Dietary habits affect fatty acid composition of visceral adipose tissue in subjects with colorectal cancer or obesity.

PURPOSE: Aim of this study was to identify a possible relationship among dietary fatty acids (FA) intake, FA adipose tissue (AT) profile and cancer condition in lean vs obese subjects affected or not by colorectal cancer (CRC). Actually, inadequate dietary habits together with physical inactivity are primary determinants of obesity and cancer risk. Changes in lipid metabolism play a crucial role in different types of cancer and key enzymes involved in lipid-metabolic pathways, such as stearoyl-coA-desaturase 1 (SCD-1), are differentially expressed in normal and cancer tissues. METHODS: Food frequency questionnaires (FFQ) were analyzed by Winfood software. FA were assessed by gas-liquid chromatography in visceral AT samples. Estimated desaturase activities were calculated as precursor FA/product FA ratio. Desaturase gene expressions were evaluated by RT-qPCR. RESULTS: Lean and obese CRC subjects showed inadequate dietary habits. In particular, lean CRC subjects showed increase in the intake of saturated FA, specifically palmitic (p = 0.0042) and stearic acid (p = 0.0091), and a corresponding reduction of monounsaturated FA consumption, in particular oleic acid (p = 0.002) with respect to lean without CRC. Estimated SCD-1 activity in AT was increased in all the groups vs lean without CRC (pANOVA = 0.029). CONCLUSIONS: Unhealthy eating habits, characterizing obese and CRC subjects, may influence the visceral AT profile and contribute to the alteration of the metabolic pathways. The quality of the diet, other than the quantity, can have a main role in the establishment of inflammatory microenvironment and in metabolic changes favouring CRC.

Interferon gamma upregulates its own gene expression in mouse peritoneal macrophages.

Interferon gamma (IFN-gamma) exerts a variety of immunoregulatory effects on several cell targets. It is generally assumed that IFN-gamma is specifically produced by T and large granular lymphocytes. In this study, we show that IFN-gamma is constitutively expressed in resting mouse peritoneal macrophages (PM). Treatment of PM with cycloheximide results in a significant accumulation of IFN-gamma mRNA, suggesting that a short-lived IFN-gamma mRNA accumulates when protein synthesis is inhibited. Moreover, treatment of PM with IFN-gamma also results in a clear-cut accumulation of this mRNA. This effect is not observed in murine lymphocytes from mesenteric lymph nodes (which instead produce IFN-gamma after phytohemagglutinin treatment) and in mouse cell lines. The treatment of PM with IFN-gamma also results in secretion of IFN-gamma after 24-48 h. The upregulation of IFN-gamma expression is also found in PM from anti-asialo GM1-treated nude mice. We suggest that the ability of PM to produce this IFN-gamma is indicative of an autocrine mechanism. The macrophage IFN-gamma may play a role in the regulation of cell differentiation and immune response.

The HIV-1 vpr protein acts as a negative regulator of apoptosis in a human lymphoblastoid T cell line: possible implications for the pathogenesis of AIDS.

Although apoptosis is considered one of the major mechanisms of CD4(+) T cell depletion in HIV-infected patients, the virus-infected cells somehow appear to be protected from apoptosis, which generally occurs in bystander cells. Vpr is an auxiliary HIV-1 protein, which, unlike the other regulatory gene products, is present at high copy number in virus particles. We established stable transfectants of CD4+ T Jurkat cells constitutively expressing low levels of vpr. These clones exhibited cell cycle characteristics similar to those of control-transfected cells. Treatment of control clones with apoptotic stimuli (i.e., cycloheximide/tumor necrosis factor alpha (TNF-alpha), anti-Fas antibody, or serum starvation) resulted in a massive cell death by apoptosis. In contrast, all the vpr-expressing clones showed an impressive protection from apoptosis independently of the inducer. Notably, vpr antisense phosphorothioate oligodeoxynucleotides render vpr-expressing cells as susceptible to apoptosis induced by cycloheximide and TNF-alpha as the control clones. Moreover, the constitutive expression of HIV-1 vpr resulted in the upregulation of bcl-2, an oncogene endowed with antiapoptotic activities, and in the downmodulation of bax, a proapoptotic factor of the bcl-2 family. Altogether, these results suggest that low levels of the endogenous vpr protein can interfere with the physiological turnover of T lymphocytes at early stages of virus infection, thus facilitating HIV persistence and, subsequently, viral spread. This might explain why apoptosis mostly occurs in bystander uninfected cells in AIDS patients.

IFN-gamma expression in macrophages and its possible biological significance.

IFN-gamma is a pleiotropic cytokine endowed with potent immunomodulatory effects whose expression was long considered to be restricted to T and NK cells. Only recently, it became evident that IFN-gamma production can also occur in other cell types, including monocyte/macrophages. However, the biological relevance of macrophage IFN-gamma is still unclear. The purpose of this article is to provide an overview of the collected evidence demonstrating IFN-gamma expression in macrophages and to discuss the possible biological significance of this cytokine production in the early phase of host response to infectious agents.

The HIV-1 vpr protein induces anoikis-resistance by modulating cell adhesion process and microfilament system assembly.

We have previously shown that CD4+ T Jurkat cells constitutively expressing low levels of the human immunodeficiency virus 1 (HIV-1) vpr protein were less susceptible to undergo apoptosis than control cells.1 In this study we have investigated the role of vpr in affecting mechanisms of importance in the control of apoptosis. Vpr-expressing clones consistently aggregated in clusters with time in culture, whereas mock-transfected cells grew as dispersed cultures. The analysis of adhesion molecules involved in cell-to-cell as well as in cell-substrate interactions showed a higher expression of cadherin and integrins alpha5 and alpha6 in vpr-transfected clones with respect to mock-transfected cells. This up-modulation was specifically blocked by cell exposure to antisense oligonucleotides targeted at the vpr. In addition, F-actin microfilament cytoskeletal organization, known to be involved in cell-cell interaction pathways and in the modulation of cell surface molecule expression, was significantly improved in vpr-expressing clones, in which filament polymerization was increased. We thus envisage that vpr viral protein can maintain cell survival via a specific activity on cytoskeleton-dependent cell adhesion pathways, i.e. by inducing anoikis-resistance. These particular effects of vpr might enhance the homing, spreading and survival of the infected lymphocytes, thus contributing to virus persistence in the course of acute HIV-1 infection.

Induction of cytokines by HIV-1 and its gp120 protein in human peripheral blood monocyte/macrophages and modulation of cytokine response during differentiation.

We previously reported that in vitro culture of human peripheral blood monocytes resulted in a time-dependent differentiation into macrophages and in an enhanced capacity for producing certain cytokines [i.e., tumor necrosis factor alpha, interleukin-6 (IL-6), and interferon-beta (IFN-beta)] in response to bacterial lipopolysaccharide (LPS). HIV-1 infection or gp120 treatment of monocyte/macrophages resulted in the induction of low levels of IFN-beta, which were very effective in restricting viral replication in 7-day cultured macrophages but not in freshly isolated cells. This enhanced response of macrophages was due to a higher sensitivity of these cells to the antiviral effect of IFN-beta. Consistent with this finding, 7-day cultured macrophages exhibited higher levels of type I IFN receptors than 1-day cultured monocytes. Treatment of monocyte/macrophages with gp120 also caused a marked increase in IL-10 secretion, regardless of the differentiation state. No IL-12 secretion was detected in monocyte/macrophage cultures treated with gp120 alone. However, consistent IL-12 secretion was found in 7-day cultured macrophages primed with IFN-beta and subsequently stimulated with gp120. Macrophages responded more efficiently than monocytes to the priming effect of IFN-beta for IL-12 production. This was consistent with a stronger antiviral response against vesicular stomatitis virus by these cells as well as with a higher expression of IFN-beta receptors. The finding that the acquisition of the macrophage phenotype is associated with an increased capacity to respond to environmental signals (such as type I and type II IFNs) underlines the importance of the differentiation process for the selection of a certain repertoire of responses that may allow these cells to have important functions in vivo.

Role of endogenous interferon-beta in the restriction of HIV replication in human monocyte/macrophages.

In vitro culture of human monocytes results in a time-dependent differentiation into macrophages. Monocyte/macrophages were infected with HIV-1Ba-L at different times after isolation and subsequent culture. When 7-day macrophages were infected in the presence of antibodies to interferon-beta (IFN-beta), a significant increase in HIV-1 p24 release was observed. This effect was not detected in 1-day monocytes. Treatment of 7-day cultured macrophages with HIV-1 rgp120 resulted in resistance to vesicular stomatitis virus infection. This rgp120-induced antiviral state was neutralized in the presence of antibodies to IFN-beta. The overall results indicate that the infection of monocyte/macrophages with HIV-1 results in the induction of IFN-beta, which, in turn, inhibits HIV-1 expression in macrophages. The finding that HIV-1 itself (possibly through its gp120) can induce a potent antiviral factor (IFN-beta) in macrophages underlines the complex physiological function of these cells in maintaining normal homeostasis in vivo in response to virus infection.

IFN-alpha and IL-18 exert opposite regulatory effects on the IL-12 receptor expression and IL-12-induced IFN-gamma production in mouse macrophages: novel pathways in the regulation of the inflammatory response of macrophages.

We characterized the IL-12 response of mouse macrophages in terms of modulation of IFN-gamma production by cytokines (IFN-alpha and IL-18) and regulation of IL-12 receptor expression. Beta1 and beta2 IL-12R chain mRNA expression increased with time in culture in the absence of exogenous stimulation, with concomitant acquisition of responsiveness to IL-12 for IFN-gamma production. Expression of the IL-12R beta1 chain mRNA was increased further following IL-12 treatment as a consequence of IFN-gamma expression. IL-12 response was regulated differentially by IFN-alpha and IL-18. Neutralization of endogenous type I IFN increased IFN-gamma secretion, whereas exogenous IFN-alpha reduced it. In contrast, IL-18 enhanced IFN-gamma mRNA accumulation and IFN-gamma secretion in IL-12-stimulated, but not -untreated, cultures. The opposite effects exerted by IFN-alpha and IL-18 mirror their mutual capacity of regulating-in a negative or positive manner, respectively-the expression of the IL-12R beta1 chain. We suggest that differential regulation of IL-12 response by IFN-alpha and IL-18 can represent previously unrecognized regulatory mechanisms for maintaining suitable levels of differentiation/activation in macrophages.

Regulation of chemokine/cytokine network during in vitro differentiation and HIV-1 infection of human monocytes: possible importance in the pathogenesis of AIDS.

The monocyte/macrophage lineage represents heterogeneous cell populations characterized by major differences in the phenotype and functional activities. These cells are a major source of soluble factors, such as cytokines and chemokines, which can both affect HIV replication and AIDS pathogenesis. Although monocytes/macrophages are unanimously considered important targets of HIV-1 infection, the HIV-induced alterations in their physiological functions at different stages of differentiation are still matter of debate. In this article, we review our data on the regulation of chemokine/cytokine network with regard to macrophage differentiation and HIV-1 infection, in comparison with studies from other groups. The ensemble of the results emphasizes that: 1) macrophages markedly differ with respect to monocytes for a variety of responses potentially important in the pathogenesis of HIV infection; and 2) the experimental conditions can influence the HIVmonocyte/macrophage interactions, reflecting the possible in vivo existence of a spectrum of responses among macrophage populations.

Inhibition of human immunodeficiency virus type 1 replication by nuclear chimeric anti-HIV ribozymes in a human T lymphoblastoid cell line.

Human immunodeficiency virus (HIV) infection represents one of the most challenging systems for gene therapy. Thanks to the extended knowledge of the molecular biology of the HIV life cycle, many different strategies have been developed including transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes, and intracellular antibody fragments. In this paper, we have tested in a human T lymphoblastoid cell line the antiviral activity of ribozymes specifically designed to co-localize inside the nucleus with the Rev pre-mRNA before it is spliced and transported to the cytoplasm. This result was obtained by inserting the ribozyme in the spliceosomal U1 small nuclear RNA (snRNA) and in a derivative that has perfect complementarity with the 5' splice site of the Rev pre-mRNA. These ribozymes were tested in human T cell clones and were shown to be very efficient in inhibiting viral replication. Not only were the p24 levels in the culture medium drastically reduced but so were the intracellular HIV transcripts. Control disabled ribozymes enabled us to show the specificity of the ribozyme activity. Therefore, these constructs have potential utility for gene therapy of HIV-1 infection.

Antiviral effect of bovine lactoferrin saturated with metal ions on early steps of human immunodeficiency virus type 1 infection.

Lactoferrin is a mammalian iron-binding glycoprotein present in many biological secretions, such as milk, tears, semen and plasma and a major component of the specific granules of polymorphonuclear leucocytes. The effect of bovine lactoferrin (BLf) in apo-form or saturated with ferric, manganese or zinc ions, on human immunodeficiency virus type 1 (HIV-1) infection in the C8166 T-cell line was studied. Both HIV-1 replication and syncytium formation were efficiently inhibited, in a dose-dependent manner, by lactoferrins. BLf in apo and saturated forms markedly inhibited HIV-1 replication when added prior to HIV infection or during the virus adsorption step, thus suggesting a mechanism of action on the HIV binding to or entry into C8166 cells. Likewise, the addition of Fe3+BLf prior to HIV infection and during the attachment step resulted in a marked reduction of the HIV-1 DNA in C8166 cells 20 h after infection. The potent antiviral effect and the high selectivity index exhibited by BLf suggest for this protein, in apo or saturated forms, an important role in inhibiting the early HIV-cell interaction, even though a post adsorption effect cannot be ruled out.

Modulations of glycerophosphorylcholine and phosphorylcholine in Friend erythroleukemia cells upon in vitro-induced erythroid differentiation: a 31P NMR study.

A 31P NMR study has been carried out on Friend erythroleukemia cells (FLC) induced to undergo erythroid differentiation in vitro. Significant levels of glycerophosphorylcholine (GroPCho) and phosphorylcholine (P-Cho) were identified both in the untreated cells and in their PCA extracts. In FLC treated 4 days in vitro with either dimethylsulfoxide (DMSO) or hexamethylenebisacetamide (HMBA), the intracellular concentration of P-Cho was markedly increased, whereas that of GroPCho appeared to be significantly reduced. HMBA was more effective than DMSO in producing this effect. The concomitant modulations of GroPCho and P-Cho in differentiated FLC suggest the hypothesis that erythroid differentiation involves modifications of the regulatory mechanisms controlling biosynthesis and catabolism of phospholipids.

IFN-beta stimulates the production of beta-chemokines in human peripheral blood monocytes. Importance of macrophage differentiation.

We investigated the effect of IFN-beta on beta-chemokine expression in differentiating human peripheral blood monocytes. MCP-1, MIP-1alpha and MIP-1beta were constitutively expressed in 1 day-cultured monocytes, and their secretion increased with time in culture despite any change in mRNA accumulation. IFN-beta treatment of differentiating monocytes resulted in a marked and dose-dependent increase of beta-chemokine secretion, which was regulated differently with respect to the differentiation stage. In particular, IFN-beta upregulated MCP-1 secretion in monocytes at all stages of differentiation although its effect was significantly higher in 1-day cultured monocytes as compared to monocyte-derived macrophages (MDM). In contrast, MIP-1alpha and MIP-1beta secretion was up-regulated by IFN-beta only in MDM. Although MCP-1, MIP-1alpha and MIP-1beta mRNA expression was up-regulated by IFN-beta in both 1 day-cultured monocytes and MDM, no correlation was found between mRNA level and protein secretion. These results suggest that the regulation of beta-chemokine secretion in monocytes/macrophages by IFN-beta occurred through different mechanisms, involving both a direct effect of this cytokine on chemokine gene expression and translational/post-translational steps of regulation more likely linked to the differentiation process. This finding reveals a novel role for this cytokine in the recruitment of specific cell types during the immune response, which may be relevant in the control of viral infections in vivo.

Downregulation of tumor necrosis factor receptors of macrophages by interferons and interleukin-1. Role of protein kinase C activation.

Freshly harvested murine peritoneal macrophages and a line of transformed murine macrophages (RAW) were used in experiments designed to investigate the effect of different interferons (IFN) and interleukin-1 (IL-1) on tumor necrosis factor (TNF) receptors. Low concentrations of IFN-gamma or somewhat higher concentrations of IFN-alpha drastically downregulated the TNF receptors of RAW cells. A similar, but less pronounced, downregulation of TNF receptors was observed in peritoneal macrophages treated with these IFNs. This downregulation could not be accounted for by an induction of TNF secretion. Furthermore, IFN-alpha and gamma interacted synergistically in downregulating TNF receptors of RAW cells. IL-1 also downregulated TNF receptors. When RAW cells were treated with inhibitors of protein kinase C, the downregulation of TNF receptors by IFNs or IL-1 was reversed, and TNF binding increased up to 2-fold over that of untreated cells. Such increase was also observed in RAW cells treated only with the inhibitor of protein kinase C, staurosporine. However, TNF receptors decreased in peritoneal macrophages treated with staurosporine. This finding was explained by activation of macrophages by staurosporine, which induced secretion of TNF. These findings indicate that protein kinase C activity regulates TNF receptors in macrophages.

The antiviral activity of tumor necrosis factor in HeLa cells is not mediated by interferons. Studies with TNF-resistant variants.

Tumor necrosis factor (TNF) has a relatively modest antiviral activity in HeLa cells, when compared to that of interferon (IFN). Addition of antisera neutralizing IFN-alpha or beta did not reduce the antiviral activity of TNF. Furthermore, we determined with a sensitive hybridization assay that less than one molecule of IFN-beta 1 mRNA could be present per TNF-treated cell. HeLa cell variants resistant to the TNF cytocidal activity were selected. These variants had TNF receptors with a dissociation constant similar to that of parental cells. Three cloned variants tested did not respond to the antiviral activity of TNF, but responded to IFN-alpha. Surprisingly, two of the variants did not respond to IFN-gamma. This suggests that the pathways mediating the cytocidal activity of TNF and the response to IFN-gamma share some common step. TNF induced transcription of IFN-beta 2 in resistant variants, indicating that this cytokine does not contribute to the antiviral activity of TNF.

Simultaneous production of IFN-gamma, IFN-alpha/beta and nitric oxide in peritoneal macrophages from TDM-treated mice.

Peritoneal macrophages (PM) were isolated from mice treated with Dimycolate of Trehalose (TDM), a glycolipid extracted from the cell wall of Mycobacterium tuberculosis. PM from TDM-treated mice (TDM-PM) were shown to secrete consistent amount of IFN-gamma, which was not detectable in control Resident-PM (Res-PM), as revealed by ELISA. In addition, biologically active IFN was detected in the supernatants of TDM-PM, whereas no IFN production was found in those of control Res-PM. The addition of specific antisera to PM cultures revealed the simultaneous production of both type I and II IFNs in TDM-PM cultures. No reciprocal regulation in the production of IFN-gamma and IFN-alpha/beta was found in these cultures. In parallel, nitric oxide (NO) production was measured in TDM-PM cultures by detecting nitrites (NO2-). TDM-PM cultures accumulated high amounts of NO2- which decreased to the level of Res-PM in the presence of NMMA, an inhibitor of NO-synthases. In vitro, neither type I nor type II IFNs were involved in the stimulation of NO production. The capacity of macrophages to simultaneously secrete IFN-gamma, IFN-alpha/beta and NO upon in vivo TDM-treatment could be of particular relevance for the defense process of innate immunity in which macrophages play a crucial role.

Spontaneous expression of interferon genes in murine peritoneal macrophages: modulation during the in vitro aging.

The expression of interferon (IFN)-beta gene and its modulation during in vitro "aging" was studied in unstimulated peritoneal macrophages (PM) explanted from lipopolysaccharide (LPS)-responsive (Lps(h)) and LPS-hyporesponsive (Lps(d)) mice. A strong direct correlation between the LPS response of PM and their capacity to express low levels of IFN-beta was found. Moreover, the decay of antiviral state during in vitro cultivation was correlated with the turnover of IFN-beta mRNA. In the light of the multiple biologic effect of IFN, the constitutive expression of IFN-beta gene in PM can play a crucial role not only in the restriction of viral replication, but also in the modulation of cell differentiation and immune response.

The glucocorticoid dexamethasone inhibits synthesis of interferon by decreasing the level of its mRNA.

Human fibroblasts were induced to secrete interferon (IFN) by treatment with the double-stranded RNA poly(inosinic).poly(cytidylic) acid or by infection with Newcastle disease virus. Treatment with 0.1-1 microM dexamethasone reduced the amount of IFN secreted by approximately 40-70%, respectively. A similar decrease in secretion of human IFN-beta was detected in dexamethasone-treated murine C127 cells that carry an IFN expression vector. These cells transcribe constitutively human IFN-beta under the control of a viral thymidine kinase promotor. Secretion of murine IFN induced by double-stranded RNA was also reduced in dexamethasone-treated C127 cells. The amount of IFN-beta mRNA present in fibroblasts and C127 cells was measured by hybridization to complementary RNA. Treatment with dexamethasone markedly reduced the level of IFN-beta mRNA present in both cells. The time course of this decrease was measured in C127 cells; 50 and 80% loss of IFN mRNA was observed after approximately 7.5 and 12 h, respectively. Murine IFN mRNA was also decreased in dexamethasone-treated C127 cells induced with double-stranded RNA. However, the rate of transcription of human IFN mRNA measured by run-on assays in isolated nuclei of dexamethasone-treated C127 cells was found to be comparable to that of control untreated cells. The finding that dexamethasone reduces the level of IFN mRNA transcribed under the control of both its own promotor and an unrelated promotor, together with the observation that dexamethasone does not apparently alter the rate of transcription of this mRNA, suggest that glucocorticoids may regulate IFN production by decreasing the level of its mRNA.