingolipids, has been implicated as a second messenger in cell growth regulation and signal transduction via calcium mobilization from internal stores. This study shows that SPP mobilizes intracellular calcium in U937 cells and demonstrates for the first time the ability of SPP to activate the transcription factor NF-kappa B in these cells. Furthermore, calcium release from the internal stores by thapsigargin (TG), an inhibitor of the endoplasmic reticulum Ca2+ pump, was associated with activation of NF-kappa B. Moreover, we have shown that while an intracellular calcium chelator BAPTA/AM was able to inhibit both SPP- and TG-induced NF-kappa B activation, it had no effect on TNF-induced NF-kappa B activation. In addition, SPP-induced NF-kappa B activation was blocked both by cyclosporin A, known to inhibit calcineurin phosphatase activity, and by the antioxidant butylated hydroxyanisole. These observations suggest that intracellular calcium mobilization is required for SPP-induced NF-kappa B activation, which may involve calcineurin- and redox-dependent mechanisms.

[Immunostimulating effect of synthetic surface-active agents in experimental tuberculosis]. / Immunostimuliruiushchee deistvie sinteticheskikh poverkhnostno- aktivnykh veshchestv pri eksperimental'nom tuberkuleze.

The influence of synthetic surfactants of different classes (BX-14, O-14, B-14) on an immunologic reactivity of mice with evolving experimental tuberculosis was studied. The use of the substances leads to an increase of the E-rosette forming cells (E-RFC) in the mice thymus gland and spleen, and stimulates the inhibited function of spleen natural killers, without modifying (except the agent O-14) the cytotoxicity level of thymus gland natural killers. The above substances can enhance the absorbing capacity of the spleen macrophages as well as their ability to reduce nitro blue tetrazolium. Attention is drawn to the possibility of using immunostimulators belonging to a group of surfactants to correct immunologic reactivity impairments caused by tuberculosis.

[An immunological analysis of the hemagglutinin from the influenza A virus (H1N1) isolated by using various detergents]. / Immunologicheskii analaiz gemaggliutinina virusa grippa A (H1N1), vydelennogo s pomoshch'iu razlichnykh detergentov.

Immunogenic properties of influenza virus hemagglutinin, isolated by new detergents O-14 (desintegron-O) and B-14 (desintegron-B) have been studied. Hemagglutinin isolated by desintegron-O has been found to be more immunogenic than virions. It has been shown that hemagglutinin isolated by desintegron-B induces a lower humoral immune response than the influenza virus.

Adenosine and homocysteine together enhance TNF-mediated cytotoxicity but do not alter activation of nuclear factor-kappa B in L929 cells.

This paper shows that a combination of adenosine and homocysteine potentiates TNF-alpha-mediated cytotoxicity, but does not modulate activation of NF-kappa B transcription factor controlling the expression of various TNF-alpha-inducible genes. Adenosine and homocysteine at concentrations (1 mM each) that enhance TNF-alpha-induced cytotoxicity accumulate S-adenosyl-L-homocysteine (AdoHcy), a potent inhibitor of S-adenosyl-L-methionine-dependent methylation reactions. In addition, preloading L929 cells with AdoHcy resulted in enhanced responses to TNF-alpha, suggesting that AdoHcy potentiates TNF-alpha-induced cytotoxicity. Moreover, the combination of adenosine and homocysteine changed the dependency of the TNF-alpha-mediated cytolysis on reactive oxygen intermediates. In the absence of adenosine and homocysteine TNF-alpha-mediated cytotoxicity was inhibited by antioxidants such as butylated hydroxyanisole and pyrrolidine dithiocarbamate. In the presence of adenosine and homocysteine, however, TNF-alpha-mediated cytotoxicity is not inhibited by these antioxidants. A L929 subclone, defective in the respiratory chain, resisted the cytotoxic action of TNF-alpha, but was rendered TNF-alpha sensitive in the presence of adenosine and homocysteine. Unlike TNF-alpha-mediated cytotoxicity, the TNF-alpha-induced activation of NF-kappa B was inhibited by antioxidants regardless whether adenosine and homocysteine were present or absent in the culture medium. In conclusion, a combination of adenosine and homocysteine selectively modulates TNF-alpha-mediated cytotoxicity without changing the TNF-alpha-induced activation of NF-kappa B. Our results could facilitate the development of strategies that permit dissection of cytotoxic and gene-activating pathways.