Catherine Ropert - Academia.edu (original) (raw)

Papers by Catherine Ropert

Research paper thumbnail of Delivery of antisense oligonucleotides by means of pH-sensitive liposomes

The concept of pH-sensitive liposomes has been applied to the delivery of an oligonucleotide comp... more The concept of pH-sensitive liposomes has been applied to the delivery of an oligonucleotide complementary to the AUG region of the env mRNA of the Friend retrovirus. The oligonucleotide-containing liposomes were prepared by using the reverse phase evaporation or the freeze-thawing methodology. They were characterized for oligonucleotide entrapment efficiency, size, oligonucleotide release at different pH and stability. Optimisation of the

Research paper thumbnail of MEK2 controls the activation of MKK3/MKK6-p38 axis involved in the MDA-MB-231 breast cancer cell survival: Correlation with cyclin D1 expression

Cellular signalling, 2016

The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. Howe... more The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis wher...

Research paper thumbnail of Cell surface fluctuations studied with defocusing microscopy

Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom... more Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional ͑2D͒ Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature ͑Laplacian of the local thickness͒ of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations ͑ruffles͒ on the surface of macrophages ͑cell of the innate immune system͒, and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

Research paper thumbnail of Impaired production of proinflammatory cytokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking functional myeloid differentiation factor …

Research paper thumbnail of Impaired production of proinflammatory cytokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking functional myeloid differentiation factor …

Research paper thumbnail of The Vaccinia Virus-Stimulated Mitogen-Activated Protein Kinase (MAPK) Pathway is Required for Virus Multiplication

Biochemical …, 2004

Early events play a decisive role in virus multiplication. We have shown previously that activati... more Early events play a decisive role in virus multiplication. We have shown previously that activation of MAPK/ERK1/2 (mitogenactivated protein kinase/extracellular-signal-regulated kinase 1/2) and protein kinase A are pivotal for vaccinia virus (VV) multiplication [de Magalhães, Andrade, Silva, Sousa, Ropert, Ferreira, Kroon, Gazzinelli and Bonjardim (2001) J. Biol. Chem. 276, 38353-38360]. In the present study, we show that VV infection provoked a sustained activation of both ERK1/2 and RSK2 (ribosomal S6 kinase 2). Our results also provide evidence that this pattern of kinase activation depends on virus multiplication and ongoing protein synthesis and is maintained independently of virus DNA synthesis. It is noteworthy that the VGF (VV growth factor), although involved, is not essential for prolonged ERK1/2 activation. Furthermore, our findings suggest that the VV-stimulated ERK1/2 activation also seems to require actin dynamics, microtubule polymerization and tyrosine kinase phosphorylation. The VV-stimulated pathway MEK/ERK1/2/RSK2 (where MEK stands for MAPK/ERK kinase) leads to phosphorylation of the ternary complex factor Elk-1 and expression of the early growth response (egr-1) gene, which kinetically paralleled the kinase activation. The recruitment of this pathway is biologically relevant, since its disruption caused a profound effect on viral thymidine kinase gene expression, viral DNA replication and VV multiplication. This pattern of sustained kinase activation after VV infection is unique. In addition, by connecting upstream signals generated at the cytoskeleton and by tyrosine kinase, the MEK/ ERK1/2/RSK2 cascade seems to play a decisive role not only at early stages of the infection, i.e. post-penetration, but is also crucial to define the fate of virus progeny.

Research paper thumbnail of In-vivo treatment with benznidazole enhances phagocytosis, parasite destruction and cytokine release by macrophages during infection with a drug-susceptible but not with a derived drug-resistant Trypansoma cruzi population

Parasite Immunology

To stuck the effect of chemotherapy on parasite-macrophage interaction we used the wild-type Y st... more To stuck the effect of chemotherapy on parasite-macrophage interaction we used the wild-type Y strain (drug-susceptible) of Trypanosoma cruzi and a drug-resistant parasite population derived from the same strain. Trypomastigotes isolated from untreated infected mice, as well as, 3 h after treatment with BZ were incubated with inflammatory macrophages and used to study phagocytosis, parasite destruction, cytokine release and reactive nitrogen intermediates (RN!) synthesis. Phagocytosis and destruction of the drug-susceptible parasites were significant/v enhanced by drug treatment. These enhancements were accompanied by an increase in cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)alpha] and RNI release by murine inflammatory macrophages primed with IFN-gamma. In contrast, BZ treatment of mice infected with drug-resistant T. cruzi population showed no effect whatsoever. The synthesis of IFN-gamma and RNI by splenocytes of mice infected with either susceptible and drug-resistant parasite populations, before and after treatment with BZ were also studied. On/v the splenocytes from mice infected with the drug-susceptible parasites treated with BZ produced high levels of IFN-gamma and RNI. Our findings indicate that BZ acts on the drug-susceptible T. cruzi parasites by enhancing the phagocytosis and the production of cytokines and RN!, thus, favouring the destruction of the intracellular parasites by the cellular compartment of the immune system.

Research paper thumbnail of Activation of TLR9 in Dendritic Cells Recruitment and Endo-Lysosomal

Research paper thumbnail of Glycosylphosphatidylinositol Anchors As Natural Immunological Adjuvants Derived From Protozoan Parasites

Infectious Disease, 2006

... Ricardo T. Gazzinelli, Catherine Ropert, Igor C. Almeida, João S. Silva, and Marco A. Campos ... more ... Ricardo T. Gazzinelli, Catherine Ropert, Igor C. Almeida, João S. Silva, and Marco A. Campos ... 4. Gazzinelli RT, Wysocka M, Hayashi S, Denkers E, Hieny S, Caspar P, Trinchieri G, Sher A. Parasite Induced IL-12 stimulates early IFN-g synthesis and resistance during acute ...

Research paper thumbnail of Cell surface fluctuations studied with defocusing microscopy

Physical Review E, 2003

Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom... more Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional ͑2D͒ Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature ͑Laplacian of the local thickness͒ of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations ͑ruffles͒ on the surface of macrophages ͑cell of the innate immune system͒, and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

Research paper thumbnail of Trypanosoma cruzi Adjuvants Potentiate T Cell-Mediated Immunity Induced by a NY-ESO-1 Based Antitumor Vaccine

PLoS ONE, 2012

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects ar... more Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4 + T and CD8 + T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-c) production by CD4 + T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-c response by CD8 + T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4 + T and CD8 + T cell responses elicited by a specific immunological adjuvant.

Research paper thumbnail of Differential Use of TLR2 and TLR9 in the Regulation of Immune Responses during the Infection with Trypanosoma cruzi

PLoS ONE, 2013

Pathogens express ligands for several TLRs that may play a role in the induction or control of th... more Pathogens express ligands for several TLRs that may play a role in the induction or control of the inflammatory response during infection. Concerning Trypanosoma cruzi, the agent of Chagas disease, we have previously characterized glycosylphosphatidylinositol (GPI) anchored mucin-like glycoproteins (tGPI-mucin) and unmethylated CpG DNA sequences as TLR2 and TLR9 agonists, respectively. Here we sought to determine how these TLRs may modulate the inflammatory response in the following cell populations: F4/80 + CD11b + (macrophages), F4/80 low CD11b + (monocytes) and MHCII + CD11c high (dendritic cells). For this purpose, TLR2 2/2 and TLR9 2/2 mice were infected with Y strain of T. cruzi and different immunological parameters were evaluated. According to our previous data, a crucial role of TLR9 was evidenced in the establishment of Th1 response, whereas TLR2 appeared to act as immunoregulator in the early stage of infection. More precisely, we demonstrated here that TLR2 was mainly used by F4/80 + CD11b + cells for the production of TNF-a. In the absence of TLR2, an increased production of IL-12/IL-23p40 and IFN-c was noted suggesting that TLR2 negatively controls the Th1 response. In contrast, TLR9 was committed to IL-12/IL-23p40 production by MHCII + CD11c high cells that constitute the main source of IL-12/IL-23p40 during infection. Importantly, a down-regulation of TLR9 response was observed in F4/ 80 + CD11b + and F4/80 low CD11b + populations that correlated with the decreased TLR9 expression level in these cells. Interestingly, these cells recovered their capacity to respond to TLR9 agonist when MHCII + CD11c high cells were impeded from producing IL-12/IL-23p40, thereby indicating possible cross-talk between these populations. The differential use of TLR2 and TLR9 by the immune cells during the acute phase of the infection explains why TLR9-but not TLR2-deficient mice are susceptible to T. cruzi infection.

Research paper thumbnail of Leishmania chagasi: lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis

Molecular and Biochemical Parasitology, 2002

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a ... more During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterized by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This glycoconjugate is polymorphic among species with variations in sugars that branch off the conserved Gal(b1,4)Man(a1)-PO 4 backbone of repeat units and the oligosaccharide cap. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly. These adaptations were explored in the context of the structure and function of LPG for the first time on a New World species, Leishmania chagasi . The distinguishing feature of LPG of procyclic L. chagasi consisted of b1,3-glucose residues that branch off the disaccharide-phosphate repeat units and also are present in the cap. Importantly, metacyclic L. chagasi significantly down-regulate the glucose substitutions in the LPG. The significance of these modifications was demonstrated in the interaction of L. chagasi with its vector Lutzomyia longipalpis. In contrast to procyclic parasites and procyclic LPG, metacyclic parasites and metacyclic LPG were unable to bind to the insect midgut. These results are consistent with the proposal that a New World Leishmania species, similar to Old World species, adapts the expression of terminally exposed sugars of its LPG to mediate parasite Á/sand fly interactions. #

Research paper thumbnail of Macrophage signaling by glycosylphosphatidylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes

Microbes and Infection, 2002

Activation of cells from the innate immune system has an important role in host resistance to ear... more Activation of cells from the innate immune system has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that have identified and structurally characterized the glycosylphosphatidylinositol (GPI) anchors, as parasite molecules responsible for the activation of cells from the macrophage lineage. We also cover the studies that have identified the receptor, signaling pathways as well as the array of genes expressed in macrophages that are activated by these glycoconjugates. We discuss the possible implications of such response on the host resistance to T. cruzi infection and the pathogenesis of Chagas disease.

Research paper thumbnail of Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Memórias do Instituto Oswaldo Cruz, 2013

Research paper thumbnail of Cellular Immunology and Immune Regulation

On the cover: Biosensor-mediated tracking of RhoA activity during Toll receptor signaling as a to... more On the cover: Biosensor-mediated tracking of RhoA activity during Toll receptor signaling as a tool for pathway elucidation. Basal RhoA GTPase activity in unstimulated lung epithelial cells is presented as CFP/FRET ratio image visualized by color-coding on a scale representing an increase of activity from purple to yellow and red. Manukyan, M., P. Nalbant, S. Luxen, K. M. Hahn, and U. G. Knaus. 2009. RhoA GTPase activation by TLR2 and TLR3 ligands: connecting via Src to NF-B.

Research paper thumbnail of Inhibition of a p38/Stress-Activated Protein Kinase-2-Dependent Phosphatase Restores Function of IL-1 Receptor-Associated Kinase-1 and Reverses Toll-Like Receptor 2- and 4-Dependent Tolerance of Macrophages

The Journal of Immunology, 2003

Research paper thumbnail of Requirement of Mitogen-Activated Protein Kinases and I B Phosphorylation for Induction of Proinflammatory Cytokines Synthesis by Macrophages Indicates Functional Similarity of Receptors Triggered by Glycosylphosphatidylinositol Anchors from Parasitic Protozoa and Bacterial Lipopolysaccharide

The Journal of Immunology, 2001

In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified ... more In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypomastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related transcription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/ SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF-␣ and IL-12 synthesis by IFN-␥-primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of IB, and the use of SN50 peptide, an inhibitor of NF-B translocation, resulted in 70% of TNF-␣ synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and IB phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates.

Research paper thumbnail of Recruitment and Endo-Lysosomal Activation of TLR9 in Dendritic Cells Infected with Trypanosoma cruzi

The Journal of Immunology, 2008

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypa... more TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-B-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes. Abbreviations used in this paper: ODN, oligodeoxynucleotide; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonium)propane; RFP, red fluorescent protein; WT, wild type; YFP, yellow fluorescent protein.

Research paper thumbnail of A Mitogenic Signal Triggered at an Early Stage of Vaccinia Virus Infection. IMPLICATION OF MEK/ERK AND PROTEIN KINASE A IN VIRUS MULTIPLICATION

Journal of Biological Chemistry, 2001

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-... more Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF ؊ ). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNAprotein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF ؊ ) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.

Research paper thumbnail of Delivery of antisense oligonucleotides by means of pH-sensitive liposomes

The concept of pH-sensitive liposomes has been applied to the delivery of an oligonucleotide comp... more The concept of pH-sensitive liposomes has been applied to the delivery of an oligonucleotide complementary to the AUG region of the env mRNA of the Friend retrovirus. The oligonucleotide-containing liposomes were prepared by using the reverse phase evaporation or the freeze-thawing methodology. They were characterized for oligonucleotide entrapment efficiency, size, oligonucleotide release at different pH and stability. Optimisation of the

Research paper thumbnail of MEK2 controls the activation of MKK3/MKK6-p38 axis involved in the MDA-MB-231 breast cancer cell survival: Correlation with cyclin D1 expression

Cellular signalling, 2016

The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. Howe... more The Ras-Raf-MEK-ERK1/2 signaling pathway regulates fundamental processes in malignant cells. However, the exact contributions of MEK1 and MEK2 to the development of cancer remain to be established. We studied the effects of MEK small-molecule inhibitors (PD98059 and U0126) and MEK1 and MEK2 knock-down on cell proliferation, apoptosis and MAPK activation. We showed a diminution of cell viability that was associated with a downregulation of cyclin D1 expression and an increase of apoptosis marker in MEK2 silenced cells; by contrast, a slight increase of cell survival was observed in the absence of MEK1 that correlated with an augment of cyclin D1 expression. These data indicate that MEK2 but not MEK1 is essential for MDA-MB-231 cell survival. Importantly, the role of MEK2 in cell survival appeared independent on ERK1/2 phosphorylation since its absence did not alter the level of activated ERK1/2. Indeed, we have reported an unrevealed link between MEK2 and MKK3/MKK6-p38 MAPK axis wher...

Research paper thumbnail of Cell surface fluctuations studied with defocusing microscopy

Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom... more Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional ͑2D͒ Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature ͑Laplacian of the local thickness͒ of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations ͑ruffles͒ on the surface of macrophages ͑cell of the innate immune system͒, and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

Research paper thumbnail of Impaired production of proinflammatory cytokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking functional myeloid differentiation factor …

Research paper thumbnail of Impaired production of proinflammatory cytokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking functional myeloid differentiation factor …

Research paper thumbnail of The Vaccinia Virus-Stimulated Mitogen-Activated Protein Kinase (MAPK) Pathway is Required for Virus Multiplication

Biochemical …, 2004

Early events play a decisive role in virus multiplication. We have shown previously that activati... more Early events play a decisive role in virus multiplication. We have shown previously that activation of MAPK/ERK1/2 (mitogenactivated protein kinase/extracellular-signal-regulated kinase 1/2) and protein kinase A are pivotal for vaccinia virus (VV) multiplication [de Magalhães, Andrade, Silva, Sousa, Ropert, Ferreira, Kroon, Gazzinelli and Bonjardim (2001) J. Biol. Chem. 276, 38353-38360]. In the present study, we show that VV infection provoked a sustained activation of both ERK1/2 and RSK2 (ribosomal S6 kinase 2). Our results also provide evidence that this pattern of kinase activation depends on virus multiplication and ongoing protein synthesis and is maintained independently of virus DNA synthesis. It is noteworthy that the VGF (VV growth factor), although involved, is not essential for prolonged ERK1/2 activation. Furthermore, our findings suggest that the VV-stimulated ERK1/2 activation also seems to require actin dynamics, microtubule polymerization and tyrosine kinase phosphorylation. The VV-stimulated pathway MEK/ERK1/2/RSK2 (where MEK stands for MAPK/ERK kinase) leads to phosphorylation of the ternary complex factor Elk-1 and expression of the early growth response (egr-1) gene, which kinetically paralleled the kinase activation. The recruitment of this pathway is biologically relevant, since its disruption caused a profound effect on viral thymidine kinase gene expression, viral DNA replication and VV multiplication. This pattern of sustained kinase activation after VV infection is unique. In addition, by connecting upstream signals generated at the cytoskeleton and by tyrosine kinase, the MEK/ ERK1/2/RSK2 cascade seems to play a decisive role not only at early stages of the infection, i.e. post-penetration, but is also crucial to define the fate of virus progeny.

Research paper thumbnail of In-vivo treatment with benznidazole enhances phagocytosis, parasite destruction and cytokine release by macrophages during infection with a drug-susceptible but not with a derived drug-resistant Trypansoma cruzi population

Parasite Immunology

To stuck the effect of chemotherapy on parasite-macrophage interaction we used the wild-type Y st... more To stuck the effect of chemotherapy on parasite-macrophage interaction we used the wild-type Y strain (drug-susceptible) of Trypanosoma cruzi and a drug-resistant parasite population derived from the same strain. Trypomastigotes isolated from untreated infected mice, as well as, 3 h after treatment with BZ were incubated with inflammatory macrophages and used to study phagocytosis, parasite destruction, cytokine release and reactive nitrogen intermediates (RN!) synthesis. Phagocytosis and destruction of the drug-susceptible parasites were significant/v enhanced by drug treatment. These enhancements were accompanied by an increase in cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)alpha] and RNI release by murine inflammatory macrophages primed with IFN-gamma. In contrast, BZ treatment of mice infected with drug-resistant T. cruzi population showed no effect whatsoever. The synthesis of IFN-gamma and RNI by splenocytes of mice infected with either susceptible and drug-resistant parasite populations, before and after treatment with BZ were also studied. On/v the splenocytes from mice infected with the drug-susceptible parasites treated with BZ produced high levels of IFN-gamma and RNI. Our findings indicate that BZ acts on the drug-susceptible T. cruzi parasites by enhancing the phagocytosis and the production of cytokines and RN!, thus, favouring the destruction of the intracellular parasites by the cellular compartment of the immune system.

Research paper thumbnail of Activation of TLR9 in Dendritic Cells Recruitment and Endo-Lysosomal

Research paper thumbnail of Glycosylphosphatidylinositol Anchors As Natural Immunological Adjuvants Derived From Protozoan Parasites

Infectious Disease, 2006

... Ricardo T. Gazzinelli, Catherine Ropert, Igor C. Almeida, João S. Silva, and Marco A. Campos ... more ... Ricardo T. Gazzinelli, Catherine Ropert, Igor C. Almeida, João S. Silva, and Marco A. Campos ... 4. Gazzinelli RT, Wysocka M, Hayashi S, Denkers E, Hieny S, Caspar P, Trinchieri G, Sher A. Parasite Induced IL-12 stimulates early IFN-g synthesis and resistance during acute ...

Research paper thumbnail of Cell surface fluctuations studied with defocusing microscopy

Physical Review E, 2003

Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom... more Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional ͑2D͒ Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature ͑Laplacian of the local thickness͒ of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations ͑ruffles͒ on the surface of macrophages ͑cell of the innate immune system͒, and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.

Research paper thumbnail of Trypanosoma cruzi Adjuvants Potentiate T Cell-Mediated Immunity Induced by a NY-ESO-1 Based Antitumor Vaccine

PLoS ONE, 2012

Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects ar... more Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4 + T and CD8 + T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-c) production by CD4 + T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-c response by CD8 + T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4 + T and CD8 + T cell responses elicited by a specific immunological adjuvant.

Research paper thumbnail of Differential Use of TLR2 and TLR9 in the Regulation of Immune Responses during the Infection with Trypanosoma cruzi

PLoS ONE, 2013

Pathogens express ligands for several TLRs that may play a role in the induction or control of th... more Pathogens express ligands for several TLRs that may play a role in the induction or control of the inflammatory response during infection. Concerning Trypanosoma cruzi, the agent of Chagas disease, we have previously characterized glycosylphosphatidylinositol (GPI) anchored mucin-like glycoproteins (tGPI-mucin) and unmethylated CpG DNA sequences as TLR2 and TLR9 agonists, respectively. Here we sought to determine how these TLRs may modulate the inflammatory response in the following cell populations: F4/80 + CD11b + (macrophages), F4/80 low CD11b + (monocytes) and MHCII + CD11c high (dendritic cells). For this purpose, TLR2 2/2 and TLR9 2/2 mice were infected with Y strain of T. cruzi and different immunological parameters were evaluated. According to our previous data, a crucial role of TLR9 was evidenced in the establishment of Th1 response, whereas TLR2 appeared to act as immunoregulator in the early stage of infection. More precisely, we demonstrated here that TLR2 was mainly used by F4/80 + CD11b + cells for the production of TNF-a. In the absence of TLR2, an increased production of IL-12/IL-23p40 and IFN-c was noted suggesting that TLR2 negatively controls the Th1 response. In contrast, TLR9 was committed to IL-12/IL-23p40 production by MHCII + CD11c high cells that constitute the main source of IL-12/IL-23p40 during infection. Importantly, a down-regulation of TLR9 response was observed in F4/ 80 + CD11b + and F4/80 low CD11b + populations that correlated with the decreased TLR9 expression level in these cells. Interestingly, these cells recovered their capacity to respond to TLR9 agonist when MHCII + CD11c high cells were impeded from producing IL-12/IL-23p40, thereby indicating possible cross-talk between these populations. The differential use of TLR2 and TLR9 by the immune cells during the acute phase of the infection explains why TLR9-but not TLR2-deficient mice are susceptible to T. cruzi infection.

Research paper thumbnail of Leishmania chagasi: lipophosphoglycan characterization and binding to the midgut of the sand fly vector Lutzomyia longipalpis

Molecular and Biochemical Parasitology, 2002

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a ... more During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterized by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This glycoconjugate is polymorphic among species with variations in sugars that branch off the conserved Gal(b1,4)Man(a1)-PO 4 backbone of repeat units and the oligosaccharide cap. LPG has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly. These adaptations were explored in the context of the structure and function of LPG for the first time on a New World species, Leishmania chagasi . The distinguishing feature of LPG of procyclic L. chagasi consisted of b1,3-glucose residues that branch off the disaccharide-phosphate repeat units and also are present in the cap. Importantly, metacyclic L. chagasi significantly down-regulate the glucose substitutions in the LPG. The significance of these modifications was demonstrated in the interaction of L. chagasi with its vector Lutzomyia longipalpis. In contrast to procyclic parasites and procyclic LPG, metacyclic parasites and metacyclic LPG were unable to bind to the insect midgut. These results are consistent with the proposal that a New World Leishmania species, similar to Old World species, adapts the expression of terminally exposed sugars of its LPG to mediate parasite Á/sand fly interactions. #

Research paper thumbnail of Macrophage signaling by glycosylphosphatidylinositol-anchored mucin-like glycoproteins derived from Trypanosoma cruzi trypomastigotes

Microbes and Infection, 2002

Activation of cells from the innate immune system has an important role in host resistance to ear... more Activation of cells from the innate immune system has an important role in host resistance to early infection with the intracellular protozoan parasite, Trypanosoma cruzi. Here we review the studies that have identified and structurally characterized the glycosylphosphatidylinositol (GPI) anchors, as parasite molecules responsible for the activation of cells from the macrophage lineage. We also cover the studies that have identified the receptor, signaling pathways as well as the array of genes expressed in macrophages that are activated by these glycoconjugates. We discuss the possible implications of such response on the host resistance to T. cruzi infection and the pathogenesis of Chagas disease.

Research paper thumbnail of Cardiomyocyte dysfunction during the chronic phase of Chagas disease

Memórias do Instituto Oswaldo Cruz, 2013

Research paper thumbnail of Cellular Immunology and Immune Regulation

On the cover: Biosensor-mediated tracking of RhoA activity during Toll receptor signaling as a to... more On the cover: Biosensor-mediated tracking of RhoA activity during Toll receptor signaling as a tool for pathway elucidation. Basal RhoA GTPase activity in unstimulated lung epithelial cells is presented as CFP/FRET ratio image visualized by color-coding on a scale representing an increase of activity from purple to yellow and red. Manukyan, M., P. Nalbant, S. Luxen, K. M. Hahn, and U. G. Knaus. 2009. RhoA GTPase activation by TLR2 and TLR3 ligands: connecting via Src to NF-B.

Research paper thumbnail of Inhibition of a p38/Stress-Activated Protein Kinase-2-Dependent Phosphatase Restores Function of IL-1 Receptor-Associated Kinase-1 and Reverses Toll-Like Receptor 2- and 4-Dependent Tolerance of Macrophages

The Journal of Immunology, 2003

Research paper thumbnail of Requirement of Mitogen-Activated Protein Kinases and I B Phosphorylation for Induction of Proinflammatory Cytokines Synthesis by Macrophages Indicates Functional Similarity of Receptors Triggered by Glycosylphosphatidylinositol Anchors from Parasitic Protozoa and Bacterial Lipopolysaccharide

The Journal of Immunology, 2001

In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified ... more In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypomastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related transcription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/ SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF-␣ and IL-12 synthesis by IFN-␥-primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of IB, and the use of SN50 peptide, an inhibitor of NF-B translocation, resulted in 70% of TNF-␣ synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and IB phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates.

Research paper thumbnail of Recruitment and Endo-Lysosomal Activation of TLR9 in Dendritic Cells Infected with Trypanosoma cruzi

The Journal of Immunology, 2008

TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypa... more TLR9 is critical in parasite recognition and host resistance to experimental infection with Trypanosoma cruzi. However, no information is available regarding nucleotide sequences and cellular events involved on T. cruzi recognition by TLR9. In silico wide analysis associated with in vitro screening of synthetic oligonucleotides demonstrates that the retrotransposon VIPER elements and mucin-like glycoprotein (TcMUC) genes in the T. cruzi genome are highly enriched for CpG motifs that are immunostimulatory for mouse and human TLR9, respectively. Importantly, infection with T. cruzi triggers high levels of luciferase activity under NF-B-dependent transcription in HEK cells cotransfected with human TLR9, but not in control (cotransfected with human MD2/TLR4) HEK cells. Further, we observed translocation of TLR9 to the lysosomes during invasion/uptake of T. cruzi parasites by dendritic cells. Consistently, potent proinflammatory activity was observed when highly unmethylated T. cruzi genomic DNA was delivered to the endo-lysosomal compartment of host cells expressing TLR9. Thus, together our results indicate that the unmethylated CpG motifs found in the T. cruzi genome are likely to be main parasite targets and probably become available to TLR9 when parasites are destroyed in the lysosome-fused vacuoles during parasite invasion/uptake by phagocytes. Abbreviations used in this paper: ODN, oligodeoxynucleotide; BMDC, bone marrow-derived dendritic cell; DC, dendritic cell; DOTAP, 1,2-dioleoyloxy-3-(trimethylammonium)propane; RFP, red fluorescent protein; WT, wild type; YFP, yellow fluorescent protein.

Research paper thumbnail of A Mitogenic Signal Triggered at an Early Stage of Vaccinia Virus Infection. IMPLICATION OF MEK/ERK AND PROTEIN KINASE A IN VIRUS MULTIPLICATION

Journal of Biological Chemistry, 2001

Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-... more Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF ؊ ). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNAprotein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF ؊ ) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.