Flavio Salazar-Onfray | Universidad de Chile (original) (raw)

Papers by Flavio Salazar-Onfray

Journal of Immunology, Jul 15, 1999

Association of HER2/neu expression with sensitivity to tumor-speci c CTL in human ovarian cancer.

Tecnologia y Produccion De Vacunas Biologicas Contra El Cancer

Molluskan hemocyanins activate in vitro the classical pathway of the human complement system, through the presence of natural serum antibodies recognizing these proteins in unsensitized donors

Frontiers in Immunology, 2015

Cancer vaccine design based on tumor cell lysates and adjuvants

European Journal of Immunology, 2016

Runx2 transcription factor regulates expression of secreted immune evasion proteins in osteosarcoma

Platelets effect in activation and differentiation of human monocyte-derived macrophages

Runx2 Transcription Factor regulates expression of immune evasion effectors genes in osteosarcoma

TAPCells based vaccination induce specific anti NY-ESO1 immune response in melanoma patients

Characterization of exosomes from human osteosarcoma cell lines

Flow Cytometry Evaluation of Gap Junction-Mediated Intercellular Communication Between Cytotoxic T Cells and Target Tumor Cells

Methods in molecular biology, 2020

Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma me... more Gap junctions (GJs) are clusters of intercellular connexin-formed channels found at the plasma membrane that allow direct communication between the cytoplasm of adjacent cells. Numerous reports have described GJs as modulators of key immunological processes, including in anti-tumor immune responses. Here, we described a simple flow cytometry method to test in vitro antigen-dependent GJ-mediated cell-to-cell coupling between cytotoxic T cells and target melanoma cells.

Lisados celulares de cáncer de vesícula biliar inducen la maduración de células dendríticas humanas

Dexamethasone inhibits tumor cell lysate induction of a pro-inflammatory dendritic cell phenotype and function modulating T cell cytokine profiles (P4246)

Journal of Immunology, May 1, 2013

Recently, we showed the effectiveness of a DCs-based immunotherapy for improving long-term surviv... more Recently, we showed the effectiveness of a DCs-based immunotherapy for improving long-term survival in patients with melanoma using an allogeneic melanoma cell lysate. Glucocorticoids are used in cancer patients being implicated in immune suppression. Here, we aim to study the effect of dexamethasone on the inhibition of the pro-inflammatory phenotype and immunogenicity capacity of melanoma cell lysate-loaded DCs. Monocytes were differentiated to DCs as previously described. Dexamethasone was given at day 1 and 2 to obtain tolerogenic DCs. DCs maturation markers and TH1, TH17 phenotype were determined by flow cytometry. T cell cytokines were measured by ELISA and proliferation evaluated by CFSE dilution assay. We show that dexametasone stimulus elicits a semi-mature DC phenotype (low MHC II, CD83, CD80 and CD86) sharing mature and immature DCs characteristics, secreting low levels of IL1β, IL-6 and IL-12 and high levels of IL-10, but not affecting DC phagocytosis capacity. TolDCs inhibited T cell proliferation and IFNγ, IL-17 and TNF-α release by T helper cells. At contrary, an augment of Treg cells could be observed. The used glucocorticoids affects the maturation of newly differentiated monocytes-derived DCs in presence of immunogenic tumor cells, affecting the activation of a correct adaptive anti tumor immunity.

Tumor Lysate Loaded Dendritic Cells Induce a T cell Specific Antitumor Response Against Gallbladder Cancer

Journal of Immunology, May 1, 2017

Introduction Although infrequent, Gallbladder cancer (GBC) has high incidence rates in South Amer... more Introduction Although infrequent, Gallbladder cancer (GBC) has high incidence rates in South America. In fact, Chile has the highest GBC incidence and mortality worldwide. Currently, surgery is the only effective treatment, and the five-year survival rate is less than 10%. Previously, we demonstrated that allogeneic melanoma cell lysates-loaded dendritic cells (DCs) provide a standardized, applicable tumor-specific vaccine; capable to induce a cell mediated immune response in melanoma patients, improving survival. Here, we investigated the effect of tumor lysates derived from gallbladder cancer cell (GCC) lines on immature DCs and their capacity to induce a T cell mediated anti GBC responses in vitro . Material and Methods DCs were stimulated with GCC lysates and analyzed by flow cytometry for a panel of surface markers. Autologous CD3 + T cells were co-cultured with these DCs for 14 days. CD4 + and CD8 + T cells were analyzed by flow cytometry for activation and chemokine receptor expression. CD8 + T cells were isolated and co-cultured with GCC lines GBd1, TGBC-2TKB and melanoma cell line Mel1 for 16hrs. IFN-γ secretion was measured by ELISPOT. Results All GCC lysates induced DCs maturation, increasing surface expression of MHC-I, MHC-II, CD80, CD83, CD86, CCR7. DCs maturated with GCC lysate induced CD3 + T cells activation, increasing expression of CD25, CD69, CXCR3, CXCR4 in both CD4 + and CD8 + T cells compared with non-stimulated lymphocytes. Additionally, CD8 + T cells co-cultured with GBC-loaded DCs, released higher amounts of IFN-γ than controls. Discussion Based on these results, DCs maturated with GCC lysates are capable to induce specific T cells activation against GBC and can be considered for future immunotherapy approaches.

C‐type lectin receptors MR and DC‐SIGN are involved in recognition of hemocyanins, shaping their immunostimulatory effects on human dendritic cells

European Journal of Immunology, 2021

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th... more Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1‐biased cell‐mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose‐rich structures. It remains unclear whether these structures influence hemocyanin‐induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C‐type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose‐binding CLRs on monocyte‐derived human DCs: MR (mannose receptor) and DC‐SIGN (DC‐specific ICAM‐3–grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose‐sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins coloc...

Journal of Immunology Research, 2019

Ovarian epithelial carcinoma (OEC) is the most frequent ovarian tumor, characterized by a high mo... more Ovarian epithelial carcinoma (OEC) is the most frequent ovarian tumor, characterized by a high mortality in advanced stages where conventional therapies are not effective. Based on the role of the immune system in the progression of this disease, immunotherapy using checkpoint blockade has been considered as a therapeutic alternative. Nevertheless, its results do not match up to the positive results in entities like melanoma and other malignancies, suggesting the need to find other therapies to be used alone or in combination. Dendritic cell- (DC-) based vaccines have shown promising results in several types of cancer, such as melanoma, prostate, and lung cancers, due to the essential role played by DCs in the activation of specific T cells, thus using other ways of activating the immune response than immune checkpoint blockade. During the last decade, we have used DC-based vaccines loaded with an allogeneic heat shock-conditioned melanoma cell lysate in the treatment of advanced st...

Frontiers in Physiology, 2019

Guequén et al. U87-CM Increases Permeability Through S-Nitrosylation endothelial barrier function... more Guequén et al. U87-CM Increases Permeability Through S-Nitrosylation endothelial barrier function in the context of glioblastoma involving S-nitrosylation of VE-cadherin and p120. Our results suggest that inhibiting S-nitrosylation may be an effective way to control and/or block damage to the endothelial barrier and prevent cancer progression.

Immunobiology, 2019

Background: Dendritic cells (DCs) are usually immunogenic, but they are also capable of inducing ... more Background: Dendritic cells (DCs) are usually immunogenic, but they are also capable of inducing tolerance under anti-inflammatory conditions. Immunotherapy based on autologous DCs loaded with an allogeneic melanoma cell lysate (TRIMEL/DCs) induces immunological responses and increases melanoma patient survival. Glucocorticoids can suppress DC maturation and function, leading to a DC-mediated inhibition of T cell responses. Methods: The effect of dexamethasone, a glucocorticoid extensively used in cancer therapies, on TRIMEL/DCs phenotype and immunogenicity was examined. Results: Dexamethasone induced a semi-mature phenotype on TRIMEL/DC with low maturation surface marker expressions, decreased pro-inflammatory cytokine induction (IL-1β and IL-12) and increased release of regulatory cytokines (IL-10 and TGF-β). Dexamethasone-treated TRIMEL/DCs inhibited allogeneic CD4 + T cell proliferation and cytokine release (IFNγ, TNF-α and IL-17). Co-culturing melanoma-specific memory tumorinfiltrating lymphocytes with dexamethasone-treated TRIMEL/DC inhibited proliferation and effector T cell activities, including cytokine secretion and anti-melanoma cytotoxicity. Conclusions: These findings suggest that dexamethasone repressed melanoma cell lysate-mediated DC maturation, generating a potent tolerogenic-like DC phenotype that inhibited melanoma-specific effector T cell activities. These results suggest that dexamethasone-induced immunosuppression may interfere with the clinical efficacy of DC-based melanoma vaccines, and must be taken into account for optimal design of cellular therapy against cancer.

Cancer Letters, 2019

Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation c... more Glioblastoma (GBM) is the brain tumor with the worst prognosis composed of a cell subpopulation called Glioblastoma Stem-like Cells (GSCs) responsible for tumor recurrence mediated by cell invasion. GSCs persist in a hypoxic microenvironment which promotes extracellular adenosine production by activating the A 3 Adenosine Receptor (A 3 AR), therefore, the aim of this study was to determine the role of extracellular adenosine and A 3 AR on GSCs invasion under hypoxia. GSCs were obtained from a U87MG cell line and primary cultures of GBM patients, and then incubated under normoxia or hypoxia. Gene expression was evaluated by RNAseq, RT-qPCR, and western blot. Cell migration was measured by spreading and transwell boyden chamber assays; cell invasion was evaluated by Matrigel-coated transwell, ex vivo brain slice, and in vivo xenograft assays. The contribution of A 3 AR on cell migration/invasion was evaluated using the A 3 AR antagonist, MRS1220. Extracellular adenosine production was higher under hypoxia than normoxia, mainly by the catalytic action of the prostatic acid phosphatase (PAP), promoting cell migration/invasion in a HIF-2-dependent process. A 3 AR blockade decreased cell migration/invasion and the expression of Epithelial-Mesenchymal Transition markers. In conclusion, high levels of extracellular adenosine production enhance cell migration/invasion of GSCs, through HIF-2/PAPdependent activation of A 3 AR under hypoxia.

Journal of Cellular Physiology, 2012

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast... more Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G 1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G 1 /S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G 1 /S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth.

Tumor recognition by cytotoxic T cells. : Definition of new tumor antigens and the effect of interleukin-10 on antigen presentation

The tumor antigens against which T cell responses have been demonstrated most frequently are not ... more The tumor antigens against which T cell responses have been demonstrated most frequently are not tumor specific but are also expressed on normal tissues. This observation has been interpreted as evidence for breaking of immunological tolerance to normal cellular proteins. In the first part of this thesis we define HLA-A2 restricted CTL epitopes from the Melanocortin 1 Receptor (MC1R) which is expressed on cells of melanocytic origin, including melanoma cells. Peptides derived from this protein were selected on the basis of HLA-A2 binding motifs and tested for their HLA-A2 binding capacity. Three high- or intermediate binding nonamers were found to induce peptide-specific CTL from PBMC of healthy HLA-A2+ donors after in vi~ro stimulation with peptide-pulsed antigen presenting cells (APC). The CTL elicited against MCIR derived peptides could recognize HLA A2+/MC1R expressing melanomas. Using a similar protocol, four new epitopes derived from the proto-oncogene Her2/neu were identified. The CTL raised from ascites fluid of patient with ovarian carcinomas against these HLA-A2+ restricted peptides could recognize naturally processed peptides from HLA-A2+ tumors and from cell lines co-transfected with the antigenic protein gene and HLA-A2. Furthermore, we screened for the expression of MC1R in normal tissues and found this protein to be express to a low degree in adrenal gland and activated monocytes. The screening of anti-melanoma CTL from TIL or PBMC of melanoma patients revealed the presence of anti-MC1R CTL precursors in 50% of the patients, indicating that MC1R is an immunodominant melanoma antigen. Taken together, our findings have implications in relation both to autoimmunity as well as immunotherapy of malignant melanomas and carcinomas. The majority of human tumors are defective in their MHC class I antigen presenting capacity. In the second part of this thesis we have studied the role of Interleukin-10 (IL-10). We showed that IL-10 inhibits antigen presentation to specific CD8+ cytotoxic T Iymphocytes in melanomas. Furthermore, we demonstrated that pre treatment of the mouse Iymphoma RMA with IL-10 resulted in a dose dependent inhibition of Iysis by CTL. However, IL-10 treatment of RMA led to an increased sensitivity to Iysis by NK cells. RMA cells showed an IL-10 dependent downregulation of H-2 expression which could be normalized by addition of H-2 binding peptides, indicating that IL-10 exerts a post transcriptional effect on H-2 expression. Oligopeptides are delivered to the secretory pathway by the TAP protein complex. Relative to normal cells, TAP-deficient cells express substantially lower levels of intracellular antigens to CTL. We demonstrated that IL-10 expression in the RMA Iymphoma and other murine tumors inhibits the TAP-dependent translocation of peptides to the endoplasmic reticulum (ER), resulting in a low expression of cell surface MHC class I molecules. This finding is explained by a down regulation of expression of TAPI and TAP2, observed in IL-10 transfected murine tumor cells. In the J558L plasmacytoma cell line constitutively expressing high levels of IL-10, an increased TAP-dependent translocation of peptides and expression of cell surface MHC class I could be induced by IL-10 anti-sense expression. The effect of IL-10 on a biological relevant system was demonstrated using the NK sensitive prototype tumor YAC-1. Our studies showed that the NK sensitivity of this cell line correlates with the capability of the cells to produce IL-10. IL-10 is the first example of a cytokine which can decrease the expression and function of the TAP1/2 molecular complex, and in more general terms the first example of a cytokine with an inhibitory effect on MHC class I mediated antigen presentation