[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.

Adenovirus-mediated wild-type-p53-gene expression sensitizes TNF-resistant tumor cells to TNF-induced cytotoxicity by altering the cellular redox state.

We have shown that the loss of p53 function contributed to resistance of tumor cells to TNF-induced cytotoxicity. In the present study, we evaluated the effect of wild-type p53 (wt-p53) expression on TNF sensitivity, by introducing wt-p53 into MCF7/Adr cells in which p53 was deleted, via a recombinant adenovirus encoding p53 (Ad-p53). Our results indicate that infection with Ad-p53 (50-100 viral particles per cell) resulted in pronounced cytotoxicity, whereas infection with 10 viral particles per cell, which was weakly toxic for the MCF7/Adr cells, sensitized these cells to TNF-induced cell death. Moreover, expression of wt-p53 in MCF7/Adr cells induced the production of reactive oxygen intermediates (ROIs) and caused glutathione (GSH) depletion, indicating disturbances in the cellular redox state. Additional treatment of cells with the anti-oxidant and glutathione (GSH) precursor N-acetylcysteine (NAC) resulted in inhibition of p53-induced ROIs production and in partial restoration of intracellular GSH levels, which was associated with the ability of NAC to inhibit p53-modulated TNF-induced cytotoxicity. Interestingly, Ad-p53 was able to inhibit TNF-induced MnSOD mRNA expression in MCF7/Adr cells, which might contribute to the sensitization of cells to the cytotoxic action of TNF. Taken together, our data strongly suggest that wt-p53 expression sensitizes TNF-resistant MCF7 cells with p53 deletion to TNF-induced cell death by a pathway that is dependent on ROIs production.

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.

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.

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 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.

Analysis of human breast adenocarcinoma MCF7 resistance to tumor necrosis factor-induced cell death. Lack of correlation between JNK activation and ceramide pathway.

Considerable progress has been made in the understanding of tumor necrosis factor (TNF) signaling; however, the molecular and biochemical basis of tumor resistance to the cytotoxic action of TNF are still not definitively identified yet. Although a role of c-Jun N-terminal kinase (JNK) pathway has been suggested as an effector in TNF signaling, its exact relative contribution and its interaction with ceramide pathway and tumor resistance to TNF remain unknown. The relationship between JNK activation and human breast adenocarcinoma MCF7 resistance acquisition to the cytotoxic action of TNF was therefore investigated. We demonstrate that TNF triggers JNK activation in both TNF-sensitive MCF7 cells and its resistant derivative, RA1/1001. In addition, when MCF7 cells were stably transfected with mitogen-activated protein kinase kinase 4 (MKK4) dominant-negative cDNA or transiently transfected with a dominant-negative c-Jun mutant (TAM 67), their susceptibility to the cytotoxic action of TNF remains comparable with control cells. We also demonstrated that JNK activation does not require ceramide generation since in MCF7 cells transfected with a dominant-negative derivative of FADD (FADD-DN), which are resistant to the cytotoxic action of TNF, TNF induced JNK activation in the absence of ceramide generation. Furthermore, our data indicate that exogenous permeable synthetic ceramide C-6 induced the killing of MCF7 cells transfected with MKK4 dominant-negative cDNA. These results provide strong evidence indicating that tumor acquisition of resistance to the cytotoxic action of TNF may occur either independently or at a level downstream of JNK activation and suggest that JNK activation is not linked to ceramide pathway in TNF-mediated apoptosis.

[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.

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.

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.

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.

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.

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1-72), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

[The functional activity of the urethral neutrophilic granulocytes in patients with chronic urethroprostatitis and its correction]. / Funktsional'naia aktivnost' uretral'nykh neitrofil'nykh granulotsitov u bol'nykh khronicheskim uretroprostatitom i ee korrektsiia.

The functional state of urethral neutrophil granulocytes in patients with chronic urethro-prostatitis, and possibility of miramistin use in complex treatment of the disease were investigated. The data revealed functional suppression of these granulocytes in the patients. Complex treatment with miramistin normalized indices of urethral phagocytic activity, on the contrary, after treatment of the patients without the agent there was no tendency to normalize urethral phagocytic function. The findings suggest that miramistin supplemented to routine antiinflammatory therapy improves curative results in patients with chronic urethro-prostatitis but the drug should be prescribed in accordance with the initial function of urethral neutrophil granulocytes.

[The effect of miramistin on the phagocytic activity of the urethral neutrophilic granulocytes in patients with chronic urethroprostatitis]. / Vliianie miramistina na fagotsitarnuiu aktivnost' uretral'nykh neitrofil'nykh granulotsitov u bol'nykh khronicheskim uretroprostatitom.

It was found that addition of mirastimin to the traditional anti-inflammatory treatment results in stimulation of the absorptive capacity of the urethral phagocytes and improves the results of treatment of patients with chronic urethroprostatitis. The immunomodulator mirastimin should be given with consideration of the initial functional state of urethral neutrophilic granulocytes.

[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.

[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.

[Effects of embryonal bone tissue on hemopoiesis]. / Vliianie émbrional'noi kostnoi tkani na hemopoéz.

The influence of human fetal bone (FB) on the hemopoietic and stromal cells were studied in the morphometric assay of the bone marrow trephine biopsy samples after a preliminary cultivation using the Marbrook system for 1-5 weeks. The rat FB effect on the hemopoiesis and survival were also studied in experiment with FB transplantation to rats with prior administration of lethal and sublethal doses of cyclophosphamide. In long-term cultivation of the bone marrow trephine biopsy samples, enhanced proliferation of the fibrous tissue and elimination of hemopoietic elements were seen. Combined cultivation of the trephine biopsy samples of the bone marrow and FB inhibited the fibrous tissue proliferation and enhanced the viability of the hemopoietic and lipid cells. Transplantation of the FB in experiment shortened the leukocytopenia period in rats after sublethal (300 mg/kg) and lethal (500 mg/kg) doses of cyclophosphamide and increased their survival.

[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.