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Cyclooxygenase (COX) is the key enzyme in prostanoid synthesis from arachidonic acid (AA). Two isoforms, named COX-1 and COX-2, are expressed in mammalian tissues. The expression of COX-2 isoform is induced by several stimuli including cytokines and mitogens, and this induction is inhibited by glucocorticoids (GCs). We have previously shown that the transcriptional induction of COX-2 occurs early after T cell receptor (TCR) triggering, suggesting functional implications of this enzyme in T cell activation. Here, we show that dexamethasone (Dex) inhibits nuclear factor of activated T cells (NFAT)-mediated COX-2 transcriptional induction upon T cell activation. This effect is dependent on the presence of the GC receptor (GR), but independent of a functional DNA binding domain, as the activation-deficient GRLS7 mutant was as effective as the wild-type GR in the repression of NFAT-dependent transcription. Dex treatment did not disturb NFAT dephosphorylation, but interfered with activation mediated by the N-terminal transactivation domain (TAD) of NFAT, thus pointing to a negative cross-talk between GR and NFAT at the nuclear level. These results unveil the ability of GCs to interfere with NFAT activation and the induction of pro-inflammatory genes such as COX-2, and explain some of their immunomodulatory properties in activated human T cells.

The Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex is considered the major receptor of the innate immune system to recognize lipopolysaccharides (LPSs). However, some atypical LPSs with different lipid A and core saccharide moiety structures and compositions than the well-studied enterobacterial LPSs can induce a TLR2-dependent response in innate immune cells. Ochrobactrum intermedium, an opportunistic pathogen, presents an atypical LPS. In this study, we found that O. intermedium LPS exhibits a weak inflammatory activity compared to Escherichia coli LPS and, more importantly, is a specific TLR4/TLR2 agonist, able to signal through both receptors. Molecular docking analysis of O. intermedium LPS predicts a favorable formation of a TLR2/TLR4/MD-2 heterodimer complex, which was experimentally confirmed by fluorescence resonance energy transfer (FRET) in cells. Interestingly, the core saccharide plays an important role in this interaction. This study reveals for the first time TLR4/TLR2 heterodimerization that is induced by atypical LPS and may help to escape from recognition by the innate immune system.

Background: COVID‐19 can generate a broad spectrum of severity and symptoms. Many studies analysed the determinants of severity but not among some types of symptoms. More importantly, very few studies analysed patients highly exposed to the virus that nonetheless remain uninfected. Methods: We analysed serum levels of ACE2, Angiotensin II and anti-Spike antibodies in 2 different cohorts at high risk of viral exposure, highly exposed but uninfected subjects, either high risk health care workers or persons cohabiting with infected close relatives and seropositive patients with symptoms. We tested the ability of the sera of these subjects to neutralize lentivirus pseudotyped with the Spike-protein. Results: We found that the serum levels of ACE2 are significantly higher in highly exposed but uninfected subjects. Moreover, sera from this seronegative persons can neutralize SARS-CoV-2 infection in cellular assays more strongly that sera from non-exposed negative controls eventhough they do not have anti-CoV-2 IgG antibodies suggesting that high levels of ACE2 in serum may somewhat protect against an active infection without generating a conventional antibody response. Finally, we show that among patients with symptoms, ACE2 levels were significantly higher in infected patients who developed cutaneous as compared with respiratory symptoms and ACE2 was also higher in those with milder symptoms. Conclusions: These findings suggest that soluble ACE2 could be used as a potential biomarker to predict SARS-CoV-2 infection risk and to discriminate COVID-19 disease subtypes., This research was funded by grants from “Ministerio de Ciencia e Innovación” (SAF2013-42850-R, SAF2016-75988-R and PID-2019104760RB-100; FEDER), “Comunidad de Madrid (S2017/BMD-3671. INFLAMUNE-CM; FEDER) to MF, Consejo Superior de Investigaciones Científica, CSIC (CSIC-COV19-108, SGL210235) to MF and UB, CRUE-Supera COVID, the European Development Regional Fund ‘‘A way to achieve Europe’’ (ERDF), Merck, Sharp & Dohme Investigator Studies Program (code MISP# IIS 60257), and Fondo Supera COVID-19 (2020-001) to SS-V. Institutional grants from “Fundación Ramón Areces” and “Banco de Santander”. This research work was also funded by the European Commission – NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global).

Colorectal cancer (CRC) is a very common life-threatening malignancy. Transcription factor-like 5 (TCFL5) has been suggested to be involved in CRC. Here, we describe the expression of four alternative transcripts of TCFL5 and their relevance in CRC. Complete deletion of all isoforms drastically decreased pro-tumoural properties such as spheroids formation and in vivo tumour growth, although increased migration in CRC cell lines. Overexpression of the two main isoforms, TCFL5_E8 and CHA, had opposite effects: TCFL5_E8 reduced proliferation and spheroids formation, while CHA increased them. TCFL5_E8 reduced in vivo tumour formation, while CHA had no effect. In addition, TCFL5_E8 and CHA have different roles in the regulation of the pluripotency-related genes SOX2 and KLF4. Both isoforms bind directly to their promoters; however, TCFL5_E8 induced SOX2 and reduced KLF4 mRNA levels, whereas CHA did the opposite. Together, our results show that TCFL5 plays an important role in the development of CRC, being however isoform-specific. This work also points to the need to analyse separately TCFL5 isoforms in cancer, due to their different and opposite functions, Ministerio de Ciencia e Innovación’ (SAF2016-75988-R and PID2019-104760RB-I00) ‘Comunidad de Madrid (S2017/BMD-3671. INFLAMUNE-CM), Fondo de Investigaciones Sanitarias’ (BIOIMID) to MF and Institutional grants from ‘Fundación Ramón Areces’ and ‘Banco de Santander

Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-04095-z.

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus‐2 (SARS‐CoV‐2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID‐19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer‐friendly non‐adherent Jurkat T cell line that stably expresses the full‐length native spike “S” protein of SARS‐CoV‐2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self‐cleaving sequence, allowing to accurately quantify the presence of anti‐S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double‐ staining with the test sera and anti‐EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti‐S antibodies with protective neutralizing activity are long‐lasting and can be detected in sera eight months after infection., This work was funded by intramural grant CSIC-COVID19-004: 202020E081 (to B.A.) and CSIC-COVID19-004: 202020E165 (to MF). L.H has been supported by an FPI fellowship from the Spanish Ministry of Science and Innovation. I.B. has been supported by an H2020-MSCA-ITN-2016 training network grant of the European Union (GA 721358).

The receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal Trypanosoma cruzi Y strain. To elucidate whether genetic diversity of the parasite was related with disease susceptibility, we further analyzed the role of SLAMF1 using 6 different Trypanosoma cruzi strains including Y. The interaction of SLAMF1 receptor with T. cruzi was evidenced by fluorescence microscopy, flow cytometry and quantitative PCR. All the strains, except VFRA, showed a decrease in parasite load in infected macrophages in Slamf1-/- compared to BALB/c. In macrophages gene expression NADPH oxidase (NOX2), and reactive oxygen species (ROS) production increased in Slamf1-/- compared to BALB/c in 5 out of 6 strains. However, Slamf1-/-macrophages infected with VFRA strain exhibited a divergent behavior, with higher parasite load, lower NOX2 expression and ROS production compared to BALB/c. Parasitological and immunological studies in vivo with Y strain showed that in the absence of SLAMF1 the immune response protected mice from the otherwise lethal Y infection favoring a proinflammatory response likely involving CD4, CD8, dendritic cells and classically activated macrophages. In the case of VFRA, no major changes were observed in the absence of SLAMF1. Thus, the results suggest that the T. cruzi affects SLAMF1-dependent ROS production, controlling parasite replication in macrophages and affecting survival in mice in a strain-dependent manner. Further studies will focus in the identification of parasite molecules involved in SLAMF1 interaction to explain the immunopathogenesis of the disease., Author summary Chagas disease, caused by Trypanosoma cruzi, is characterized by an acute phase, with low mortality, and after many years without any sign of disease, patients develop a symptomatic chronic phase, characterized by cardiomyopathy and/or digestive mega syndromes. These differences have been attributed to the high genetic variability of this parasite. We have shown that the receptor Signaling Lymphocyte-Activation Molecule Family 1 (SLAMF1) controls susceptibility to Infection by the lethal T. cruzi Y strain. Here we studied in detail the immunopathogenic role of SLAMF1 using 6 genetically diverse strains of T. cruzi using in vitro and in vivo approaches. Our results indicate an important role of SLAMF1 in T. cruzi infection which is parasite strain-dependent. We found that parasites interact with SLAMF1 in macrophages affecting NADPH oxidase (NOX2) expression and reactive oxygen species (ROS) production 5 out of 6 strains tested. Y and VFRA strains showed a divergent behavior in vitro and the role of SLAMF1 in the in vivo infection was also strikingly different. The Y strain caused 70% mortality in BALB/c mice but not in Slamf1-/- mice. The proinflammatory response was stronger in the last, suggesting that SLAMF1 was repressing protective immune responses of mice infected with the Y strain. In contrast, for VFRA, SLAMF1 deficiency resulted in 100% survival of BALB/c mice, without major changes in the immune response in the absence of SLAMF1. Thus, the results indicate that SLAMF1 receptor interacts with T. cruzi, affecting parasite replication and ROS production in macrophages as well as the adaptive immune response in mice in a parasite strain-dependent manner. Future studies will focus in understanding the immunopathogenic role of SLAMF1 during T. cruzi infection.

The glycolytic enzyme and bacterial virulence factor of Listeria monocytogenes, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Lmo2459), ADP-ribosylated the small GTPase, Rab5a, and blocked phagosome maturation. This inhibitory activity localized within the NAD binding domain of GAPDH at the N-terminal 1–22 peptides, also conferred listeriosis protection when used in dendritic cell-based vaccines. In this study, we explore GAPDH of Listeria, Mycobacterium, and Streptococcus spp. taxonomic groups to search for epitopes that confer broad protection against pathogenic strains of these bacteria. GAPDH multivalent epitopes are selected if they induce inhibitory actions and wide-ranging immune responses. Proteomic isolation of GAPDH from dendritic cells infected with Listeria, Mycobacterium, or Streptococcus confirmed similar enzymatic, Rab5a inhibitory and immune stimulation abilities. We identified by bioinformatics and functional analyses GAPDH N-terminal 1–22 peptides from Listeria, Mycobacterium, and Streptococcus that shared 95% sequence homology, enzymatic activity, and B and T cell immune domains. Sera obtained from patients or mice infected with hypervirulent pathogenic Listeria, Mycobacterium, or Streptococcus presented high levels of anti-GAPDH 1–22 antibodies and Th2 cytokines. Monocyte derived dendritic cells from healthy donors loaded with GAPDH 1–22 peptides from Listeria, Mycobacterium, or Streptococcus showed activation patterns that correspond to cross-immunity abilities. In summary, GAPDH 1–22 peptides appeared as putative candidates to include in multivalent dendritic based vaccine platforms for Listeria, Mycobacterium, or Streptococcus.

Allergic contact dermatitis, also known as contact hypersensitivity, is a frequent T-cell‒mediated inflammatory skin disease characterized by red, itchy, swollen, and cracked skin. It is caused by the direct contact with an allergen and/or irritant hapten. Galectin-1 (Gal-1) is a β-galactoside‒binding lectin, which is highly expressed in several types of immune cells. The role of endogenous Gal-1 in contact hypersensitivity is not known. We found that Gal-1‒deficient mice display more sustained and prolonged skin inflammation than wild-type mice after oxazolone treatment. Gal-1‒deficient mice have increased CD8

[Background]: Psoriasis is a frequent inflammatory skin disease that is mainly mediated by IL-23, IL-1β, and IL-17 cytokines. Although psoriasis is a hyperproliferative skin disorder, the possible role of amino acid transporters has remained unexplored., [Objective]: We sought to investigate the role of the essential amino acid transporter L-type amino acid transporter (LAT) 1 (SLC7A5) in psoriasis., [Methods]: LAT1 floxed mice were crossed to Cre-expressing mouse strains under the control of keratin 5, CD4, and retinoic acid receptor–related orphan receptor γ. We produced models of skin inflammation induced by imiquimod (IMQ) and IL-23 and tested the effect of inhibiting LAT1 (JPH203) and mammalian target of rapamycin (mTOR [rapamycin])., [Results]: LAT1 expression is increased in keratinocytes and skin-infiltrating lymphocytes of psoriatic lesions in human subjects and mice. LAT1 deletion in keratinocytes does not dampen the inflammatory response or their proliferation, which could be maintained by increased expression of the alternative amino acid transporters LAT2 and LAT3. Specific deletion of LAT1 in γδ and CD4 T cells controls the inflammatory response induced by IMQ. LAT1 deletion or inhibition blocks expansion of IL-17–secreting γ4+δ4+ and CD4 T cells and dampens the release of IL-1β, IL-17, and IL-22 in the IMQ-induced model. Moreover, inhibition of LAT1 blocks expansion of human γδ T cells and IL-17 secretion by human CD4 T cells. IL-23 and IL-1β stimulation upregulates LAT1 expression and induces mTOR activation in IL-17+ γδ and TH17 cells. Deletion or inhibition of LAT1 efficiently controls IL-23– and IL-1β–induced phosphatidylinositol 3-kinase/AKT/mTOR activation independent of T-cell receptor signaling., [Conclusion]: Targeting LAT1-mediated amino acid uptake is a potentially useful immunosuppressive strategy to control skin inflammation mediated by the IL-23/IL-1β/IL-17 axis., Supported by grants SAF2017-82886-R (to F.S.-M.), PI17/01972 (to E.D.), and SAF2013-42850-R (to M.F.) from the Spanish Ministry of Economy and Competitiveness; CAM (S2017/BMD-3671-INFLAMUNE-CM) from the Comunidad de Madrid (to F.S.-M.); and CIBERCV, BIOIMID PIE13/041 from Instituto de Salud Carlos III and Fundacion La Marato TV3 (20152330 31). The project leading to these results has also received funding from FUNDACION BBVA A EQUIPOS DE INVESTIGACION CIENTIFICA 2018 and from ‘‘la Caixa’’ Banking Foundation under the project code HR17-00016 (to F.S.-M.) and from Agencia Estatal de Investigacion, Fondo Europeo de Desarrollo Regional, European Union.

Summary Galectin-3 is the best-characterized member of galectins, an evolutionary conserved family of galactoside-binding proteins that play central roles in infection and immunity, regulating inflammation, cell migration and cell apoptosis. Differentially expressed by cells and tissues with immune privilege, they bind not only to host ligands, but also to glycans expressed by pathogens. In this regard, we have previously shown that human galectin-3 recognizes several genetic lineages of the protozoan parasite Trypanosoma cruzi, the causal agent of Chagas’ disease or American trypanosomiasis. Herein we describe a molecular mechanism developed by T. cruzi to proteolytically process galectin-3 that generates a truncated form of the protein lacking its N-terminal domain – required for protein oligomerization – but still conserves a functional carbohydrate recognition domain (CRD). Such processing relies on specific T. cruzi proteases, including Zn-metalloproteases and collagenases, and ultimately conveys profound changes in galectin-3-dependent effects, as chemical inhibition of parasite proteases allows galectin-3 to induce parasite death in vitro. Thus, T. cruzi might have established distinct mechanisms to counteract galectin-3-mediated immunity and microbicide properties. Interestingly, non-pathogenic T. rangeli lacked the ability to cleave galectin-3, suggesting that during evolution two genetically similar organisms have developed different molecular mechanisms that, in the case of T. cruzi, favoured its pathogenicity, highlighting the importance of T. cruzi proteases to avoid immune mechanisms triggered by galectin-3 upon infection. This study provides the first evidence of a novel strategy developed by T. cruzi to abrogate signalling mechanisms associated with galectin-3-dependent innate immunity.

Biomarkers of infection with consistent discriminating power for diagnosis and/or prognosis are keystones for efficient therapeutic management of diseases. The protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease (CD), exhibits high clinical and genetic diversity, making it difficult to define biomarkers. In animal models of infection, as well as in patients, many different outcomes have been described. Thus, pathophysiogenesis parameters were highly variable in patients and even in inbred animals, which impeded reliable one-dimensional diagnosis/prognosis. To help solve those problems, an in-depth analysis of the similarities and differences in the CD caused by different parasite strains or different patient conditions is needed. Multidimensional statistics may overcome the high variability for each individual parameter in patients and even in inbred animals, revealing some pathophysiological patterns that accurately allow diagnosis of clinical and physiopathological characteristics. Here, we describe this type of method and its application to T. cruzi infection.

Cancer is one of the most diagnosed diseases in developed countries. Inflammation is a common response to different stress situations including cancer and infection. In those processes, the family of mitogen-activated protein kinases (MAPKs) has an important role regulating cytokine secretion, proliferation, survival, and apoptosis, among others. MAPKs regulate a large number of extracellular signals upon a variety of physiological as well as pathological conditions. MAPKs activation is tightly regulated by phosphorylation/dephosphorylation events. In this regard, the dual-specificity phosphatase 10 (DUSP10) has been described as a MAPK phosphatase that negatively regulates p38 MAPK and c-Jun N-terminal kinase (JNK) in several cellular types and tissues. Several studies have proposed that extracellular signal-regulated kinase (ERK) can be also modulated by DUSP10. This suggests a complex role of DUSP10 on MAPKs regulation and, in consequence, its impact in a wide variety of responses involved in both cancer and inflammation. Here, we review DUSP10 function in cancerous and immune cells and studies in both mouse models and patients that establish a clear role of DUSP10 in different processes such as inflammation, immunity, and cancer., Ministerio de Ciencia e Innovación (SAF2013-42850-R and SAF2016-75988-R) Comunidad de Madrid (S2017/BMD-3671. INFLAMUNE-CM), Fondo de Investigaciones Sanitarias (BIOIMID) to M.F. K.S. was recipient of a Spanish Association Against Cancer oncology investigator grant (AECC AIO), We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

NF-κB-inducing kinase (NIK, MAP3K14) is best known as the apical kinase that triggers non-canonical NF-κB activation and by its role in the immune system. Recent data indicate a role for NIK expressed by non-lymphoid cells in cancer, kidney disease, liver injury, glucose homeostasis, osteosarcopenia, vascular calcification, hematopoiesis, and endothelial function. The spectrum of NIK-associated disease now ranges from immunodeficiency (when NIK is defective) to autoimmunity, cancer, sterile inflammation, fibrosis, and metabolic disease when NIK is overactive. The development of novel small-molecule NIK inhibitors has paved the way to test NIK targeting to treat disease in vivo, and may eventually lead to NIK targeting in the clinic. In addition, NIK activators are being explored for specific conditions such as myeloid leukemia.

The protozoan Entamoeba gingivalis resides in the oral cavity and is frequently observed in the periodontal pockets of humans and pets. This species of Entamoeba is closely related to the human pathogen Entamoeba histolytica, the agent of amoebiasis. Although E. gingivalis is highly enriched in people with periodontitis (a disease in which inflammation and bone loss correlate with changes in the microbial flora), the potential role of this protozoan in oral infectious diseases is not known. Periodontitis affects half the adult population in the world, eventually leads to edentulism, and has been linked to other pathologies, like diabetes and cardiovascular diseases. As aging is a risk factor for the disorder, it is considered an inevitable physiological process, even though it can be prevented and cured. However, the impact of periodontitis on the patient's health and quality of life, as well as its economic burden, are underestimated. Commonly accepted models explain the progression from health to gingivitis and then periodontitis by a gradual change in the identity and proportion of bacterial microorganisms in the gingival crevices. Though not pathognomonic, inflammation is always present in periodontitis. The recruitment of leukocytes to inflamed gums and their passage to the periodontal pocket lumen are speculated to fuel both tissue destruction and the development of the flora. The individual contribution to the disease of each bacterial species is difficult to establish and the eventual role of protozoa in the fate of this disease has been ignored. Following recent scientific findings, we discuss the relevance of these data and propose that the status of E. gingivalis be reconsidered as a potential pathogen contributing to periodontitis.

Cyclooxygenase2 (COX2) has been associated with cell growth, invasiveness, tumor progression and metastasis of colorectal carcinomas. However, the downstream prostaglandin (PG)-PG receptor pathway involved in these effects is poorly characterized. We studied the PG-pathway in gene expression databases and we found that PTGS2 (prostaglandin G/H synthase and cyclooxygenase) and PTGES (prostaglandin E synthase) are co-expressed in human colorectal tumors. Moreover, we detected that COX2 and microsomal Prostaglandin E synthase 1 (mPGES1) proteins are both up-regulated in colorectal human tumor biopsies. Using colon carcinoma cell cultures we found that COX2 overexpression significantly increased mPGES1 mRNA and protein. This up-regulation was due to an increase in early growth response 1 (EGR1) levels and its transcriptional activity. EGR1 was induced by COX2-generated PGF. A PGF receptor antagonist, or EGR1 silencing, inhibited the mPGES1 induction by COX2 overexpression. Moreover, using immunodeficient mice, we also demonstrated that both COX2- and mPGES1- overexpressing carcinoma cells were more efficient forming tumors. Our results describe for the first time the molecular pathway correlating PTGS2 and PTGES in colon cancer progression. We demonstrated that in this pathway mPGES1 is induced by COX2 overexpression, via autocrine PGs release, likely PGF, through an EGR1-dependent mechanism. This signaling provides a molecular explanation to PTGS2 and PTGES association and contribute to colon cancer advance, pointing out novel potential therapeutic targets in this oncological context., Ministerio de Ciencia e Innovación (SAF2010– 18733, SAF2013–42850-R), Comunidad de Madrid S2010/ BMD-2332, RED RICET RD12/0018/004, PIE13/00041 and an institutional grant from Fundación Ramon Areces to MF. FIS PS12/00094 to MLGB

Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting millions of people worldwide. One of the major causes of mortality in the disease is the cardiomyopathy observed in chronic patients, despite the low number of parasites detected in cardiac tissue. Galectin-3, a carbohydrate-binding protein with affinity for β-galactoside-containing glycoconjugates, is upregulated upon infection, and it has been recently involved in the pathophysiology of heart failure.We investigated the role of galectin-3 in systemic and local responses in a murine model of T. cruzi infection, using knockout animals. Molecular mechanisms underlying galectin-3-dependent inflammatory responses were further assessed in cultured dendritic cells in vitro.Mice deficient for galectin-3 have elevated blood parasitemia levels and impaired cytokine production during infection. Remarkably, galectin-3 promotes cellular infiltration in the heart of infected mice and subsequent collagen deposition and cardiac fibrosis. Furthermore, we show that an unbalanced Toll-like receptor expression on antigen-presenting cells may be the cause of the impaired immune response observed in galectin-3-deficient mice in vivo.These results suggest that galectin-3 is strongly involved in Chagas disease, not only in the immune response against T. cruzi, but also in mediating cardiac tissue damage.

The extreme genetic diversity of the protozoan Trypanosoma cruzi has been proposed to be associated with the clinical outcomes of the disease it provokes: Chagas disease (CD). To address this question, we analysed the similarities and differences in the CD pathophysiogenesis caused by different parasite strains. Using syngeneic mice infected acutely or chronically with 6 distant parasite strains, we integrated simultaneously 66 parameters: parasite tropism (7 parameters), organ and immune responses (local and systemic; 57 parameters), and clinical presentations of CD (2 parameters). While the parasite genetic background consistently impacts most of these parameters, they remain highly variable, as observed in patients, impeding reliable one-dimensional association with phases, strains, and damage. However, multi-dimensional statistics overcame this extreme intra-group variability for each individual parameter and revealed some pathophysiological patterns that accurately allow defining (i) the infection phase, (ii) the infecting parasite strains, and (iii) organ damage type and intensity. Our results demonstrated a greater variability of clinical outcomes and host responses to T. cruzi infection than previously thought, while our multi-parametric analysis defined common pathophysiological patterns linked to clinical outcome of CD, conserved among the genetically diverse infecting strains., Ministerio de Ciencia e Innovación” (SAF2013-42850-R); “Fondo de Investigaciones Sanitarias” (PI12/00289); “Red de Investigación de Centros de Enfermedades Tropicales” (RICET 2010RD12/0018/0004); European Union (HEALTH-FE-2008-22303, ChagasEpiNet); “Universidad Autónoma de Madrid” and “Comunidad de Madrid” (CC08-UAM/SAL-4440/08); AECID Cooperation with Argentine

[Background] Changas disease is caused by the protozoan Trypanosoma cruzi and is characterized by heart failure and sudden death. Identifying which factors are involved in evolution and treatment response is actually challenging. Thus, the aim of this work was to determine the Th1/Th17 (IL-6, IL-2, TNF, IL-17 and IFN-γ) and Th2 (IL-4 and IL-10) serum profile in Venezuelan Chagasic patients stratified according amiodarone treatment, hypertension and arrhythmias., [Methods] Sera from 38 chagasic patients were analyzed to determine the level of cytokines by Multiplexed Bead-Based Immunoassays. ANOVA test was applied to determine differences for each group. Additionally, a Linear Discriminant Analysis (LDA) was applied to observe the accuracy of different cytokines to discriminate between the groups., [Results] The levels of several cytokines were significantly higher in the high-risk of sudden death and untreated group. LDA showed that IL-2, IFN-γ and IL-10 were the best cytokines for discriminating between high-risk of sudden death and untreated patients versus low-risk of sudden death, treated and control groups., [Conclusions] High IL-2 levels seem to identify patients with high-risk of sudden death and seems adequate as treatment efficacy marker. To our knowledge, this is the first report about the anti-inflammatory role of the amiodarone in Chagas disease, suggesting an inmunomodulatory effect that may be exploited as coadjutant therapy in chronic Chagas disease., This work was supported by MF grant (SN/2013) and NG grant (CEAL-AL/2015-12) from “UAM-Banco Santander” for collaboration with Latin America; by NG grants from “Fondo de Investigaciones Sanitarias/FEDER” (PS09/00538 and PI12/00289), MINECO/FEDER (SAF2015-63868-R) “Universidad Autónoma de Madrid” and “Comunidad de Madrid” (CC08-UAM/SAL-4440/08); by MF grants from “Ministerio de Ciencia e Innovación” (SAF2010-17833 and SAF2013-42850-R), MINECO/FEDER (SAF2016-75998-R), “Red de Investigación de Centros de Enfermedades Tropicales” (RICET RD12/0018/0004; RICET RD16/0027/0006); European Union (HEALTH-FE-2008-22303, ChagasEpiNet, HOMIN - 317057 - FP7-PEOPLE-2012-ITN); AECID (A/025417/09 and A/031735/10), Comunidad de Madrid (S-2010/BMD-2332), Proyecto Excelencia BIOIMID - IIS La Princesa and “Fundación Ramón Areces”; and by HR and AM grants from Instituto Venezolano de Investigaciones Científicas (IVIC-1365).

The design and optimization of protective equipment and devices such as exoskeletons and prosthetics have the potential to be enhanced by the ability of accurately measure the positions of the bones during movement. Existing technologies allow a quite precise measurement of motion—mainly by using coordinate video-cameras and skin-mounted markers—but fail in directly measuring the bone position. Alternative approaches, as fluoroscopy, are too invasive and not usable during extended lapses of time, either for cost or radiation exposure. An approach to solve the problem is to combine the skin-glued markers with ultrasound technology in order to obtain the bone position by measuring at the same time the marker coordinates in 3D space and the depth of the echo from the bone. Given the complex structure of the bones and the tissues, the echoes from the ultrasound transducer show a quite complex structure as well. To reach a good accuracy in determining the depth of the bones, it is of paramount importance the ability to measure the time-of-flight (TOF) of the pulse with a high level of confidence. In this paper, the performance of several methods for determining the TOF of the ultrasound pulse has been evaluated when they are applied to the problem of measuring the bone depth. Experiments have been made using both simple setups used for calibration purposes and in real human tissues to test the performance of the algorithms. The results show that the method used to process the data to evaluate the time-of-flight of the echo signal can significantly affect the value of the depth measurement, especially in the cases when the verticality of the sensor with respect to the surface causing the main echo cannot be guaranteed. Finally, after testing several methods and processing algorithms for both accuracy and repeatability, the proposed cumulative kurtosis algorithm was found to be the most appropriate in the case of measuring bone depths in vivo with ultrasound sensors at frequencies around 5 MHz.

Peritoneal dissemination is a frequent metastatic route for cancers of the ovary and gastrointestinal tract. Tumour cells metastasize by attaching to and invading through the mesothelial cell (MC) monolayer that lines the peritoneal cavity. Metastases are influenced by carcinoma-associated fibroblasts (CAFs), a cell population that derives from different sources. Hence, we investigated whether MCs, through mesothelial-mesenchymal transition (MMT), were a source of CAFs during peritoneal carcinomatosis and whether MMT affected the adhesion and invasion of tumour cells. Biopsies from patients with peritoneal dissemination revealed the presence of myofibroblasts expressing mesothelial markers in the proximity of carcinoma implants. Prominent new vessel formation was observed in the peritoneal areas harbouring tumour cells when compared with tumour-free regions. The use of a mouse model of peritoneal dissemination confirmed the myofibroblast conversion of MCs and the increase in angiogenesis at places of tumour implants. Treatment of omentum MCs with conditioned media from carcinoma cell cultures resulted in phenotype changes reminiscent of MMT. Adhesion experiments demonstrated that MMT enhanced the binding of cancer cells to MCs in a β1-integrin-dependent manner. Scanning electron microscopy imaging showed that the enhanced adhesion was mostly due to increased cell-cell interaction and not to a mere matrix exposure. Invasion assays suggested a reciprocal stimulation of the invasive capacity of tumour cells and MCs. Our results demonstrate that CAFs can derive from mesothelial cells during peritoneal metastasis. We suggest that MMT renders the peritoneum more receptive for tumour cell attachment/invasion and contributes to secondary tumour growth by promoting its vascularization. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., Ministerio de Economıa y Competitividad (SAF2010–21249); Comunidad Autonoma de Madrid (S2010/BMD-2321); Ministerio de Economıa y Competitividad (SAF2010-18733); Instituto de Salud Carlos III (PI10/00101); Fondo de Investigaciones Sanitarias (FIS); Fundacion Mutua Madrilena; Digna-Biotech; Asociacion Espanola Contra el Cancer; Fundación Ramón Areces

Nowadays, coffee beans are almost exclusively used for the preparation of the beverage. The sustainability of coffee production can be achieved introducing new applications for the valorization of coffee by-products. Coffee silverskin is the by-product generated during roasting, and because of its powerful antioxidant capacity, coffee silverskin aqueous extract (CSE) may be used for other applications, such as antiaging cosmetics and dermaceutics. This study aims to contribute to the coffee sector’s sustainability through the application of CSE to preserve skin health. Preclinical data regarding the antiaging properties of CSE employing human keratinocytes and Caenorhabditis elegans are collected during the present study. Accelerated aging was induced by tert-butyl hydroperoxide (t-BOOH) in HaCaT cells and by ultraviolet radiation C (UVC) in C. elegans. Results suggest that the tested concentrations of coffee extracts were not cytotoxic, and CSE 1 mg/mL gave resistance to skin cells when oxidative damage was induced by t-BOOH. On the other hand, nematodes treated with CSE (1 mg/mL) showed a significant increased longevity compared to those cultured on a standard diet. In conclusion, our results support the antiaging properties of the CSE and its great potential for improving skin health due to its antioxidant character associated with phenols among other bioactive compounds present in the botanical material, The authors are grateful for the financial support from the SUSCOFFEE Project (AGL2014-57239-R) and the NATURAGE Project (AGL2010-17779). This work was partially funded by a Santander Small and Medium Enterprises Work Placement Grant in Beacon Biomedicine. Amaia Iriondo is a fellow of the FPI predoctoral program of the Ministry of Economy and Competitiveness (BES-2015-072191). Konstantinos Stamatakis is a recipient of an Asociación Española Contra el Cancer fellowship., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3e subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activationwith an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against amouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrumof human T cell-mediated autoimmune and inflammatory diseases.

Endothelial cells (EC) are able to actively control vascular permeability, coagulation, blood pressure and angiogenesis. Most recently, a role for endothelial cells in the immune response has been described. Therefore, the endothelium has a dual role controlling homeostasis but also being the first line for host defence and tissue damage repair thanks to its ability to mount an inflammatory response. Endothelial cells have been shown to express pattern-recognition receptors (PRR) including Toll-like receptors (TLR) that are activated in response to stimuli within the bloodstream including pathogens and damage signals. TLRs are strategic mediators of the immune response in endothelial cells but they also regulate the angiogenic process critical for tissue repair. Nevertheless, endothelial activation and angiogenesis can contribute to some pathologies. Thus, inappropriate endothelial activation, also known as endothelial dysfunction, through TLRs contributes to tissue damage during autoimmune and inflammatory diseases such as atherosclerosis, hypertension, ischemia and diabetes associated cardiovascular diseases. Also TLR induced angiogenesis is required for the growth of some tumors, atherosclerosis and rheumatoid arthritis, among others. In this review we discuss the importance of various TLRs in modulating the activation of endothelial cells and their importance in immunity to infection and vascular disease as well as their potential as therapeutic targets.

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings. Sin financiación 4.557 JCR (2016) Q2, 57/190 Cell Biology, 39/151 Immunology UEM

Chagas disease is caused by the protozoan Trypanosoma cruzi. This is an endemic disease in the Americas, but increased migration to Europe has made it emerge in countries where it was previously unknown, being Spain the second non endemic country in number of patients. T. cruzi is a parasite with a wide genetic diversity, which has been grouped by consensus into 6 Discrete Typing Units (DTUs) affecting humans. Some authors have linked these DTUs either to a specific epidemiological context or to the different clinical presentations. Our main objective was to describe the T. cruzi DTUs identified from a population of chronically infected Latin American migrants attending a reference clinic in Madrid. 149 patients meeting this condition were selected for the study. Molecular characterization was performed by an algorithm that combines PCR of the intergenic region of the mini exon-gene, the 24S? and 18S regions of rDNA and the variable region of the satellite DNA. A descriptive analysis was performed and associations between geographical/clinical data and the different DTUs were tested. DTUs could be determined in 105 out of 149 patients, 93.3% were from Bolivia, 67.7% were women and median age was 35 years (IQR 29-44). The most common DTU found was TcV (58; 55.2%), followed by TcIV (17; 16.2%), TcII (10; 9.5%) and TcI (4; 3.8%). TcIII and TcVI were not identified from any patient, and 15.2% patients presented mixed infections. In addition, we determined DTUs after treatment in a subset of patients. In 57% patients had different DTUs before and after treatment. DTUs distribution from this study indicates active transmission of T. cruzi is occurring in Bolivia, in both domestic and sylvatic cycles. TcIV was confirmed as a cause of chronic human disease. The current results indicate no correlation between DTU and any specific clinical presentation associated with Chagas disease, nor with geographical origin. Treatment with benznidazole does not always clear T. cruzi's genetic material from blood, and DTUs detected in the same patient may vary over time indicating that polyparasitism is frequent. © 2016 Elsevier B.V.

Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway., This study was supported by grant ISCIII-RETIC REDinREN/RD06/0001, by grants Fundación Mutua Madrileña (FMM2008 – 004) and Proyecto CAMUAH (CCG08-UAH/BIO-4237) to M. Alique, by grant Ministerio de Educación (SAF 2007 623471) to M. Rodríguez-Puyol, by grant Fondo de Investigaciones Sanitarias (FISS-PI070695) to D. Rodríguez-Puyol, and by grants Ministerio de Ciencia e Innovación (SAF2007-61716; BFU2007-62659/ BMC), ISCIII-RETIC RED RECAVA RD06/0014/1013, Comunidad Autónoma de Madrid, (S-SAL-0159/2006), CAM-UAM (CCG08-UAM/BIO-4299), and European Union (EICOSANOX LSH-CT-2004-005033) to M. Fresno and M. A. Iñiguez.

Background— Experimental autoimmune myocarditis (EAM), a mouse model of post–infectious cardiomyopathy, reflects mechanisms of inflammatory cardiomyopathy in humans. EAM is characterized by an infiltration of inflammatory cells into the myocardium that can be followed by myocyte fibrosis, edema, and necrosis, leading to ventricular wall dysfunction and heart failure. Different data indicate that CD69 exerts an important immunoregulatory effect in vivo. However, the possible role of CD69 in autoimmune myocarditis has not been studied. Methods and Results— We have explored the role of the leukocyte regulatory molecule CD69 in the inflammation that leads to cardiac dysfunction after myocardial injury in EAM. We have found that after induction of EAM, the draining lymph nodes from CD69-deficient mice developed an exacerbated Th17 inflammatory response, resulting in increases in the numbers of infiltrating leukocytes in the myocardium. In the chronic phase of EAM, transthoracic echocardiography revealed a significantly reduced left ventricular fractional shortening and a decreased ejection fraction in CD69-deficient mice, indicative of an impaired cardiac contractility. This condition was accompanied by a greater extent of myocardial fibrosis, an elevated number of sinus pauses on ECG, and an enhanced ratio of heart weight to body weight in CD69 −/− mice. Moreover, both bone marrow transplantation and adoptive transfer of Th17 cells isolated from immunized CD69 −/− mice with EAM into naive wild-type recipients reproduced the severity of the disease, demonstrating that CD69 exerts its function within the lymphocyte compartment. Conclusion— Our findings indicate that CD69 negatively regulates heart-specific Th17 responses, cardiac inflammation, and heart failure progression in EAM.

Anti-inflammatory efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) has been related to their properties as inhibitors of cyclooxygenase (COX)-mediated prostaglandin (PG) synthesis. However, recent studies have suggested that variations of the in vivo anti-inflammatory actions among different NSAIDs could not be solely explained by COX inhibition. Here, we have analyzed the effects on T cell activation of novel 4,5-dihydro-3 trifluoromethyl pyrazole anti-inflammatory drugs with different potencies as COX-2 inhibitors, namely E-6087, E-6232, E-6231, E-6036 and E-6259 as well as the chemically related COX-2 inhibitor Celecoxib. These drugs inhibited mitogen-mediated T cell proliferation as well as Interleukin (IL)-2, tumor necrosis factor (TNF)-α and Interferon (IFN)-γ synthesis by activated T cells, independently of their ability to inhibit COX-2 enzymatic activity. Immunosuppressive effects of these drugs seem to be due to their interference on transcription factor activation as induced transcription from Nuclear Factor (NF)-κB and Nuclear Factor of Activated T cells (NFAT)-dependent enhancers was inhibited in a dose-dependent manner, being the latter effect the most sensitive to the action of those compounds. Both NFAT dephosphorylation, required for its nuclear translocation, as well as transcriptional activity of a GAL4-NFAT chimera were diminished in the presence of these compounds. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory actions of NSAIDs, which may have important implications in anti-inflammatory therapy, through inhibition of NFAT.

Both amyloid-β peptide 1-42 (Aβ1-42) formation and cyclooxygenase-2 (COX-2) have been involved in the pathogenesis of Alzheimer's disease (AD), a devastating neurological disorder. However, the relationship between Aβ1-42 and COX-2 is unclear. We found that the addition of Aβ1-42 to astrocytoma cultures induced COX-2 mRNA and protein and PGE2 synthesis in primary human astrocytes and in human astrocytoma cell lines. Moreover, Aβ1-42 induced COX-2 promoter transcription. Deletion of nuclear factor-κB (NF-κB) sites of the promoter diminished Aβ1-42-COX-2 dependent transcription. In agreement with this, Aβ1-42 induced transcription of NF-κB reporter gene. In contrast, Aβ1-42 neither did not induce NFAT not AP-1 factors activation suggesting that both NFAT and AP-1 was not necessary to control COX-2 transcription induced by Aβ1-42. Over expression of NF-κB inhibitory subunit, IκB, completely abrogated Aβ1-42-induced COX-2 activity in U-87 cells, whereas the opposite effect was shown when p65/rel A NF-κB was over expressed. In addition, Aβ1-42 induced p65/rel A subunit translocation to the nucleus and binding to the distal site of the COX-2 promoter. The importance of NF-κB in COX-2 induction and PGE2 synthesis by Aβ1-42 was corroborated by using the pharmacological inhibitor of the NF-κB pathway, PDTC. In addition, Aβ1-42 treated astrocytoma supernatants were toxic for neuroblastoma cells, an effect which was blocked by PDTC. Summing up, our results indicate that Aβ1-42 was able to induce COX-2 and PGE2 synthesis in astrocytic cells through a NF-κB dependent mechanism. This may have implicated in our understanding of AD pathology.

Background Donor T lymphocytes are directly responsible for graft-versus-host disease. Molecules important in T-cell function may, therefore, be appropriate targets for graft-versus-host disease therapy and/or prophylaxis. Here we analyzed whether nuclear factor-κ B inducing kinase might have a role in graft-versus-host disease.Design and Methods We studied the expression of nuclear factor-κ B inducing kinase in human samples from patients with graft-versus-host disease. We also explored the effect of nuclear factor-κ B inducing kinase in a murine model of graft-versus-host disease using donor cells from aly/aly mice (deficient in nuclear factor-κ B inducing kinase) and C57BL/6 mice (control).Results We detected expression of nuclear factor-κ B inducing kinase in T-lymphocytes in the pathological lesions of patients with acute graft-versus-host disease. Mice transplanted with aly/aly T lymphocytes did not develop graft-versus-host disease at all, while mice receiving C57BL/6 cells died of a lethal form of the disease. Deficiency of nuclear factor-κ B inducing kinase did not affect the engrafting ability of donor T cells, but severely impaired their expansion capacity early after transplantation, and aly/aly T cells showed a higher proportion of apoptosis than did C57BL/6 T cells. Effector T lymphocytes were the T-cell subset most affected by nuclear factor-κ B inducing kinase deficiency. We also detected lower amounts of inflammatory cytokines in the serum of mice receiving aly/aly T cells than in the serum of mice receiving C57BL/6 T cells.Conclusions Our results show that nuclear factor-κ B inducing kinase has a role in graft-versus-host disease by maintaining the viability of activated alloreactive T lymphocytes.

Purpose: 2-(3,4-dihydroxyphenil)-ethanol (DPE), a polyphenol present in olive oil, has been found to attenuate the growth of colon cancer cells, an effect presumably related to its anti-inflammatory activity. Experimental Design: To further explore the effects of DPE on angiogenesis and tumor growth we investigated the in vivo efficacy of DPE in a HT-29 xenograft model and in vitro activities in colon cancer cells exposed to interleukin-1β (IL-1β) and prostaglandin E-2 (PGE-2). Results: DPE (10 mg/kg/day for 14 days) inhibited tumor growth, reducing vessel lumina and blood perfusion to tumor, and diminished expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and microsomal prostaglandin-E synthase-1 (mPGEs-1). In vitro, DPE (100 μmol/L) neither affected cell proliferation nor induced apoptosis in HT-29 and WiDr cells. DPE prevented the IL-1β–mediated increase of mPGEs-1 expression and PGE-2 generation, as it did the silencing of HIF-1α. Moreover, DPE blocked mPGEs-1–dependent expression of VEGF and inhibited endothelial sprouting induced by tumor cells in a coculture system. PGE-2 triggers a feed-forward loop involving HIF-1α, which impinges on mPGEs-1 and VEGF expression, events prevented by DPE via extracellular signal–related kinase 1/2. The reduction of PGE-2 and VEGF levels, caused by DPE, was invariably associated with a marked decrease in HIF-1α expression and activity, independent of proteasome activity, indicating that the DPE effects on tumor growth and angiogenesis are dependent on the inhibition of HIF-1α translation. Conclusions: We show that the in vivo DPE antitumor effect is associated with anti-inflammatory and antiangiogenic activities resulting from the downregulation of the HIF-1α/mPGEs-1/VEGF axis. Clin Cancer Res; 16(16); 4207–16. ©2010 AACR.

Neurodegenerative and neuroinflammatory disorders are commonly associated with local chemokine release. In other way, emerging data indicate that the prostaglandin E2 (PGE(2)), one of the major prostaglandins produced in the brain, play a central role in several pathological diseases. In this study, we investigated the relationship between CXCL12, cyclooxygenase (COX)-2 and PGE(2) in human brain cells. CXCL12 induced COX-2 and secretion of PGE(2) in a dose-dependent manner in human astrocytes. This induction was abolished by treatment with pertussis toxin and AMD3100, confirming the role of CXCR4 signaling. The nuclear factor-kappaB involvement was confirmed by using pyrrolidine dithiocarbamate, and with transient transfection assays. Over-expression of inhibitory proteins of nuclear factor-kappaB abrogated COX-2 induction, and CXCL12 induced p65/relA translocation. Culture supernatants from CXCL12-treated astrocytes reduced viability of neuroblastoma cells, and COX inhibitors abrogated this toxicity. Therefore, the relationship between chemokines and PGs could differentially influence the pathogenic network responsible for neurodegeneration.

The protozoan parasiteTrypanosoma cruzihas evolved sophisticated systems to evade the immune response. An important requirement for a productive immune response is recruitment of the appropriate immune cells from the bloodstream to the sites of infection. Here, we show that a mucin expressed and secreted by the metacyclic infective form ofT. cruzi, AgC10, is able to interfere with L-selectin-mediated monocyte adhesion. Thus, incubation of U937 monocytic cells stably expressing L-selectin (U937LAM) with AgC10 strongly reduced their adhesion on P-selectin under flow, which is dependent on L-selectin. This treatment also results in a significant inhibition by AgC10 of U937LAM and human primary monocyte adhesion to activated vascular endothelium. This effect was specific for L-selectin, because vascular cell adhesion molecule 1 (VCAM-1)-mediated adhesion was not affected by AgC10 pretreatment. This effect of AgC10 is likely due to its ability to induce L-selectin shedding from the monocyte membrane, since pharmacologic blocking of this shedding prevents AgC10 activity. This is the first description of a mechanism that prevents leukocyte adhesion to the endothelium by a parasite and represents a new potential countermeasure to evade the generation of a correct immune response.

BackgroundThe multidrug resistance proteins (MRPs) form a subfamily within the ATP binding cassette transporters that confer resistance to a variety of structurally unrelated compounds. MRP4 has been reported to transport antiretroviral drugs out of cells in an active process. Although the main therapeutic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are their ability to inhibit cyclooxygenase activity, in recent years, some pharmacological effects independent of this action have been described, such as inhibition of the activity of MRP4.MethodsDetection of MRP4 expression was carried out by Western blot analysis, immunofluorescence and flow cytometry in peripheral blood lymphocytes (PBLs). Cells were infected with HIV type-1NL4.3isolate, and treated with anti-retroviral drugs plus different NSAIDs. Agp24 was measured by ELISA 3 days post-infection. Intracellular [3H] zidovudine (AZT) was quantified by a scintiller counter. Expression of different cell markers was assessed by flow cytometry.ResultsNSAIDs, as well as probenecid, were able to potentiate the antiretroviral effect of several nucleoside reverse transcriptase inhibitors (NRTIs). PBLs expressed MRP4 and treatment with ibuprofen did not affect this expression. However, MRP4 expression increased following phytohaemaglutinin and AZT treatment. This decrease of Agp24 was correlated with an increase in the intracellular AZT concentration. This effect was unrelated to changes on expression of CD4, CXCR4, cell viability or activation. Interestingly, patients treated with highly active antiretroviral therapy, who had a detectable viral load, presented a higher expression of MRP4 than those with an undetectable viral load.ConclusionsNSAIDs can improve the antiretroviral activity of NRTIs, increasing their intracellular concentration by blocking MRP4. This finding could have implications for success of antiviral therapy.

We have investigated the role of intracellular HIV-1 Tat on CXCR4 expression on T cells. We found that stable or doxycycline-regulated expression of HIV-1 Tat on Jurkat T cells results in lower cell surface expression of CXCR4, but not of other chemokine receptors. This effect was not due to an alteration in CXCR4 transcription, and total CXCR4 levels remained unaltered. Rather, when cells were treated with CXCL12/Stromal Cell-Derived Factor 1, a faster downmodulation of CXCR4 was observed although resurfacing was unaffected. Similar effect was seen in peripheral human T cells transiently transfected with Tat. At the molecular level Tat did not alter cellular levels of G-coupled receptor kinases 2 and 6 and beta-arrestin, proteins involved in CXCR4 downregulation. Neither Tat significantly affected phosphatidylinositol 3-kinase activation in response to CXCL12. Interestingly, in Jurkat cell clones stably expressing both Protein kinase (PK)-Czeta and HIV-1 Tat, CXCL12 induced a faster CXCR4 internalization than in cells only expressing HIV-1 Tat. In contrast in Jurkat cell stably expressing a dominant negative PKCzeta, Tat enhancement of CXCR4 internalization was abrogated. Thus, our results show a new function of HIV-1 Tat, its ability to regulate CXCR4 expression via PKCzeta. The significance of those results is discussed.

HIV-2 invades CNS and causes neurological disease as well as HIV-1 does. Induction of COX-2 in CNS of HIV-1 infected people has been proposed as a cause of cognitive impairment, so we tested whether HIV-2 may cause damage by a similar mechanism. COX-2 mRNA and protein expression were induced in human astrocytes upon interaction with HIV-2, being this induction abrogated by CXCR4 antagonists. HIV-2 induced COX-2 promoter transcription and deletion of the two NF-kappaB binding sites of the promoter abrogated this induction. Neither AP-1 nor NFAT seem to be involved in COX-2 transcriptional activation. Overexpression of IkappaBalpha completely abrogated COX-2 induction, and transfection of p65/relA NF-kappaB induced COX-2 transcription. Interestingly, HIV-2 activated NF-kappaB by inducing p65/relA transactivating activity through Ser536 phosphorylation. Moreover, the astrocyte activation induced by HIV-2 was abrogated by different COX inhibitors. In summary, HIV-2 induces COX-2 in human astrocytes depending on CXCR4 coreceptor.

We have previously described that IFN-gamma induces cyclooxygenase 2 and inducible NO synthase expression by a mechanism that involved endogenously produced TNF-alpha. In this study, we report that TNF-alpha production is induced by IFN-gamma treatment in the murine macrophage cell line RAW 264.7. TNF-alpha mRNA levels are increased in cells treated with IFN-gamma in a time-dependent manner and IFN-gamma also increased human TNF-alpha promoter-dependent transcription. Two regions in the TNF-alpha promoter seem to be responsible for the IFN-gamma response: a distal region between -1311 and -615 bp of the human TNF-alpha promoter, and a proximal region located between -95 and -36 bp upstream of the transcriptional start. In contrast, IFN-gamma stimulation induces the expression of the transcription factors IRF-1 and IRF-8. Overexpression of these transcription factors produces an increase in the transcriptional activity of the human TNF-alpha promoter. There is a correlation between the regions of the TNF-alpha promoter responsible of the transcriptional activation elicited by IRF-1 and IRF-8 and those required for IFN-gamma response. In addition, IRF-1 and IRF-8 are recruited to the TNF-alpha promoter in IFN-gamma-treated RAW 264.7 cells, as demonstrated by chromatin immunoprecipitation assays. Moreover, overexpression of IRF-1 and IRF-8 induces TNF-alpha production in unstimulated RAW 264.7 macrophages, comparable to the production of TNF-alpha elicited by IFN-gamma stimulation, and silencing of IRF-1 and/or IRF-8 with specific small interfering RNAs, decreases IFN-gamma-elicited TNF-alpha production. In summary, IFN-gamma treatment induces TNF-alpha expression at transcriptional level requiring the coordinate action of IRF-1 and IRF-8.

Prostanoids, including prostaglandins (PGs) and thromboxanes (TXs) are synthesized from arachidonic acid by the combined action of cyclooxygenases (COXs) and PG and TX synthases. Finally after their synthesis, prostanoids are quickly released to the extracellular medium exerting their effects upon interaction with prostanoid receptors present in the neighbouring cells. These agents exert important actions in the cardiovascular system, modulating vascular homeostasis and participating in the pathogenesis of vascular diseases as thrombosis and atherosclerosis. Among prostanoids, Tromboxane (TX)A(2), a potent platelet activator and vasoconstrictor and prostacyclin (PGI2), a platelet inhibitor and vasodilator, are the most important in controlling vascular homeostasis. Although multiple studies using pharmacological inhibitors and genetically deficient mice have demonstrated the importance of prostanoid-mediated actions on cardiovascular physiology, further analysis on the prostanoid mediated actions in the vascular system are required to better understand the benefits and risks for the use of COX inhibitors in cardiovascular diseases.

Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs. The cleavage and polyadenylation specificity factor (CPSF) is critical for this process and its 73 kDa subunit (CPSF-73) mediates cleavage coupled to polyadenylation and histone pre-mRNA processing. Using CPSF-73 over-expression and siRNA-mediated knockdown experiments, this study identifies CPSF-73 as an important regulatory protein that represses the basal transcriptional activity of the HIV-1 LTR promoter. Similar results were found with over-expression of the CPSF-73 homologue RC-68, but not with CPSF 100 kDa subunit (CPSF-100) and RC-74. Chromatin immunoprecipitation assays revealed the physical interaction of CPSF-73 with the HIV-1 LTR promoter. Further experiments revealed indirect CPSF-73 binding to the region between -275 to -110 within the 5' upstream region. Functional assays revealed the importance for the 5' upstream region (-454 to -110) of the LTR for CPSF-73-mediated transcription repression. We also show that HIV-1 Tat protein interacts with CPSF-73 and counteracts its repressive activity on the HIV-1 LTR promoter. Our results clearly show a novel function for CPSF-73 and add another candidate protein for explaining the molecular mechanisms underlying HIV-1 latency.

Nuclear factor of activated T cells (NFAT) plays a prominent role in gene transcription during the immune response. Growing evidence demonstrates the implication of inducible phosphorylation of the transactivation domain (TAD) of NFAT in transcriptional activation of genes. We have analyzed the regulation of NFATc2 activation by Cot/Tpl2 and protein kinase C zeta (PKCzeta) in T cells. Our results show that PKCzeta and Cot/Tpl2 cooperate in regulating the transactivation activity mediated by the amino-terminal domain of NFATc2. Neither Cot/Tpl2 kinase nor PKCzeta-mediated induction of the transactivation activity of NFATc2 was affected by cyclosporin-A treatment, supporting a calcineurin independent route in the signaling pathways mediating NFATc2 activation. Co-precipitation experiments showed physical interaction among Cot/Tpl2, PKCzeta and NFATc2. Analysis of the transactivation activity of deletions in the N-terminal region of NFATc2, suggested the involvement of amino acids 52-64 of NFATc2 in the induction of its transactivating function by PKCzeta. This kinase in vitro phosphorylates NFATc2 and deletion and mutational studies identified Ser53 and Ser56 (of the SPPS motif) as substrates for PKCzeta. Thus, our results suggest that PKCzeta phosphorylation of Ser53 and Ser56 in the N-terminal TAD from NFATc2 potentiates its transactivating function in human T cells.

Human immunodeficiency virus-1 gp120 alters astroglial function, which compromises the function of the nearby of neuronal cells contributing to the cognitive impairment in human immunodeficiency virus-1 infection. Cyclooxygenase (COX)-2 has been involved in this process, although the intracellular pathways and second messengers involved are yet unknown. We have investigated the role of gp120-induced COX-2 in the astrocytoma human cell line U-87, and the different pathways involved in this activation. COX-2 mRNA and protein expression were detected in gp120-stimulated cells. Moreover, gp120 induces COX-2 promoter transcription. The effect of gp120 was abrogated by a neutralizing antibody against the chemokine receptor CXCR4 neutralizing antibody. Analysis of the promoter show that deletion or mutation of a proximal nuclear factor (NF)-kappaB site completely abrogated gp120-dependent transcription. NF-kappaB but neither Activating protein-1 nor nuclear factor of activated T-cells-dependent transcription was induced by gp120, as shown by reporter and electrophoretic mobility shift assays. In addition, transfection assays with the NF-kappaB inhibitor, IkappaBalpha, prevented gp120-mediated COX-2 induction. In contrast, there was no inhibition of COX-2 promoter transcription by expressing a dominant negative c-Jun, or nuclear factor of activated T-cells constructs. The antioxidant pyrrolidine dithiocarbamate inhibited COX-2 protein expression and COX-2 transcriptional activity induced by gp120. Thus, our results indicate that gp120 induced COX-2 transcription through NF-kappaB activation in astrocytoma cells.

Chagas disease, caused by Trypanosoma cruzi, affects several million people in Central and South America. About 30% of chronic patients develop cardiomyopathy probably caused by parasite persistence and/or autoimmunity. While several cross-reactive antibodies generated during mammal T. cruzi infection have been described, very few cross-reactive T cells have been identified. We performed adoptive transfer experiments of T cells isolated from chronically infected mice. The results showed the generation of cardiac pathology in the absence of parasites. We also transferred cross-reactive SAPA-specific T cells and observed unspecific alterations in heart repolarization, cardiac inflammatory infiltration, and tissue damage.

Background Progesterone levels are higher in placental barriers during pregnancy, but the effect of progesterone on human immunodeficiency virus type 1 (HIV-1) infection in placental cells has not been addressed. We hypothesize that progesterone may affect HIV infection. Methods Purified trophoblastic cells and trophoblastic cell lines were infected or transfected with HIV-1, and the effect of progesterone was analyzed. Viral replication was measured by viral p24 or viral load quantification. Nuclear factor kappa -B (NF- kappa B) or long terminal repeat (LTR)-dependent transcription was measured by luciferase assays. Expression of chemokine receptors was analyzed by flow cytometry. Tumor necrosis factor (TNF) messenger RNA was assessed by reverse-transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR. Results Progesterone inhibits HIV-1 replication in placental cells at the concentration found in the placental interface, at a postentry step, and does not affect cell surface expression of chemokine receptors. Progesterone did not inhibit basal or induced LTR transcription or NF- kappa B activation. TNF synthesis in placental cells is induced by HIV-1 infection that, in an autocrine manner, activates viral replication, because neutralizing anti-TNF antibodies block it. Progesterone inhibits the induction of TNF synthesis by viral infection and virus or gp-120-induced TNF transcription. Conclusion Our results demonstrate that progesterone inhibits HIV-1 replication in placental cells by reducing TNF levels, which are required for optimal viral replication.

It has been shown that phosphorylation of p65/RelA and c-Rel plays a role in the regulation of transcriptional activity of NF-kappaB independent on IkappaB degradation. In this study, we show that anti CD3/CD28 activation induces the transactivation activity of both p65/RelA and c-Rel in T cells using Gal4 dependent assays. Moreover, protein kinase C (PKC)zeta, Cot kinase and NF-kappaB-inducing kinase (NIK) seem to be involved in those processes in a different manner. Thus, transfection of dominant negative forms of Cot and PKCzeta inhibits CD3/CD28 induction of Gal4-p65 transactivation, whereas the kinase inactive versions of the 3 kinases inhibit induction of Gal4-c-Rel. Cot induction of Gal4-c-Rel transactivating activity seems to be mediated sequentially through PKCzeta and NIK activation, since dominant negative form of NIK blocks Cot and PKCzeta induction, whereas kinase inactive PKCzeta only blocks Cot activity. In contrast, the contribution of NIK to the transactivation function of p65/RelA seems to be negligible and more importantly NIK-KD did not inhibit induction by Cot and PKCzeta. Besides, the enhancing effect of Cot on Gal4-p65 was not decreased in mouse embryo fibroblasts from NIK deficient aly/aly mice in contrast with a greatest reduction on Gal4-c-Rel. By using Ser to Ala mutants in p65 and c-Rel transactivation domains, PKCzeta and NIK activities seem to be dependent of a restricted set of Ser in both proteins. In contrast, the enhancing effect of Cot seems to be less dependent of a particular set of Ser residues being partially abrogated by mutation of several Ser residues.

Visceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years., Ministerio de Economía y Competitividad (www.mineco.gob.es) grants AGL2010-16078/GAN to RBF and CYTED 214RT0482 to RMR; Instituto de Salud Carlos III (www.isciii.es) grants PI12/00104 to RMR and RICET RD12/0018/0004 to MF; Junta de Castilla y León (www.jcyl.es) grants Gr238 and LE182U13; European Commision (cordis.europa.eu/home_es. html), grant HOMIN - 317057-FP7-PEOPLE-2012-ITN and BIOIMID (http://www.fundacionareces.es)

Inflammation plays an important role in the pathophysiology of Chagas disease, caused by Trypanosoma cruzi. Prostanoids are regulators of homeostasis and inflammation and are produced mainly by myeloid cells, being cyclooxygenases, COX-1 and COX-2, the key enzymes in their biosynthesis from arachidonic acid (AA). Here, we have investigated the expression of enzymes involved in AA metabolism during T. cruzi infection. Our results show an increase in the expression of several of these enzymes in acute T. cruzi infected heart. Interestingly, COX-2 was expressed by CD68+ myeloid heart-infiltrating cells. In addition, infiltrating myeloid CD11b+Ly6G- cells purified from infected heart tissue express COX-2 and produce prostaglandin E2 (PGE2) ex vivo. T. cruzi infections in COX-2 or PGE2- dependent prostaglandin receptor EP-2 deficient mice indicate that both, COX-2 and EP-2 signaling contribute significantly to the heart leukocyte infiltration and to the release of chemokines and inflammatory cytokines in the heart of T. cruzi infected mice. In conclusion, COX-2 plays a detrimental role in acute Chagas disease myocarditis and points to COX-2 as a potential target for immune intervention., This work was supported by (NG) grants from “Fondo de Investigaciones Sanitarias” (PS09/00538 and PI12/00289); “Universidad Autónoma de Madrid” and “Comunidad de Madrid” (CC08-UAM/SAL-4440/08); by (MF) grants from “Ministerio de Ciencia e Innovación” (SAF2010-17833); “Red de Investigación de Centros de Enfermedades Tropicales” (RICET RD12/0018/0004); European Union (HEALTH-FE-2008-22303, ChagasEpiNet); AECID Cooperation with Argentine (A/025417/09 and A/031735/10), Comunidad de Madrid (S-2010/BMD- 2332) and “Fundación Ramón Areces”. NAG was recipient of a ISCIII Ph.D. fellowship financed by the Spanish “Ministerio de Sanidad”. CCM and HC were recipients of contracts from SAF2010-17833 and PI060388, respectively.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

Human immunodeficiency virus type 1 (HIV-1) Tat affects cellular gene expression through modulation of the activity of different transcription factors. Here, the role of Tat in the cooperation between nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) transcription factors was investigated. Constitutive or transient Tat expression in Jurkat T cells enhanced cooperative NFAT/AP-1- but not AP-1-dependent transcription independent of its ability to transactivate the HIV-1 LTR. The enhancing effect of Tat took place after nuclear translocation of NFAT. Furthermore, transactivation of an NFAT/AP-1 reporter by transfection of NFAT and c-Jun was strongly enhanced by simultaneous Tat transfection. Moreover, intracellular Tat expression increased the binding of NFAT/AP-1 complexes to the interleukin 2 promoter without significantly altering NFAT- and AP-1-independent binding. HIV-1 Tat interacted with NFAT but not c-Jun. These results indicate that Tat interacts with NFAT, affecting its cooperation with AP-1, without altering independent binding of these transcription factors to DNA.

Transcriptional induction of cyclooxygenase-2 (COX-2) occurs early after T cell receptor triggering and has functional implications in inflammation. Here, we show that phosphodiesterase (PDE)-4 inhibitors block COX-2 induction and prostaglandin synthesis in activated T cells. COX-2 inhibition by PDE4 inhibitors occurs mainly at the transcriptional level. Two response elements for the nuclear factor of activated T cells (NFAT) in the COX-2 promoter were required for inhibition by these drugs. PDE4 inhibitors did not affect NFAT nuclear translocation upon T cell activation; rather they prevented NFAT binding to DNA and induction of the transactivation function of GAL4-NFAT. These effects seem to be cAMP/PKA independent as they were not mimicked by the permeable analog dBcAMP or by forskolin, neither can be reverted by the PKA inhibitors H89 or KT-5720. These results may explain some of the anti-inflammatory properties of PDE4 inhibitors through the blockade of NFAT-mediated transactivation of pro-inflammatory genes such as COX-2.