Imunotrofismo placentário:o papel das citocinas no desenvolvimento embrionário

Orientadora: Maria da Graça BicalhoMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciencias Biológicas. Curso de Graduaçao em Ciencias Biológica

Flavonoids as molecules with Anti-Zika virus activity

Zika virus (ZIKV) is an arthropod-born virus that is mainly transmitted to humans by mosquitoes of the genus Aedes spp. Since its first isolation in 1947, only a few human cases had been described until large outbreaks occurred on Yap Island (2007), French Polynesia (2013), and Brazil (2015). Most ZIKV-infected individuals are asymptomatic or present with a self-limiting disease and nonspecific symptoms such as fever, myalgia, and headache. However, in French Polynesia and Brazil, ZIKV outbreaks led to the diagnosis of congenital malformations and microcephaly in newborns and Guillain-Barré syndrome (GBS) in adults. These new clinical presentations raised concern from public health authorities and highlighted the need for anti-Zika treatments and vaccines to control the neurological damage caused by the virus. Despite many efforts in the search for an effective treatment, neither vaccines nor antiviral drugs have become available to control ZIKV infection and/or replication. Flavonoids, a class of natural compounds that are well-known for possessing several biological properties, have shown activity against different viruses. Additionally, the use of flavonoids in some countries as food supplements indicates that these molecules are nontoxic to humans. Thus, here, we summarize knowledge on the use of flavonoids as a source of anti-ZIKV molecules and discuss the gaps and challenges in this area before these compounds can be considered for further preclinical and clinical trials

Amblyomma sculptum Salivary PGE2 Modulates the Dendritic Cell-Rickettsia rickettsii Interactions in vitro and in vivo

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host's blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo

Extracellular Vesicles Shed By Trypanosoma cruzi Potentiate Infection and Elicit Lipid Body Formation and PGE2 Production in Murine Macrophages

During the onset of Trypanosoma cruzi infection, an effective immune response is necessary to control parasite replication and ensure host survival. Macrophages have a central role in innate immunity, acting as an important trypanocidal cell and triggering the adaptive immune response through antigen presentation and cytokine production. However, T. cruzi displays immune evasion mechanisms that allow infection and replication in macrophages, favoring its chronic persistence. One potential mechanism is the release of T. cruzi strain Y extracellular vesicle (EV Y), which participate in intracellular communication by carrying functional molecules that signal host cells and can modulate the immune response. The present work aimed to evaluate immune modulation by EV Y in C57BL/6 mice, a prototype resistant to infection by T. cruzi strain Y, and the effects of direct EV Y stimulation of macrophages in vitro. EV Y inoculation in mice prior to T. cruzi infection resulted in increased parasitemia, elevated cardiac parasitism, decreased plasma nitric oxide (NO), reduced NO production by spleen cells, and modulation of cytokine production, with a reduction in TNF-α in plasma and decreased production of TNF-α and IL-6 by spleen cells from infected animals. In vitro assays using bone marrow-derived macrophages showed that stimulation with EV Y prior to infection by T. cruzi increased the parasite internalization rate and release of infective trypomastigotes by these cells. In this same scenario, EV Y induced lipid body formation and prostaglandin E2 (PGE2) production by macrophages even in the absence of T. cruzi. In infected macrophages, EV Y decreased production of PGE2 and cytokines TNF-α and IL-6 24 h after infection. These results suggest that EV Y modulates the host response in favor of the parasite and indicates a role for lipid bodies and PGE2 in immune modulation exerted by EVs

Immature Dendritic Cells Generated from Cryopreserved Human Monocytes Show Impaired Ability to Respond to LPS and to Induce Allogeneic Lymphocyte Proliferation

<div><p>Dendritic cells play a key role in the immune system, in the sensing of foreign antigens and triggering of an adaptive immune response. Cryopreservation of human monocytes was investigated to understand its effect on differentiation into immature monocyte-derived dendritic cells (imdDCs), the response to inflammatory stimuli and the ability to induce allogeneic lymphocyte proliferation. Cryopreserved (crp)-monocytes were able to differentiate into imdDCs, albeit to a lesser extent than freshly (frh)-obtained monocytes. Furthermore, crp-imdDCs had lower rates of maturation and cytokine/chemokine secretion in response to LPS than frh-imdDCs. Lower expression of Toll-like receptor 4 (at 24 and 48 h) and higher susceptibility to apoptosis in crp-imdDCs than in fresh cells would account for the impaired maturation and cytokine/chemokine secretion observed. A mixed leukocyte reaction showed that lymphocyte proliferation was lower with crp-imdDCs than with frh-imdDCs. These findings suggested that the source of monocytes used to generate human imdDCs could influence the accuracy of results observed in studies of the immune response to pathogens, lymphocyte activation, vaccination and antigen sensing. It is not always possible to work with freshly isolated monocytes but the possible effects of freezing/thawing on the biology and responsiveness of imdDCs should be taken into account.</p></div

Cryopreservation interferes with the allogeneic lymphoproliferative response.

<p>(A) Representative histogram of autologous and allogeneic lymphocyte proliferation of one donor (frh-imdDCs (black), crp-imdDCs (blue) and Lyn-stained lymphocytes without DC stimulation (red)). (B) Frh- (circles) and crp-imdDCs (squares) were co-cultured with autologous (negative control) or allogeneic lymphocytes stained with CellTracker™ Green CMFDA in a 1∶10 ratio of imdDCs:lymphocytes. After five days of co-culture lymphocytes proliferation was analyzed by flow cytometry. Data was analyzed by Student’s <i>t</i> test and the values shown are the means ± SDs of six individual donors. ***<i>p</i>≤0.001. frh: freshly obtained, crp: cryopreserved, imdDCs: immature monocyte derived dendritic cells, Lyn: stained lymphocytes without DC stimulation, Aut lyn: autologous lymphocytes, All lyn: allogeneic lymphocytes.</p

Phenotypic analyses of frh- and crp-imdDCs.

<p>Frh-imdDCs (black), crp-imdDCs (blue) and isotype-matched controls (red) were analyzed for the expression of CD11c/CD14 and CD11c/HLA-DR. (A) Representative dot blot analyses for the expression of CD11c/CD14 and CD11c/HLA-DR from one blood donor. (B) Data from six independent cultures of frh- (circles) and crp-imdDCs (squares). Data was analyzed by Student’s <i>t</i> test and the values shown are the means ± SDs of six individual donors. (C and D) Expression of IL-4 (CD124) and GM-CSF (CD116) receptors on CD11c⁺ cells during monocytes differentiation to imdDCs. Frh-monocytes (circles) and crp-monocytes (squares) were induced to differentiate to imdDCs and growth factors receptors were analyzed at 0, 4 and 7 days post-differentiation (dpd). Data was analyzed using one-way ANOVA followed by a Bonferroni test; values represent means ± SDs of six individual donors. *<i>p</i>≤0.05, frh: freshly obtained, crp: cryopreserved, imdDCs: immature monocyte derived dendritic cells.</p

Maturation of frh- and crp-imdDCs after stimulation with LPS.

<p>Expression of CD40, CD80, CD83 and CD86 on frh- and crp-imdDCs after treatment with LPS (1 µg/mL) at 0 (imdDCs), 6, 24 and 48 h. Histogram of one individual donor (representative of six). frh: freshly obtained, crp: cryopreserved, imdDCs: immature monocyte derived dendritic cells.</p

Apoptosis of frh- and crp-imdDCs.

<p>Analysis of apoptosis (Annexin V⁺/PI^-) in frh- (circles) and crp-imdDCs (squares) at 0, 6, 24 and 48 h. Data was analyzed by Student’s <i>t</i> test and the values shown are the means ± SDs of six individual donors. *<i>p</i>≤0.05.</p

Experimental tuberculosis: Designing a better model to test vaccines against tuberculosis

Experimental models of infection are good tools for establishing immunological parameters that have an effect on the host-pathogen relationship and also for designing new vaccines and immune therapies. In this work, we evaluated the evolution of experimental tuberculosis in mice infected with increasing bacterial doses or via distinct routes. We showed that mice infected with low bacterial doses by the intratracheal route were able to develop a progressive infection that was proportional to the inoculum size. In the initial phase of disease, mice developed a specific Th1-driven immune response independent of inoculum concentration. However, in the late phase, mice infected with higher concentrations exhibited a mixed Th1/Th2 response, while mice infected with lower concentrations sustained the Th1 pattern. Significant IL-10 concentrations and a more preeminent T regulatory cell recruitment were also detected at 70 days post-infection with high bacterial doses. These results suggest that mice infected with higher concentrations of bacilli developed an immune response similar to the pattern described for human tuberculosis wherein patients with progressive tuberculosis exhibit a down modulation of IFN-gamma production accompanied by increased levels of IL-4. Thus, these data indicate that the experimental model is important in evaluating the protective efficacy of new vaccines and therapies against tuberculosis. (C) 2010 Elsevier Ltd. All rights reserved.FAPESP: Foundation for the Support of Research in the State of Sao Paulo[03/11303-2]FAPESP: Foundation for the Support of Research in the State of Sao Paulo[05/01995-0]CNPq, Council for Scientific and Technological DevelopmentRede-TB, Brazilian Tuberculosis Research Networ