Millennium Institute for Fundamental and Applied Biology

Santiago, Chile

Millennium Institute for Fundamental and Applied Biology

Santiago, Chile
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Sauma D.,University of Chile | Espejo P.,University of Chile | Ramirez A.,University of Chile | Fierro A.,Transplantation Unit | And 4 more authors.
Scandinavian Journal of Immunology | Year: 2011

The Notch signalling pathway has recently been linked to T helper 1 (Th1)/T helper 2 (Th2) cell polarization via a mechanism involving differential expression of Notch ligands, Delta-like and Jagged, in antigen-presenting cells. However, whether stimuli other than pathogen-derived factors are involved in the regulation of Notch ligand expression in dendritic cells (DCs) remains unknown. Here, we address the effect of T helper cells (Th1 and Th2) on Delta-like 4 and Jagged 2 expression in bone marrow-derived DCs. We demonstrate that both Th1 and Th2 cells induce Delta-like 4 mRNA expression in DCs, in a process that is, in part, mediated by CD40 signalling. In contrast, only Th2 cells induce a significant increase in Jagged 2 mRNA levels in DCs. Additionally, we show that IL-4, a hallmark Th2 cytokine, plays a role in Jagged 2 expression, as evidenced by the fact that cholera toxin, a Th2-promoting stimulus, induces Jagged 2 mRNA expression in DCs only in the presence of IL-4. Finally, we demonstrate that DCs also express Notch 1 and that this expression is downregulated by IL-4. These data suggest that Notch ligands are differentially regulated in DCs: Delta-like 4 is regulated by T helper cells and by pathogen-derived Th1 stimuli, whereas Jagged 2 is regulated by Th2 cells and pathogen-derived Th2-promoting stimuli. Based on our results, we propose that the positive feedback loop that Th2 cells exert on T cell polarization may involve the induction of Jagged 2 expression in DCs. © 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.

Sauma D.,University of Chile | Ramirez A.,University of Chile | Alvarez K.,University of Chile | Rosemblatt M.,University of Chile | And 3 more authors.
Scandinavian Journal of Immunology | Year: 2012

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd.

Jorgensen T.N.,University of Colorado at Denver | Jorgensen T.N.,Cleveland Clinic | Alfaro J.,University of Chile | Alfaro J.,University of Virginia | And 16 more authors.
Journal of Immunology | Year: 2010

Autoantibodies are of central importance in the pathogenesis of Ab-mediated autoimmune disorders. The murine lupus susceptibility locus Nba2 on chromosome 1 and the syntenic human locus are associated with a loss of immune tolerance that leads to antinuclear Ab production. To identify gene intervals within Nba2 that control the development of autoantibody-producing B cells and to determine the cellular components through which Nba2 genes accomplish this, we generated congenic mice expressing various Nba2 intervals where genes for the FcγR, SLAM, and IFN-inducible families are encoded. Analysis of congenic strains demonstrated that the FcγR and SLAM intervals independently controlled the severity of autoantibody production and renal disease, yet are both required for lupus susceptibility. Deregulated homeostasis of terminally differentiated B cells was found to be controlled by the FcγR interval where FcγRIIb-mediated apoptosis of germinal center B cells and plasma cells was impaired. Increased numbers of activated plasmacytoid dendritic cells that were distinctly CD19+ and promoted plasma cell differentiation via the proinflammatory cytokines IL-10 and IFNα were linked to the SLAM interval. These findings suggest that SLAM and FcγR intervals act cooperatively to influence the clinical course of disease through supporting the differentiation and survival of autoantibody-producing cells. Copyright © 2010 by The American Association of Immunologists, Inc.

Hernandez P.P.,University of Chile | Undurraga C.,University of Chile | Gallardo V.E.,University of Chile | Mackenzie N.,University of Chile | And 4 more authors.
Biological Research | Year: 2011

Copper is an essential ion that forms part of the active sites of many proteins. At the same time, an excess of this metal produces free radicals that are toxic for cells and organisms. Fish have been used extensively to study the effects of metals, including copper, present in food or the environment. It has been shown that different metals induce different adaptive responses in adult fish. However, until now, scant information has been available about the responses that are induced by waterborne copper during early life stages of fish. Here, acute toxicity tests and LC50 curves have been generated for zebrafish larvae exposed to dissolved copper sulphate at different concentrations and for different treatment times. We determined that the larvae incorporate and accumulate copper present in the medium in a concentration dependent manner, resulting in changes in gene expression. Using a transgenic fish line that expresses enhanced green fluorescent protein (EGFP) under the hsp70 promoter, we monitored tissue-specific stress responses to waterborne copper by following expression of the reporter. Furthermore, TUNEL assays revealed which tissues are more susceptible to cell death after exposure to copper. Our results establish a framework for the analysis of whole-organism management of excess external copper in developing aquatic animals.

Diskin S.,Tufts University | Chen W.-S.,Tufts University | Cao Z.,Tufts University | Gyawali S.,Tufts University | And 6 more authors.
PLoS ONE | Year: 2012

Purpose: The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling. Methods: Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-β1 integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined. Principal Findings: We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-β1 integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632. Conclusions/Significance: The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow. © 2012 Diskin et al.

Escobar S.,Andrés Bello University | Escobar S.,Institute Acuicultura Of Torre Of La Sal Iats | Fuentes E.N.,Andrés Bello University | Poblete E.,Andrés Bello University | And 7 more authors.
Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology | Year: 2011

Insulin-like growth factor-1 and insulin-like growth factor-1 receptor (IGF-1 and IGF-1R) play main roles in vertebrate growth and development. In fish, besides contributing to somatic growth, both molecules exhibit pleiotropic functions. We isolated complete cDNAs sequences encoding for both IGF-1 and IGF-1R in the Chilean flounder by using RT-PCR and rapid amplification of cDNAs ends (RACE) techniques. We analyzed gene expression in pre-metamorphic larvae and different organs of juvenile fish through whole mount in situ hybridization and RT-PCR, respectively. In addition, we studied the presence of calcified skeletal structures in pre-metamorphic larvae through the fluorescent chromophore calcein. The IGF-1 cDNA sequence displays an open reading frame of 558 nucleotides, encoding a 185 amino acid preproIGF-1. Moreover, IGF-1R contains an open reading frame spanning 4239 nucleotides, rendering a 702 amino acid subunit alpha and a 676 amino acid subunit beta. The deduced mature IGF-1 and IGF-1R exhibited high sequence identities with their corresponding orthologs in fishes, especially those domains involved in biological activity. RT-PCR showed expression of IGF-1 and IGF-1R transcripts in all studied tissues, consistent with their pleiotropic functions. Furthermore, we observed IGF-1 expression in notochord and IGF-1R expression in notochord, somites and head in larvae of 8 and 9. days post fertilization. Complementarily, we detected in larvae of 8. days post fertilization the presence of calcified skeletal structures in notochord and head. Interestingly, both mRNAs and calcified structures were found in territories such as notochord, an embryonic midline structure essential for the pattern of surrounding tissues as nervous system and mesoderm. Our results suggest that IGF-1 and its receptor play an important role in the development of the nervous system, muscle and bone-related structures during larval stages. © 2011 Elsevier Inc.

Oro Stica M.L.,University of Santiago de Chile | Oro Stica M.L.,Chilean Center of Nanosciences and Nanotechnology | Zuniga L.M.,Millennium Institute for Fundamental and Applied Biology | Utz D.,University of Santiago de Chile | And 7 more authors.
Reproduction | Year: 2013

Mating shut down a 2-methoxyestradiol (2ME) nongenomic action necessary to accelerate egg transport in the rat oviduct. Herein, we investigated whether tumour necrosis factor-a (TNF-a) participates in this mating effect. In unmated and mated rats, we determined the concentration of TNF-a in the oviductal fluid and the level of the mRNA for Tnf-a (Tnf ) and their receptors Tnfrsf1a and Tnfrsf1b in the oviduct tissues. The distribution of the TNFRSF1A and TNFRSF1B proteins in the oviduct of unmated and mated was also assessed. Finally, we examined whether 2ME accelerates oviductal egg transport in unmated rats that were previously treated with a rat recombinant TNF-a alone or concomitant with a selective inhibitor of the NF-kB activity. Mating increased TNF-a in the oviductal fluid, but Tnf transcript was not detected in the oviduct. The mRNA for TNF-a receptors as well as their distribution was not affected by mating, although they were mainly localized in the endosalpinx. Administration of TNF-a into the oviduct of unmated rats prevented the effect of 2ME on egg transport. However, the NF-kB activity inhibitor did not revert this effect of TNF-a. These results indicate that mating increased TNF-a in the oviductal fluid, although this not associated with changes in the expression and localization of TNF-a receptors in the oviductal cells. Furthermore, TNF-a mimicked the effect of mating on the 2ME-induced egg transport acceleration, independently of the activation of NF-kB in the oviduct.We concluded that TNF-a is the signal induced by mating to shut down a 2ME nongenomic action in the rat oviduct. © 2013 Society for Reproduction and Fertility.

Parada-Bustamante A.,University of Santiago de Chile | Parada-Bustamante A.,Millennium Institute for Fundamental and Applied Biology | Orihuela P.A.,University of Santiago de Chile | Orihuela P.A.,Chilean Center of Nanosciences and Nanotechnology | And 10 more authors.
Reproduction | Year: 2010

Estradiol (E2) accelerates oviductal egg transport through intraoviductal non-genomic pathways in unmated rats and through genomic pathways in mated rats. This shift in pathways has been designated as intracellular path shifting (IPS), and represents a novel and hitherto unrecognized effect of mating on the female reproductive tract. We had reported previously that IPS involves shutting down the E2 non-genomic pathway up- and downstream of 2-methoxyestradiol. Here, we evaluated whether IPS involves changes in the genomic pathway too. Using microarray analysis, we found that a common group of genes changed its expression in response to E2 in unmated and mated rats, indicating that an E2 genomic signaling pathway is present before and after mating; however, a group of genes decreased its expression only in mated rats and another group of genes increased its expression only in unmated rats. We evaluated the possibility that this difference is a consequence of an E2 non-genomic signaling pathway present in unmated rats, but not in mated rats. Mating shuts down this E2 non-genomic signaling pathway up- and downstream of cAMP production. The Star level is increased by E2 in unmated rats, but not in mated rats. This is blocked by the antagonist of estrogen receptor ICI 182 780, the adenylyl cyclase inhibitor SQ 22536, and the catechol-O-methyltransferase inhibitor, OR 486. These results indicate that the E2-induced gene expression profile in the rat oviduct differs before and after mating, and this difference is probably mediated by an E2 non-genomic signaling pathway operating on gene expression only in unmated rats. © 2010 Society for Reproduction and Fertility.

Jarpa E.,University of Santiago de Chile | Babul M.,University of Santiago de Chile | Calderon J.,University of Santiago de Chile | Gonzalez M.,University of Santiago de Chile | And 8 more authors.
Lupus | Year: 2011

Psychiatric diagnosis in patients with systemic lupus erythematosus (SLE) is controversial: variations have been reported in frequency, diagnostic assays, associations with disease activity, autoantibodies, and contributing social factors. Eighty-three consecutive non-selected Chilean patients with SLE were evaluated for: (i) 26 common mental disorders according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV), using the Mini-International Neuropsychiatric Interview (MINI-plus); (ii) psychological suffering measured by Hospital Anxiety and Depression Scale (HADS); (iii) ACR 1999 neuropsychiatric (NP)SLE criteria; (iv) SLE disease activity (SLEDAI-2K); (v) cumulative damage (SLICC/ACR); and (vi) anti-ribosomal P antibodies by enzyme-linked immunoassay and immunoblot. Psychiatric diagnoses occurred in 44.6% of patients; the most frequent (21.7%) was major depressive episode (MDE). No association with lupus activity was observed in patients with a DSM-IV diagnosis or MDE or psychological suffering. ACR 1999 NPSLE criteria were present in 42.2% of patients, the majority corresponding to mood (28.9%) or anxiety disorders (15.6%). Suicidal risk was present in 9.6% of patients. Anti-ribosomal P antibodies (13.3%) were not associated with DSM-IV diagnosis. Severe psychiatric disorders in SLE are common and not associated with disease activity. © The Author(s), 2011.

Rios M.,Unidad University | Parada-Bustamante A.,Unidad University | Velasquez L.A.,University of Santiago de Chile | Velasquez L.A.,Chilean Center of Nanosciences and Nanotechnology | And 5 more authors.
Reproductive Biology and Endocrinology | Year: 2011

Background: Mating changes the mechanism by which E2 regulates oviductal egg transport, from a non-genomic to a genomic mode. Previously, we found that E2 increased the expression of several genes in the oviduct of mated rats, but not in unmated rats. Among the transcripts that increased its level by E2 only in mated rats was the one coding for an s100 calcium binding protein G (s100 g) whose functional role in the oviduct is unknown.Methods: Herein, we investigated the participation of s100 g on the E2 genomic effect that accelerates oviductal transport in mated rats. Thus, we determined the effect of E2on the mRNA and protein level of s100 g in the oviduct of mated and unmated rats. Then, we explored the effect of E2on egg transport in unmated and mated rats under conditions in which s100 g protein was knockdown in the oviduct by a morpholino oligonucleotide against s100 g (s100 g-MO). In addition, the localization of s100 g in the oviduct of mated and unmated rats following treatment with E2was also examined.Results: Expression of s100 g mRNA progressively increased at 3-24 h after E2 treatment in the oviduct of mated rats while in unmated rats s100 g increased only at 12 and 24 hours. Oviductal s100 g protein increased 6 h following E2and continued elevated at 12 and 24 h in mated rats, whereas in unmated rats s100 g protein increased at the same time points as its transcript. Administration of a morpholino oligonucleotide against s100 g transcript blocked the effect of E2on egg transport in mated, but not in unmated rats. Finally, immunoreactivity of s100 g was observed only in epithelial cells of the oviducts of mated and unmated rats and it was unchanged after E2 treatment.Conclusions: Mating affects the kinetic of E2-induced expression of s100 g although it not changed the cellular localization of s100 g in the oviduct after E2. On the other hand, s100 g is a functional component of E2 genomic effect that accelerates egg transport. These findings show a physiological involvement of s100 g in the rat oviduct. © 2011 Ríos et al; licensee BioMed Central Ltd.

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