Search

Objective: The aim of this study was to study the effect of ω-3 supplementation on the nutritional status and the immune and inflammatory profiles of patients with gastric cancer during antineoplastic pretreatment., Methods: This was a randomized, open, controlled longitudinal study with intervention in outpatient patients with gastric cancer. Sixty-eight patients were randomized into two groups and received either a formula enriched with ω-3 (intervention group [IG]) or standard formula without ω-3 (control group) for 30 d consecutively. Nutritional status (based on patient-generated subjective global assessment, bioimpedance, and anthropometric measurements) and immune and inflammatory parameters were collected before and after supplementation. Results were expressed as frequency, median, and interquartile intervals and were compared by non-parametric test. P < 0.05 was considered statistically significant., Results: Thirty-four patients were included in each group. Of the patients, 64.7% were men, 44.1% were older than 60 years, and 45.6% had stage III disease. There was an increase in C-reactive protein in the control group before and after supplementation, in addition to the worsening in some anthropometric parameters, such as arm muscle area and arm muscle circumference. There was maintenance of the immune profile in both groups. An increase in weight gain was observed in the IG but not in the control group (1.2 [0.9-9] versus 0.7 kg [0.4-1.3]; P = 0.03), as was a reduction of interleukin-6 (5.7 [4.1-6.4] versus 6.3 pg/mL [5.6-8.6]; P = 0.03) and a maintenance of nutritional status, after supplementation., Conclusions: Supplementation with ω-3 leads to weight gain, reduction in the inflammatory profile, and maintenance of the nutritional and immune profiles of these patients, but further studies are needed to examine changes in body composition., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Exposure to ionizing radiation greatly increases the risk of developing papillary thyroid carcinoma (PTC), especially during childhood, mainly due to gradual inactivation of DNA repair genes and DNA damages. Recent molecular characterization of PTC revealed DNA methylation deregulation of several promoters of DNA repair genes. Thus, epigenetic silencing might be a plausible mechanism for the activity loss of tumor suppressor genes in radiation-induced thyroid tumors. Herein, we investigated the impact of ionizing radiation on global methylation and CpG islands within promoter regions of homologous recombination (HR) and non-homologous end joining (NHEJ) genes, as well as its effects on gene expression, using two well-established normal differentiated thyroid cell lines (FRTL5 and PCCL3). Our data reveal that X-ray exposure promoted G 2 /M arrest in normal thyroid cell lines. The FRTL5 cells displayed a slower kinetics of double-strand breaks (DSB) repair and a lower long interspersed nuclear element-1 (LINE-1) methylation than the PCCL3 cells. Nevertheless, acute X-ray exposure does not alter the expression of genes involved in HR and NHEJ pathways, apart from the downregulation of Brca1 in thyroid cells. On the other hand, HR and NHEJ gene expressions were upregulated in radiation-induced senescent thyroid cells. Taken together, these data suggest that FRTL5 cells intrinsically have less efficient DNA DSB repair machinery than PCCL3 cells, as well as genomic instability, which could predispose the FRTL5 cells to unrepaired DSB lesions and, therefore, gene mutations.

This study evaluated the effect of an immune-modulatory diet on patients with gastric cancer and identified the parameters associated with postoperative outcomes. This was a single-arm prospective intervention study. At baseline, patients were assessed for nutrition (Patient-Generated Subjective Global Assessment), inflammatory markers (albumin, C-reactive protein, and interleukin 6 [IL-6]), and immune markers (percentage NK, CD4, CD8, and CD4:CD8 ratio); they also received nutrition counseling and high-calorie/protein supplement. A week before surgery, they were assessed for nutrition and inflammatory/immune markers and started on an immune-modulatory supplement until the day before surgery, when they were evaluated again. On the second postoperative day, patients were assessed for inflammatory/immune parameters, and a final nutrition evaluation was performed until the day of discharge. Complications were recorded daily and up to 30 days after discharge. Thirty-seven patients (60 ± 10 years old) were included, and 57% were classified as malnourished. Maintenance of nutrition and immune parameters occurred throughout the study period, but we found a preoperative increase in C-reactive protein (0.1-1.5 mg/dL) and IL-6 (2.0-14.2 pg/mL) and a postoperative increase in the CD4:CD8 ratio (2.3 ± 1.0). Complications and death were seen in 35%, especially patients with higher preoperative IL-6 (2.2-46 pg/mL), lower CD4:CD8 ratio (1.7 ± 0.5), and lower protein (1.2 ± 0.5 g/kg/d) and calorie intake (1552 ± 584 kcal/kg/d). The high-calorie/protein supplementation with the immune-modulating diet was able to maintain the nutrition and immune status of patients with gastric cancer.

CD4 T cell activation and differentiation mechanisms constitute a complex and intricate signaling network involving several regulatory proteins. IRF2BP2 is a transcriptional repressor that is involved in gene-expression regulation in very diverse biologic contexts. Information regarding the IRF2BP2 regulatory function in CD4 T lymphocytes is very limited and suggests a role for this protein in repressing the expression of different cytokine genes. Here, we showed that Irf2bp2 gene expression was decreased in CD4 T cells upon activation. To investigate the possible regulatory roles for IRF2BP2 in CD4 T cell functions, this protein was ectopically expressed in murine primary-activated CD4 T lymphocytes through retroviral transduction. Interestingly, ectopic expression of IRF2BP2 led to a reduction in CD25 expression and STAT5 phosphorylation, along with an impaired proliferative capacity. The CD69 expression was also diminished in IRF2BP2-overexpressing cells, whereas CD44 and CD62L levels were not altered. In vivo, transferred, IRF2BP2-overexpressing, transduced cells displayed an impaired expansion capacity compared with controls. Furthermore, overexpression of IRF2BP2 in differentiated Th cells resulted in slightly reduced IL-4 and pro-TGF-β production in Th2 and iT regs but had no effect on IFN-γ or IL-17 expression in Th1 and Th17 cells, respectively. Taken together, our data suggest a role for IRF2BP2 in regulating CD4 T cell activation by repressing proliferation and the expression of CD25 and CD69 induced by TCR stimuli., (© Society for Leukocyte Biology.)

Nuclear factor of activated T cells (NFAT) 2 null mutant mice die in utero of cardiac failure, precluding analysis of the role of NFAT2 in lymphocyte responses. Only the NFAT2 -/- /Rag-1 -/- chimeric mice model gave insight into the role of NFAT2 transcription factor in T lymphocyte development, activation, and differentiation. As reports are mainly focused on the role of NFAT2 in CD4 + T lymphocytes activation and differentiation, we decided to investigate NFAT2's impact on CD8 + T lymphocyte responses. We report that NFAT2 is phosphorylated and inactive in the cytoplasm of naive CD8 + T cells, and upon TCR stimulation, it is dephosphorylated and translocated into the nucleus. To study the role of NFAT2 in CD8 + T responses, we employed NFAT2 fl/fl CD4-Cre mice with NFAT2 deletion specifically in T cells. Interestingly, the absence of NFAT2 in T cells resulted in increased percentage of non-conventional innate-like CD8 + T cells. These cells were CD122 + , rapid producer of interferon gamma (IFN-γ) and had characteristics of conventional memory CD8 + T cells. We also observed an expansion of PLZF + expressing CD3 + thymocyte population in the absence of NFAT2 and increased IL-4 production. Furthermore, we found that CD8 + T lymphocytes deficient in NFAT2 had reduced activation, proliferation, and IFN-γ and IL-2 production at suboptimal TCR strength. NFAT2 absence did not significantly influence differentiation of CD8 + T cells into cytotoxic effector cells but reduced their IFN-γ production. This work documents NFAT2 as a negative regulator of innate-like CD8 + T cells development. NFAT2 is required for complete CD8 + T cell responses at suboptimal TCR stimulation and regulates IFN-γ production by cytotoxic CD8 + T cells in vitro .

The NFAT family of transcription factors has been primarily related to T cell development, activation, and differentiation. Further studies have shown that these ubiquitous proteins are observed in many cell types inside and outside the immune system, and are involved in several biological processes, including tumor growth, angiogenesis, and invasiveness. However, the specific role of the NFAT1 family member in naive B cell proliferation remains elusive. Here, we demonstrate that NFAT1 transcription factor controls Cyclin E expression, cell proliferation, and tumor growth in vivo. Specifically, we show that inducible expression of NFAT1 inhibits cell cycle progression, reduces colony formation, and controls tumor growth in nude mice. We also demonstrate that NFAT1-deficient naive B lymphocytes show a hyperproliferative phenotype and high levels of Cyclin E1 and E2 upon BCR stimulation when compared to wild-type B lymphocytes. NFAT1 transcription factor directly regulates Cyclin E expression in B cells, inhibiting the G1/S cell cycle phase transition. Bioinformatics analysis indicates that low levels of NFAT1 correlate with high expression of Cyclin E1 in different human cancers, including Diffuse Large B-cell Lymphomas (DLBCL). Together, our results demonstrate a repressor role for NFAT1 in cell cycle progression and Cyclin E expression in B lymphocytes, and suggest a potential function for NFAT1 protein in B cell malignancies.

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.

Lithium is a well-established non-competitive inhibitor of glycogen synthase kinase-3β (GSK-3β), a kinase that is involved in several cellular processes related to cancer progression. GSK-3β is regulated upstream by PI3K/Akt, which is negatively modulated by PTEN. The role that lithium plays in cancer is controversial because lithium can activate or inhibit survival signaling pathways depending on the cell type. In this study, we analyzed the mechanisms by which lithium can modulate events related to colorectal cancer (CRC) progression and evaluated the role that survival signaling pathways such as PI3K/Akt and PTEN play in this context. We show that the administration of lithium decreased the proliferative potential of CRC cells in a GSK-3β-independent manner but induced the accumulation of cells in G2/M phase. Furthermore, high doses of lithium increased apoptosis, which was accompanied by decreased proteins levels of Akt and PTEN. Then, cells that were induced to overexpress PTEN were treated with lithium; we observed that low doses of lithium strongly increased apoptosis. Additionally, PTEN overexpression reduced proliferation, but this effect was minor compared with that in cells treated with lithium alone. Furthermore, we demonstrated that PTEN overexpression and lithium treatment separately reduced cell migration, colony formation, and invasion, and these effects were enhanced when lithium treatment and PTEN overexpression were combined. In conclusion, our findings indicate that PTEN overexpression and lithium treatment cooperate to reduce the malignancy of CRC cells and highlight lithium and PTEN as potential candidates for studies to identify new therapeutic approaches for CRC treatment., (© 2015 Wiley Periodicals, Inc.)

The NFAT (nuclear factor of activated T cells) family of transcription factors is composed of four calcium-responsive proteins (NFAT1 to -4). The NFAT2 (also called NFATc1) gene encodes the isoforms NFAT2α and NFAT2β that result mainly from alternative initiation exons that provide two different N-terminal transactivation domains. However, the specific roles of the NFAT2 isoforms in cell physiology remain unclear. Because previous studies have shown oncogenic potential for NFAT2, this study emphasized the role of the NFAT2 isoforms in cell transformation. Here, we show that a constitutively active form of NFAT2α (CA-NFAT2α) and CA-NFAT2β distinctly control death and transformation in NIH 3T3 cells. While CA-NFAT2α strongly induces cell transformation, CA-NFAT2β leads to reduced cell proliferation and intense cell death through the upregulation of tumor necrosis factor alpha (TNF-α). CA-NFAT2β also increases cell death and upregulates Fas ligand (FasL) and TNF-α in CD4(+) T cells. Furthermore, we demonstrate that differential roles of NFAT2 isoforms in NIH 3T3 cells depend on the N-terminal domain, where the NFAT2β-specific N-terminal acidic motif is necessary to induce cell death. Interestingly, the NFAT2α isoform is upregulated in Burkitt lymphomas, suggesting an isoform-specific involvement of NFAT2 in cancer development. Finally, our data suggest that alternative N-terminal domains of NFAT2 could provide differential mechanisms for the control of cellular functions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Multidrug resistance (MDR) is considered a multifactorial event that favors cancer cells becoming resistant to several chemotherapeutic agents. Numerous mechanisms contribute to MDR, such as P-glycoprotein (Pgp/ABCB1) activity that promotes drug efflux, overexpression of inhibitors of apoptosis proteins (IAP) that contribute to evasion of apoptosis, and oncogenic pathway activation that favors cancer cell survival. MDR molecules have been identified in membrane microparticles (MP) and can be transferred to sensitive cancer cells. By co-culturing MP derived from MDR-positive cells with recipient cells, we showed that sensitive cells accumulated Pgp, IAP proteins and mRNA. In addition, MP promoted microRNA transfer and NFκB and Yb-1 activation. Therefore, our results indicate that MP can induce a multifactorial phenotype in sensitive cancer cells., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.

Interferon- (IFN-) γ is an essential cytokine for immunity against intracellular pathogens and cancer. IFN-γ expression by CD4 T lymphocytes is observed only after T helper (Th) 1 differentiation and there are several studies about the molecular mechanisms that control Ifng expression in these cells. However, naïve CD8 T lymphocytes do not produce large amounts of IFN-γ, but after TCR stimulation there is a progressive acquisition of IFN-γ expression during differentiation into cytotoxic T lymphocytes (CTL) and memory cells, which are capable of producing high levels of this cytokine. Differential gene expression can be regulated from the selective action of transcriptional factors and also from epigenetic mechanisms, such as DNA CpG methylation or posttranslational histone modifications. Recently it has been recognized that epigenetic modification is an integral part of CD8 lymphocyte differentiation. This review will focus on the chromatin status of Ifng promoter in CD8 T cells and possible influences of epigenetic modifications in Ifng gene and conserved noncoding sequences (CNSs) in regulation of IFN-γ production by CD8 T lymphocytes.

The NFAT family of transcription factors plays a central role in the regulation of cytokine gene expression during the immune response. NFAT functions have been extensively explored in lymphocyte activation and differentiation, but the involvement of NFAT proteins in dendritic cells (DCs) is still not well known. Here, we investigated the role of the NFAT1 transcription factor in murine DCs. Initially, we demonstrated by western blot that the NFAT1 protein is present in splenic DCs and is rapidly activated upon calcium influx. We then used NFAT1-deficient mice (NFAT1-/-) to investigate whether NFAT1 influences the ability of DCs to induce Th differentiation. Our data demonstrated that NFAT1-/- DCs showed an increased capacity to differentiate CD4 T cells to the Th1 phenotype. CD4 cells that were primed in vitro with NFAT1-/- DCs had increased IFN-γ production. The same results were observed when the CD4 cells were primed in vivo through the sensitization of NFAT1-/- mice with ovalbumin. Furthermore, our results demonstrated that the cytokine IL-12 is one of the factors involved in this process because its production is increased in NFAT1-/- mice, and neutralizing anti-IL-12 antibodies almost completely eliminated the IFN-γ production. These results demonstrated that the NFAT1 transcription factor regulates specific functions in DCs that are involved in CD4 differentiation, suggesting that the inhibition of NFAT1 in DCs may be used as a therapy to modulate specific immune responses., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The altered expressions of claudin proteins have been reported during the tumorigenesis of colorectal cancer. However, the molecular mechanisms that regulate these events in this cancer type are poorly understood. Here, we report that epidermal growth factor (EGF) increases the expression of claudin-3 in human colorectal adenocarcinoma HT-29 cells. This increase was related to increased cell migration and the formation of anchorage-dependent and anchorage-independent colonies. We further showed that the ERK1/2 and PI3K-Akt pathways were involved in the regulation of these effects because specific pharmacological inhibition blocked these events. Genetic manipulation of claudin-1 and claudin-3 in HT-29 cells showed that the overexpression of claudin-1 resulted in decreased cell migration; however, migration was not altered in cells that overexpressed claudin-3. Furthermore, the overexpression of claudin-3, but not that of claudin-1, increased the tight junction-related paracellular flux of macromolecules. Additionally, an increased formation of anchorage-dependent and anchorage-independent colonies were observed in cells that overexpressed claudin-3, while no such changes were observed when claudin-1 was overexpressed. Finally, claudin-3 silencing alone despite induce increase proliferation, and the formation of anchoragedependent and -independent colonies, it was able to prevent the EGF-induced increased malignant potential. In conclusion, our results show a novel role for claudin-3 overexpression in promoting the malignant potential of colorectal cancer cells, which is potentially regulated by the EGF-activated ERK1/2 and PI3K-Akt pathways.

Breast cancer is the leading cause of deaths in women around the world. Resistance to therapy is the main cause of treatment failure and still little is known about predictive biomarkers for response to systemic therapy. Increasing evidence show that Survivin and XIAP overexpression is closely associated with chemoresistance and poor prognosis in breast cancer. However, their impact on resistance to doxorubicin (dox), a chemotherapeutic agent widely used to treat breast cancer, is poorly understood. Here, we demonstrated that dox inhibited cell viability and induced DNA fragmentation and activation of caspases-3, -7 and -9 in the breast cancer-derived cell lines MCF7 and MDA-MB-231, regardless of different p53 status. Dox exposure resulted in reduction of Survivin and XIAP mRNA and protein levels. However, when we transfected cells with a Survivin-encoding plasmid, we did not observe a cell death-resistant phenotype. XIAP and Survivin silencing, either alone or in combination, had no effect on breast cancer cells sensitivity towards dox. Altogether, we demonstrated that breast cancer cells are sensitive to the chemotherapeutic agent dox irrespective of Survivin and XIAP expression levels. Also, our findings suggest that dox-mediated modulation of Survivin and XIAP might sensitize cells to taxanes when used in a sequential regimen., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Nuclear factor of activated T cells (NFAT) was described as an activation and differentiation factor in T cells. NFAT1 protein is expressed in several cell types and has been implicated in the control of the cell cycle, death and migration. Overexpression or activation of NFAT1 has been demonstrated to induce cell death in different cell types, such as T lymphocytes, Burkitt's lymphoma, and fibroblasts. Although these findings indicate a role for NFAT1 transcription factor in control of cell death, the precise mechanisms involved in this process regulated by NFAT1 are still poorly understood. The Ras/Raf/MEK/ERK pathway is activated by many growth factors and cytokines that are important in driving proliferation and preventing apoptosis and is widely implicated in cell transformation and cancer development. We show that NFAT1 protein can cooperate with Ras/Raf/MEK/ERK, but not with the JNK, p38 or NFκB pathways in cell death induction. NFAT1 can induce a cell death pathway consistent with apoptosis, which can be shifted to programmed necrosis by caspase inhibitors. Finally, through screening genes involved in cell death regulation, although we determined that TNF-α, TRAIL and PAK7 genes were up-regulated, only TNF-α expression was responsible for cell death in this context. These data suggest that NFAT1 protein activation can shift oncogenic Ras/Raf/MEK/ERK signaling to acting as a tumor suppressor pathway. These data support a potential role for regulating NFAT1 expression in gene therapy in tumors that display an activated Ras pathway, which could lead to more specific, target-directed TNF-α expression and, thus, tumor suppression., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chronic inflammation is a risk factor for the development of colon cancer, providing genotoxic insults, growth and pro-angiogenic factors that can promote tumorigenesis and tumor growth. Immunomodulatory agents can interfere with the inflammation that feeds cancer, but their impact on the transformed cell is poorly understood. The calcium/calcineurin signaling pathway, through activation of NFAT, is essential for effective immune responses, and its inhibitors cyclosporin A (CsA) and FK506 are used in the clinics to suppress immunity. Moreover, the kinases GSK3β and mTOR, modulated by PI-3K/Akt, can inhibit NFAT activity, suggesting a cross-talk between the calcium and growth factor signaling pathways. Both NFAT and mTOR activity have been associated with tumorigenesis. We therefore investigated the impact of calcineurin and PI-3K/mTOR inhibition in growth of human colon carcinoma cells. We show that despite the efficient inhibition of NFAT1 activity, FK506 promotes tumor growth, whereas CsA inhibits it due to a delay in cell cycle progression and induction of necroptosis. We found NFκB activation and mTORC1 activity not to be altered by CsA or FK506. Similarly, changes to mitochondrial homeostasis were equivalent upon treatment with these drugs. We further show that, in our model, NFAT1 activation is not modulated by PI3K/mTOR. We conclude that CsA slows cell cycle progression and induces necroptosis of human carcinoma cell lines in a TGFβ-, NFAT-, NFκB- and PI3K/mTOR-independent fashion. Nevertheless, our data suggest that CsA, in addition to its anti-inflammatory capacity, may target transformed colon and esophagus carcinoma cells without affecting non-transformed cells, promoting beneficial tumoristatic effects.

A number of physiological processes in both normal and cancer cells are regulated by the proto-oncogene c-Myc. Among them, processes such as cell cycle regulation, apoptosis, angiogenesis and metastasis are also controlled by the nuclear factor of activated T cells (NFAT) family of transcription factors. It is already known that NFAT upregulates c-Myc expression by binding to an element located in the minimal c-Myc promoter. However, the importance of other NFAT sites in the context of the full promoter has not been evaluated. In this work, we demonstrate that the regulation of c-Myc by NFAT1 is more complex than previously conceived. In addition to the proximal site, NFAT1 directly binds to distal sites in the c-Myc promoter with different affinities. Promoter deletions and site-directed mutagenesis of NFAT binding sites in HEK293T cells suggest that in NFAT1-mediated transactivation, some NFAT elements are negative and dominant and others are positive and recessive. Furthermore, we demonstrate that cooperation with partner proteins, such as p300, enhances NFAT1-mediated transactivation of the c-Myc promoter. At last, the newly identified sites are also responsive to NFAT2 in HEK293T cells. However, in NIH3T3 cells, the regulation mediated by NFAT proteins is not dependent on the known NFAT sites, including the site previously described. Thus, our data suggest that the contribution of NFAT to the regulation of c-Myc expression may depend on a balance between the binding to positive and negative NFAT-responsive elements and cooperation with transcriptional cofactors, which may differ according to the context and/or cell type.

The proteins belonging to the nuclear factor of activated T cells (NFAT) family of transcription factors are expressed in several cell types and regulate genes involved in differentiation, cell cycle and apoptosis. NFAT proteins share two conserved domains, the NFAT-homology region (NHR) and a DNA-binding domain (DBD). The N- and C-termini display two transactivation domains (TAD-N and TAD-C) that have low sequence similarity. Due to the high sequence conservation in the NHR and DBD, NFAT members have some overlapping roles in gene regulation. However, several studies have shown distinct roles for NFAT proteins in the regulation of cell death. The TAD-C shows low sequence similarity among NFAT family members, but its contribution to specific NFAT1-induced phenotypes is poorly understood. Here, we described at least two regions of NFAT1 TAD-C that confer pro-apoptotic activity to NFAT1. These regions extend from amino acids 699 to 734 and 819 to 850 of NFAT1. We also showed that the NFAT1 TAD-C is unable to induce apoptosis by itself and requires a functional DBD. Furthermore, we showed that when fused to NFAT1 TAD-C, NFAT2, which is associated with cell transformation, induces apoptosis in fibroblasts. Together, these results suggest that the NFAT1 TAD-C includes NFAT death domains that confer to different NFAT members the ability to induce apoptosis.

The nuclear factor of activated T cells (NFAT) family of transcription factors is expressed in a wide range of cell types and regulates genes involved in cell cycle, differentiation, and apoptosis. NFAT proteins share two well-conserved regions, the regulatory domain and the DNA binding domain. The N- and C-terminal ends are transactivation sites and show less sequence similarity, whereas their molecular functions remain poorly understood. Here, we identified a transcriptional repressor, interferon regulatory factor 2 binding protein 2 (IRF-2BP2), which specifically interacts with the C-terminal domain of NFAT1 among the NFAT family members. IRF-2BP2 was described as a corepressor by inhibiting both enhancer-activated and basal transcription. Gene reporter assays demonstrated that IRF-2BP2 represses the NFAT1-dependent transactivation of NFAT-responsive promoters. The ectopic expression of IRF-2BP2 in CD4 T cells resulted in decreased interleukin-2 (IL-2) and IL-4 production, supporting a repressive function of IRF-2BP2 for NFAT target genes. Furthermore, NFAT1 and IRF-2BP2 colocalized in the nucleus in activated cells, and the mutation of a newly identified nuclear localization signal in the IRF-2BP2 rendered it cytoplasmic, abolishing its repressive effect on NFAT1 activity. Collectively, our data demonstrate that IRF-2BP2 is a negative regulator of the NFAT1 transcription factor and suggest that NFAT1 repression occurs at the transcriptional level.

Lithium is a specific inhibitor of GSK3-β, and hence, an activator of the Wnt/β-catenin pathway, whereas the epidermal growth factor (EGF) has been linked to malignant transformation in epithelial cancer cells. Both pathways are aberrantly activated in most colorectal cancers (CRCs). However, the relationship between them in modulating events related to the progression of this cancer type remains to be defined. In this study, we investigated whether the Wnt/β-catenin and EGFR pathways converge to modulate the malignant potential of CRC. We used Caco-2 cells, a well-established model used to study CRC, and treatments with lithium chloride, as a modulator of the Wnt/β-catenin pathway, and with EGF as an inducer of EGFR signaling. We found that both agents altered the subcellular distribution of claudin-1 and β-catenin, two important proteins of the apical junctional complex, but not their abundance in the cell. Nuclear stabilization of β-catenin, a marker of Wnt pathway activation, was observed after treatment with both compounds. However, lithium, but not EGF, inhibited GSK3-β, indicating that these agents modulate this enzyme in a differential fashion. Furthermore, EGF treatment increased the proliferative and migratory capacity but did not alter the colony formation potential of these cells. Surprisingly, lithium, known to activate the Wnt/β-catenin pathway, inhibited the increased proliferation by arresting cells in the G2/M phase as well as the cell migration promoted by EGF, as demonstrated by the combined treatment with these agents. Lithium treatment alone reduced the cell colony formation. Thus, our findings suggest that lithium plays an important role in regulating cellular events related to tumor progression in CRC.

Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-β in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.

Ovarian carcinoma is one of the most aggressive gynecological diseases and generally diagnosed at advanced stages. Osteopontin (OPN) is one of the proteins overexpressed in ovarian cancer and is involved in tumorigenesis and metastasis. Alternative splicing of OPN leads to 3 isoforms, OPNa, OPNb, and OPNc. However, the expression pattern and the roles of each of these isoforms have not been previously characterized in ovarian cancer. Herein, we have evaluated the expression profiling of OPN isoforms in ovarian tumor and nontumor samples and their putative roles in ovarian cancer biology using in vitro and in vivo functional assays. OPNa and OPNb were expressed both in tumor and nontumor ovarian samples, whereas OPNc was specifically expressed in ovarian tumor samples. The isoform OPNc significantly activated OvCar-3 cell proliferation, migration, invasion, anchorage-independent growth and tumor formation in vivo. Additionally, we have also shown that some of the OPNc-dependent protumorigenic roles are mediated by PI3K/Akt signaling pathway. OPNc stimulated immortalized ovarian epithelial IOSE cell proliferation, indicating a role for this isoform in ovarian cancer tumorigenesis. Functional assays using OPNc conditioned medium and an anti-OPNc antibody have shown that most cellular effects observed herein were promoted by the secreted OPNc. According to our data, OPNc-specific expression in ovarian tumor samples and its role on favoring different aspects of ovarian cancer progression suggest that secreted OPNc contributes to the physiopathology of ovarian cancer progression and tumorigenesis. Altogether, the data open possibilities of new therapeutic approaches for ovarian cancer that selectively down regulate OPNc, altering its properties favoring ovarian tumor progression.

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., (© 2011 Blackwell Publishing Ltd.)

Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4(+) T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation.

Accumulation of lipid droplets (also known as lipid bodies or adiposomes) within leukocytes, epithelial cells, hepatocytes and other non-adipocytic cells is a frequently observed phenotype in infectious, neoplastic and other inflammatory conditions. Lipid droplet biogenesis is a regulated cellular process that culminates in the compartmentalization of lipids and of an array of enzymes, protein kinases and other proteins, suggesting that lipid droplets are inducible organelles with roles in cell signaling, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Enzymes involved in eicosanoid synthesis are localized at lipid droplets and lipid droplets are sites for eicosanoid generation in cells during inflammation and cancer. In this review, we discuss the current evidence related to the biogenesis and function of lipid droplets in cell metabolism and signaling in inflammation and cancer. Moreover, the potential of lipid droplets as markers of disease and targets for novel anti-inflammatory and antineoplastic therapies will be discussed., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Allergic asthma is a chronic inflammatory disease of the lung whose incidence and morbidity continues to rise in developed nations. Despite being a hallmark of asthma, the molecular mechanisms that determine airway hyperresponsiveness (AHR) are not completely established. Transcription factors of the NFAT family are involved in the regulation of several asthma-related genes. It has been shown that the absence of NFAT1 leads to an increased pleural eosinophilic allergic response accompanied by an increased production of Th2 cytokines, suggesting a role for NFAT1 in the regulation of allergic diseases. Herein, we analyze NFAT1-/- mice to address the role of NFAT1 in a model of allergic airway inflammation and its influence in AHR. NFAT1-/- mice submitted to airway inflammation display a significant exacerbation of several features of the allergic disease, including lung inflammation, eosinophilia, and serum IgE levels, which were concomitant with elevated Th2 cytokine production. However, in spite of the increased allergic phenotype, NFAT1-/- mice failed to express AHR after methacholine aerosol. Refractoriness of NFAT1-/- mice to methacholine was confirmed in naïve mice, suggesting that this refractoriness occurs in an intrinsic way, independent of the lung inflammation. In addition, NFAT1-/- mice exhibit increased AHR in response to serotonin inhalation, suggesting a specific role for NFAT1 in the methacholine pathway of bronchoconstriction. Taken together, these data add support to the interpretation that NFAT1 acts as a counterregulatory mechanism to suppress allergic inflammation. Moreover, our findings suggest a novel role for NFAT1 protein in airway responsiveness mediated by the cholinergic pathway.

Nuclear factor of activated T cells (NFAT) was first described as an activation and differentiation transcription factor in lymphocytes. Several in vitro studies suggest that NFAT family members are redundant proteins. However, analysis of mice deficient for NFAT proteins suggested different roles for the NFAT family of transcription factors in the regulation of cell proliferation and apoptosis. NFAT may also regulate several cell cycle and survival factors influencing tumor growth and survival. Here, we demonstrate that two constitutively active forms of NFAT proteins (CA-NFAT1 and CA-NFAT2 short isoform) induce distinct phenotypes in NIH 3T3 cells. Whereas CA-NFAT1 expression induces cell cycle arrest and apoptosis in NIH 3T3 fibroblasts, CA-NFAT2 short isoform leads to increased proliferation capacity and induction of cell transformation. Furthermore, NFAT1-deficient mice showed an increased propensity for chemical carcinogen-induced tumor formation, and CA-NFAT1 expression subverted the transformation of NIH 3T3 cells induced by the H-rasV12 oncogene. The differential roles for NFAT1 are at least partially due to the protein C-terminal domain. These results suggest that the NFAT1 gene acts as a tumor suppressor gene and the NFAT2 short isoform acts gene as an oncogene, supporting different roles for the two transcription factors in tumor development.

Lipid bodies (lipid droplets) are emerging as dynamic organelles involved in lipid metabolism and inflammation. Increased lipid body numbers have been described in tumor cells; however, its functional significance in cancer has never been addressed. Here, we showed increased number of lipid bodies in tumor tissues from patients with adenocarcinoma of colon submitted to surgical resection when compared with an adjacent normal tissue. Accordingly, increased numbers of lipid bodies were observed in human colon adenocarcinoma cell lines and in a H-rasV12-transformed intestinal epithelial cell line (IEC-6 H-rasV12) compared with nontransformed IEC-6 cells. The functions of lipid bodies in eicosanoid synthesis in cancer cells were investigated. CACO-2 cells have increased expression of cyclooxygenase-2 (COX-2) when compared with IEC-6 cells. We showed by immunolocalization that, in addition to perinuclear stain, COX-2 and prostaglandin E (PGE) synthase present punctate cytoplasmic localizations that were concordant with adipose differentiation-related protein-labeled lipid bodies. The colocalization of COX-2 at lipid bodies was confirmed by immunoblot of subcellular fractionated cells. Direct localization of PGE(2) at its synthesis locale showed that lipid bodies are sources of eicosanoids in the transformed colon cancer cells. Treatment with either aspirin or the fatty acid synthase inhibitor C75 significantly reduced the number of lipid bodies and PGE(2) production in CACO-2 and in IEC-6 H-rasV12 cells with effects in cell proliferation. Together, our results showed that lipid bodies in colon cancer cells are dynamic and functional active organelles centrally involved in PGE(2) synthesis and may potentially have implications in the pathogenesis of adenocarcinoma of colon.

The NFAT (Nuclear Factor of Activated T cells) family of transcription factors plays a central role in the regulation of several genes related to the immune response. Recently, NFAT proteins have been implicated in the proliferation and differentiation of different cell types. Previous studies have shown that NFAT1-deficient mice display lymphocyte hyperproliferation, shortened cell cycle duration, and cyclin overexpression. Here, we demonstrate that cyclin A2 expression is upregulated in the absence of NFAT1 in lymphocytes. Ectopic expression of NFAT1 in CHO cells decreases cyclin A2 levels. Indeed, NFAT1 binds to a consensus binding site found at the mouse cyclin A2 promoter in vitro and in vivo. Luciferase reporter assays show that NFAT1 downregulates cyclin A2 expression by directly binding to the cyclin A2 promoter. Together, these results indicate that the NFAT1 transcription factor represses cyclin A2 expression in lymphocytes, and may act as a silencer of gene transcription during the cell cycle.

Macrophage migration inhibitory factor (MIF) is increased in asthmatic patients and plays a critical role in the pathogenesis of asthma. We show here that mice lacking MIF failed to develop airway hyper-responsiveness (AHR), tissue eosinophilia, and mucus metaplasia. Analysis of the bronchoalveolar fluids revealed a substantial reduction of IL-13, eotaxin and cysteinyl-leukotrienes. The lack of these cardinal features of asthma in MIF(-/-) mice occurs regardless of high concentrations of IL-4 in the lung and OVA-specific IgE in the serum. Antigen-specific lymphocyte proliferation and IL-13 production were similarly increased in the draining lymph nodes of OVA-immunized and challenged MIF(-/-) mice compared to WT, but were reduced in the spleen of MIF(-/-), thus indicating differential roles of MIF in these compartments. Stimulation of naive CD4(+) cells with anti-CD3 antibody demonstrated that MIF(-/-) cells produced increased amounts of IFN-gamma and IL-4 compared to WT CD4(+) cells. Finally, treatment of sensitized BALB/c mice with neutralizing anti-MIF antibody abrogated the development of ARH and airway inflammation without affecting the production of Th2 cytokines or IgE. The present study demonstrates that MIF is required for allergic inflammation, adding important elements to our knowledge of asthma pathogenesis and suggesting that neutralization of MIF might be of therapeutic value in asthma.

CD8+ T lymphocytes are excellent sources of IFN-gamma; however, the molecular mechanisms that dictate IFN-gamma expression upon TCR stimulation in these cells are not completely understood. In this study, we evaluated the involvement of NFAT1 in the regulation of IFN-gamma gene expression in murine CD8+ T cells and its relevance during Th differentiation. We show that CD8+, but not CD4+, T cells, represent the very first source of IFN-gamma upon primary T cell activation, and also that the IFN-gamma produced by naive CD8+ T cells may enhance CD4+ Th1 differentiation in vitro. TCR stimulation rapidly induced IFN-gamma expression in CD8+ T lymphocytes in a cyclosporin A-sensitive manner. Evaluation of CD8+ T cells showed that calcium influx alone was sufficient to activate NFAT1 protein, transactivate IFN-gamma gene promoter, and induce IFN-gamma production. In fact, NFAT1-deficient mice demonstrated highly impaired IFN-gamma production by naive CD8+ T lymphocytes, which were totally rescued after retroviral transduction with NFAT1-encoding vectors. Moreover, NFAT1-dependent IFN-gamma production by the CD8+ T cell compartment was crucial to control a Th2-related response in vivo, such as allergic inflammation. Consistently, CD8alpha- as well as IFN-gamma-deficient mice did not mount a Th1 immune response and also developed in vivo allergic inflammation. Our results clearly indicate that IFN-gamma production by CD8+ T cells is dependent of NFAT1 transcription factor and may be an essential regulator of Th immune responses in vivo.

Allergic diseases have been closely related to Th2 immune responses, which are characterized by high levels of interleukin (IL) IL-4, IL-5, IL-9 and IL-13. These cytokines orchestrate the recruitment and activation of different effector cells, such as eosinophils and mast cells. These cells along with Th2 cytokines are key players on the development of chronic allergic inflammatory disorders, usually characterized by airway hyperresponsiveness, reversible airway obstruction, and airway inflammation. Accumulating evidences have shown that altering cytokine-producing profile of Th2 cells by inducing Th1 responses may be protective against Th2-related diseases such as asthma and allergy. Interferon-gamma (IFN-gamma), the principal Th1 effector cytokine, has shown to be crucial for the resolution of allergic-related immunopathologies. In fact, reduced production of this cytokine has been correlated with severe asthma. In this review, we will discuss the role of IFN-gamma during the generation of immune responses and its influence on allergic inflammation models, emphasizing its biologic properties during the different aspects of allergic responses.

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.

Dendritic cells play a major role in the induction of both innate and acquired immune responses against pathogenic invaders. These cells are also able to sense endogenous activation signals liberated by injured tissues even in the absence of infection. In the present work, we demonstrate that kinins mobilize dendritic cells to produce IL-12 through activation of the B(2) bradykinin receptor subtype and that bradykinin-induced IL-12 responses are tightly regulated both by angiotensin-converting enzyme, a kinin-degrading peptidase, and by endogenous IL-10. Using a mouse model of allergic inflammation, we further show that addition of bradykinin to OVA during immunization results in decreased eosinophil infiltration on Ag challenge. The latter effect was demonstrated to be due to IL-12-driven skewing of Ag-specific T cell responses to a type 1 cytokine profile. Our data thus indicate that kinin peptides can serve as danger signals that trigger dendritic cells to produce IL-12 through activation of B(2) bradykinin receptors.

Upon antigen stimulation, lymphocytes enter in cell cycle and proliferate, and most of the activated T cells die by apoptosis. Many of the proteins that regulate lymphocyte activation are Under the control of transcription factors belonging to the NFAT family. As previously demonstrated, NFATC2-/- mice consistently showed a marked increase in lymphocyte proliferation. Here, we evaluate the role of NFATC2 in regulating lymphocyte proliferation and its involvement in the control of cell cycle progression during lymphocyte activation. NFATC2-/- lymphocytes, including CD4+ T cells and B cells, hyperproliferated upon stimulation when compared with NFATC2+/+ cells. Analysis of cell death demonstrated that NFATC2-/- lymphocytes displayed an increased rate of apoptosis after antigen stimulation in addition to the hyperproliferation. Cell cycle analysis after antigen stimulation showed that NFATC2-/- cultures contained more cycling cells when compared with NFATC2+/+ cultures, which is related to a shortening in time of cell division upon activation. Furthermore, hyperproliferation of NFATC2-/- lymphocytes is correlated to an overexpression of cyclins A2, B1, E, and F. Taken together, our results suggest that the NFATC2 transcription factor plays an important role in the control of cell cycle during lymphocyte activation and may act as an inhibitor of cell proliferation in normal cells.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.