Lipid droplet biogenesis and COX-2 pathway activation are triggered by Barrett's esophagus and adenocarcinoma, but not esophageal squamous cell carcinoma risk factors.

Esophageal cancer (EC) is an aggressive disease, presenting two main histological subtypes: adenocarcinoma (EAC) and squamous cell carcinoma (ESCC). The two EC subtypes widely differ concerning virtually all factors. ESCC development is mainly associated with tobacco and alcohol abuse, whereas obesity and chronic gastroesophageal reflux disease (GERD) are important risk factors not only for EAC, but also for for Barrett's esophagus (BE), an intestinal metaplasia that precedes EAC. Obesity triggers ectopic lipid droplets (LD) accumulation in non-adipose tissues. LD are organelles involved in cell metabolism, signaling, proliferation and production of inflammatory mediators. Therefore, the aim of this work was to investigate LD occurrence and role in EC. This study shows progressive LD levels increase along EAC development, in esophageal samples from non-obese through obese individuals, as well as BE, and EAC patients, whereas no significant changes were observed in ESCC samples, when compared to non-tumor samples. Additionally, in order to mimic BE and EAC risk factors exposure, a non-tumor esophageal cell line was incubated with oleic acid (OA) and acidified medium and/or deoxycholic acid (DCA), revealing a significant increment in LD amount as well as in COX-2 and CXCL-8 expression, and in IL-8 secretion. Further, COX-2 expression and LD amount presented a significant positive correlation and were detected co-localized in EAC, but not in ESCC, suggesting that LD may be the site for eicosanoid production in EAC. In conclusion, this study shows that obesity, and BE- and EAC-associated inflammatory stimuli result in a gradual increase of LD, that may be responsible for orchestrating inflammatory mediators' production and/or action, thus contributing to BE and EAC genesis and progression.

Cell cycle and apoptosis regulation by NFAT transcription factors: new roles for an old player.

The NFAT (nuclear factor of activated T cells) family of transcription factors consists of four Ca(2+)-regulated members (NFAT1-NFAT4), which were first described in T lymphocytes. In addition to their well-documented role in T lymphocytes, where they control gene expression during cell activation and differentiation, NFAT proteins are also expressed in a wide range of cells and tissue types and regulate genes involved in cell cycle, apoptosis, angiogenesis and metastasis. The NFAT proteins share a highly conserved DNA-binding domain (DBD), which allows all NFAT members to bind to the same DNA sequence in enhancers or promoter regions. The same DNA-binding specificity suggests redundant roles for the NFAT proteins, which is true during the regulation of some genes such as IL-2 and p21. However, it has become increasingly clear that different NFAT proteins and even isoforms can have unique functions. In this review, we address the possible reasons for these distinct roles, particularly regarding N- and C-terminal transactivation regions (TADs) and the partner proteins that interact with these TADs. We also discuss the genes regulated by NFAT during cell cycle regulation and apoptosis and the role of NFAT during tumorigenesis.

Lidocaine-derivative JMF2-1 prevents ovalbumin-induced airway inflammation by regulating the function and survival of T cells.

BACKGROUND: Inhalation of the local anaesthetic lidocaine has been suggested to be beneficial for asthmatics, but airway anaesthesia is unpleasant and may exacerbate bronchoconstriction. Our previous study showed that inhalation of the lidocaine analogue JMF2-1 can elicit the anti-inflammatory properties of lidocaine without anaesthesia. This prompted further research on the mechanism of action and putative therapeutic application of JMF2-1. OBJECTIVE: We tested the hypothesis that JMF2-1 would prevent allergen-induced lung inflammation and airway hyperresponsiveness (AHR) by modulating T cell function in vivo and in vitro. Methods Local and systemic changes in leucocyte levels, cytokine production and lung mechanics were examined in a murine model of lung inflammation. JMF2-1 (0.05-2%) or saline was aerosolized twice a day during the ovalbumin (OVA)-provocation period (19-21 days post-sensitization). Analyses were performed 24 h after the final challenge. Primary cultured lymph node cells were used to assess the effects of JMF2-1 (100-600 µm) at the cellular level. RESULTS: OVA challenge resulted in lung recruitment of CD4(+) T cells and eosinophils, increased generation of inflammatory cytokines and AHR to inhaled methacholine within 24 h. These changes were prevented by JMF2-1 nebulization, and occurred in parallel with an increase in the number of apoptotic cells in the lung. JMF2-1 treatment did not alter levels of CD4(+) or CD8(+) T cells in the thymus or lymph nodes of naïve mice, although it inhibited OVA-induced IL-13 production and the lymphocyte proliferative response in vitro. It also induced apoptosis of OVA-activated lymphocytes in a mechanism sensitive to z-VAD, indicating that JMF2-1 mediates caspase-dependent apoptosis. CONCLUSION: Inhalation of JMF2-1 prevents the cardinal features of asthma by reducing T(H) 2 cytokine generation and lung eosinophilic inflammatory infiltrates via local inhibition of T cell function and survival. JMF2-1 may represent a novel therapeutic alternative for asthma control with distinct advantages over local anaesthetics.

NFAT transcription factors: from cell cycle to tumor development.

The nuclear factor of activated T cells (NFAT) family of transcription factors has been primarily identified in immune cells; however, these proteins have been recently found to be functionally active in several other non-immune cell types. NFAT proteins are activated upon different stimuli that lead to increased intracellular calcium levels. Regardless of their widely known cytokine gene expression properties, NFATs have been shown to regulate other genes related to cell cycle progression, cell differentiation and apoptosis, revealing a broader role for these proteins in normal cell physiology. Several reports have addressed the participation of NFATs in many aspects of malignant cell transformation and tumorigenic processes. In this review, we will discuss the involvement of the different NFAT family members in the regulation of cell cycling, differentiation and tumor formation, and also its implications on oncogenesis. Better understanding the mechanisms by which NFATs regulate cell cycle and tumor-related events should be relevant for the development of rational anti-cancer therapies.

NFAT transcription factors: from cell cycle to tumor development

The nuclear factor of activated T cells (NFAT) family of transcription factors has been primarily identified in immune cells; however, these proteins have been recently found to be functionally active in several other non-immune cell types. NFAT proteins are activated upon different stimuli that lead to increased intracellular calcium levels. Regardless of their widely known cytokine gene expression properties, NFATs have been shown to regulate other genes related to cell cycle progression, cell differentiation and apoptosis, revealing a broader role for these proteins in normal cell physiology. Several reports have addressed the participation of NFATs in many aspects of malignant cell transformation and tumorigenic processes. In this review, we will discuss the involvement of the different NFAT family members in the regulation of cell cycling, differentiation and tumor formation, and also its implications on oncogenesis. Better understanding the mechanisms by which NFATs regulate cell cycle and tumor-related events should be relevant for the development of rational anti-cancer therapies.

Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity.

NFAT transcription factors are highly phosphorylated proteins that are regulated by the calcium-dependent phosphatase calcineurin. We show by mass spectrometry that NFAT1 is phosphorylated on fourteen conserved phosphoserine residues in its regulatory domain, thirteen of which are dephosphorylated upon stimulation. Dephosphorylation of all thirteen residues is required to mask a nuclear export signal (NES), cause full exposure of a nuclear localization signal (NLS), and promote transcriptional activity. An inducible phosphorylation site in the transactivation domain contributes to transcriptional activity. Our data suggest that dephosphorylation promotes NFAT1 activation by increasing the probability of an active conformation, in a manner analogous to that by which depolarization increases the open probability of voltage-gated ion channels. This conformational switch paradigm may explain modification-induced functional changes in other heavily phosphorylated proteins.

Costimulatory action of glycoinositolphospholipids from Trypanosoma cruzi: increased interleukin 2 secretion and induction of nuclear translocation of the nuclear factor of activated T cells 1.

The effects of the glycoinositolphospholipids (GIPLs) fromTrypanosoma cruzi on T lymphocyte activation were investigated in a mouse T cell hybridoma (DO-11.10). Purified GIPLs from T. cruzi strains Y and G markedly increased IL-2 mRNA transcripts and IL-2 secretion induced by mitogenic anti-CD3 and anti-Thy1 mAbs. This costimulatory function was also revealed by the induction of IL-2 secretion after the simultaneous addition of the T. cruzi GIPLs and either the calcium ionophore A23187 or phorbol ester. The capacity of the GIPL molecule to induce an increase in cytoplasmic calcium levels was also demonstrated. After exposure of T cell hybridoma to GIPL, the nuclear transcription factor NFAT1 became partially dephosphorylated, and its nuclear localization was demonstrated both in the T cell hybridoma and in Balb/c CD3(+) cells. These results demonstrate that T. cruzi GIPL molecules are capable of signaling to T cells and therefore could be valuable tools for the study of T cell activation, besides playing a potential role in subverting the T lymphocyte immune response during T. cruzi infection.

Chromatin-based regulatory mechanisms governing cytokine gene transcription.

On initial contact with antigen, naive T cells differentiate and acquire effector characteristics, including the ability to transcribe specific cytokine genes rapidly and at high levels on subsequent exposure to antigen. Several effector T-cell subsets showing distinct patterns of cytokine gene transcription have been described. The patterns of cytokine expression in response to pathogenic challenges have a significant impact on the outcome of immune and inflammatory reactions. Here we review recent studies suggesting that the ability of naive T cells to differentiate into specific cytokine-expressing cells is regulated by epigenetic changes in the accessibility and chromatin structure of cytokine genetic loci. Antigen and cytokine stimulation of naive T cells activates diverse intracellular signaling pathways, which result in chromatin remodeling and demethylation of cytokine genes. These changes are likely to increase, in a stable and heritable fashion, the accessibility of these genes to the basal transcriptional machinery. Chromatin-based regulatory mechanisms may explain several features of cytokine gene expression in effector versus naive T cells, including their monoallelic expression, coordinate regulation, and stable maintenance in memory T cells. The hypothesis of epigenetic changes occurring during T-cell differentiation provides a framework for a comprehensive understanding of cytokine expression by T cells.

Molecular regulation of cytokine gene expression during the immune response.

Cytokine expression by immune system cells plays an important role in the regulation of the immune response. On first encounter with antigen, naive CD4+ T helper (Th) cells differentiate into cytokine-producing effector cells. Two types of effector cells characterized by their distinct expression of cytokine profiles have been described. Th1 cells produce IL-2 and IFN-gamma, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13. In many pathological situations, the balance between Th1 and Th2 immune responses determines the outcome of diverse immunologically mediated clinical syndromes including infectious, autoimmune, and allergic diseases. However, the molecular basis for the tissue-specific expression of Th1/Th2-like cytokines has remained elusive. In this review we evaluate the possible in vivo role of different transcription factors and transcriptional mechanisms in T cell differentiation and the immune response.

Regulation of allergic inflammation and eosinophil recruitment in mice lacking the transcription factor NFAT1: role of interleukin-4 (IL-4) and IL-5.

Transcription factors of the NFAT (nuclear factor of activated T cells) family regulate the expression of many genes encoding immunoregulatory cytokines and cell surface proteins during the immune response. The NFAT protein NFAT1 (NFATp) is expressed and functional in T cells, B cells, mast cells, and natural killer cells. Here we report a detailed analysis of the enhanced eosinophil responses of NFAT1-deficient mice, observed in an in vivo model of allergic inflammation. In addition to the pleural eosinophilia described previously, NFAT1-/- mice that have been sensitized with antigen display a significant increase, relative to wild-type mice, in the numbers of eosinophils in bone marrow and peripheral blood. After restimulation with antigen in vitro, antigen-responsive T cells from the draining lymph nodes of NFAT1-/- mice show increased expression of mRNA encoding the Th2 cytokines interleukin-4 (IL-4), IL-5, and IL-13. Consistent with this finding, there is a pronounced increase in the levels of IL-5 and IL-13 in the pleural cavities of sensitized NFAT1-/- mice after allergen challenge in vivo. Furthermore, development of eosinophilia depends on overexpression of IL-4 and IL-5, because it is strongly inhibited by administration of neutralizing antibodies to either of these cytokines. These results indicate that NFAT1-deficient mice are prone to develop a classically allergic phenotype characterized by eosinophilia and increased production of Th2 cytokines. Thus, the presence of NFAT1 might inhibit the allergic response, perhaps by interfering with the development of Th2 immune responses, and the lack or dysfunction of NFAT1 could potentially underlie certain cases of atopic disease.

Role of the cyclosporin-sensitive transcription factor NFAT1 in the allergic response.

Proteins belonging to the NFAT (nuclear factor of activated T cells) family of transcription factors are expressed in most immune cell types, and play a central role in the transcription of cytokine genes, such as IL-2, IL-4, IL-5, IL-13, IFN-gamma, TNF-alpha, and GM-CSF. The activity NFAT proteins is regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a target for inhibition by CsA and FK506. Recently, two different groups have described that mice lacking the NFAT1 transcription factor show an enhanced immune response, with tendency towards the development of a late Th2-like response. This review evaluates the possible role of NFAT proteins in the Th2 immune response and in the eosinophil-mediated allergic response.

Down-regulation of IL-4 gene transcription and control of Th2 cell differentiation by a mechanism involving NFAT1.

Transcription factors of the NFAT family play a critical role in the immune response by activating the expression of cytokines and other inducible genes in antigen-stimulated cells. Here we show that a member of this family, NFAT1, is involved in down-regulating the late phase of IL-4 gene transcription, thus inhibiting T helper 2 responses. Whereas stimulated T cells from wild-type mice show a transient increase and then a rapid decline in the steady-state levels of IL-4 mRNA in vitro, the levels of IL-4 gene transcripts in NFAT1-deficient T cells are maintained at high levels under the same conditions. Consistent with this observation, NFAT1-/- mice are more susceptible to infection with Leishmania major. This report provides evidence that NFAT proteins regulate not only the initiation but also the termination of gene transcription.

An enhanced immune response in mice lacking the transcription factor NFAT1.

Transcription factors of the NFAT family are thought to play a major role in regulating the expression of cytokine genes and other inducible genes during the immune response. The role of NFAT1 was investigated by targeted disruption of the NFAT1 gene. Unexpectedly, cells from NFAT1 -/- mice showed increased primary responses to Leishmania major and mounted increased secondary responses to ovalbumin in vitro. In an in vivo model of allergic inflammation, the accumulation of eosinophils and levels of serum immunoglobulin E were increased in NFAT1 -/- mice. These results suggest that NFAT1 exerts a negative regulatory influence on the immune response.

Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity.

NFAT1 (previously termed NFATp) is a cytoplasmic transcription factor involved in the induction of cytokine genes. We have previously shown that the dephosphorylation of NFAT1, accompanied by its nuclear translocation and increased DNA binding activity, is regulated by calcium- and calcineurin-dependent mechanisms, as each of these hallmarks of NFAT1 activation is elicited by ionomycin and blocked by the immunosuppressive drugs cyclosporin A and FK506 (Shaw, K.T.-Y., Ho, A.M., Raghavan, A., Kim, J., Jain, J., Park, J., Sharma, S., Rao, A., and Hogan, P.G. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11205-11209). Here we show that the activation state of NFAT1 in T cells is remarkably sensitive to the level of calcineurin activity. Addition of cyclosporin A, even in the presence of ongoing ionomycin stimulation, results in rephosphorylation of NFAT1, its reappearance in the cytoplasm, and a return of its DNA binding activity to low levels. Similar effects are observed upon removal of ionomycin or addition of EGTA. We also demonstrate a direct interaction between calcineurin and NFAT1 that is consistent with a direct enzyme-substrate relation between these two proteins and that may underlie the sensitivity of NFAT1 activation to the level of calcineurin activity. The NFAT1-calcineurin interaction, which involves an N-terminal region of NFAT1 conserved in other NFAT family proteins, may provide a target for the design of novel immunosuppressive drugs.

Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of a Leishmania mdr1 gene.

A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

Enteric parasites and HIV infection: occurrence in AIDS patients in Rio de Janeiro, Brazil

The occurrence of intestinal parasites, its relation with the transmission mechanism of HIV, and the clinical state of the AIDS patients, were analyzed in 99 Group IV patients (CDC, 1986), treated at "Hospital Universitário Pedro Ernesto" (HUPE), between 1986 and 1988. The group consisted of 79 (79.8%) patients whose HIV transmission mechanism took place through sexual contact and of 16 (20.2%) who were infected through blood. Feces samples from each patient were examined by four distincts methods (Faust et al, Kato-Katz, Baermann-Moraes and Baxby et al.). The moste occuring parasites were: Cryptosporidium sp., Entamoeba coli and Endolimax nana (18.2%), Strongyloides stercoralis and Giardia lambia (15.2%). E. histolytica and/or E. hartmanni (13.1%), Ascaris lumbricoides (11.1%) and Isospora belli (10.1%). Furthermore, 74.7% of the patients carried at least one species. Intestinal parasites were found in 78.5% of the patients who acquired the HIV through sexual intercourse and in 56,3% of those infected by blood contamination. The difference, was not statistically significant (p > 0.05). In the group under study, the increase of the occurrence of parasitc infections does not seem to depend on the acquisiton of HIV through sexual contact. It appears that in developing countries, the dependancy is more related to the classic mechanisms of parasites transmission and its endemicity

Enteric parasites and HIV infection: occurrence in AIDS patients in Rio de Janeiro, Brazil.

The occurrence of intestinal parasites, its relation with the transmission mechanism of HIV, and the clinical state of the AIDS patients, were analyzed in 99 Group IV patients (CDC, 1986), treated at "Hospital Universitário Pedro Ernesto" (HUPE), between 1986 and 1988. The group consisted of 79 (79.8%) patients whose HIV transmission mechanism took place through sexual contact and of 16 (20.2%) who were infected through blood. Feces samples from each patient were examined by four distincts methods (Faust et al., Kato-Katz, Baermann-Moraes and Baxby et al.). The most occurring parasites were: Cryptosporidium sp., Entamoeba coli and Endolimax nana (18.2%), Strongyloides stercoralis and Giardia lamblia (15.2%), E. histolytica and/or E. hartmanni (13.1%), Ascaris lumbricoides (11.1%) and Isospora belli (10.1%). Furthermore, 74.7% of the patients carried at least one species. Intestinal parasites were found in 78.5% of the patients who acquired the HIV through sexual intercourse and in 56.3% of those infected by blood contamination. The difference, was not statistically significant (p greater than 0.05). In the group under study, the increase of the occurrence of parasitic infections does not seem to depend on the acquisition of HIV through sexual contact. It appears that in developing countries, the dependency is more related to the classic mechanisms of parasites transmission and its endemicity.