Coelho SVA, Neris RLS, Papa MP, Schnellrath LC, Meuren LM, Tschoeke DA, Leomil L, Verçoza BRF, Miranda M, Thompson FL, Da Poian AT, Souza TML, Carneiro FA, Damaso CR, Assunção-Miranda I, de Arruda LB. Development of standard methods for Zika virus propagation, titration, and purification. J Virol Methods. 2017 Aug;246:65-74.
DOI: 10.1016/j.jviromet.2017.04.011
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The emergence of Zika virus (ZIKV) infection has stimulated several research groups to study and collaborate to understand virus biology and pathogenesis. These efforts may assist with the development of antiviral drugs, vaccines and diagnostic tests, as well as to promote advancements in public health policies. Here, we aim to develop standard protocols for propagation, titration, and purification of ZIKV strains, by systematically testing different cell types, kinetics, multiplicity of infection and centrifugation protocols. ZIKV produces a productive infection in human, non-human primate, and rodents-derived cell lines, with different efficacies. The highest yield of ZIKV-AFR and ZIKV-BR infectious progeny was obtained at 7 days post infection in C6/36 cells (7 × 107 and 2 × 108 PFU/ml, respectively). However, high titers of ZIKV-AFR could be obtained at earlier time points in Vero cells (2.5 × 107 PFU/ml at 72 hpi), whereas ZIKV-BR titers reached 108 PFU/ml at 4dpi in C6/36 cells. High yield of purified virus was obtained by purification through a discontinuous sucrose gradient. This optimized procedure will certainly contribute to future studies of virus structure and vaccine development. Beyond the achievement of efficient virus propagation, the normalization of these protocols will also allow different laboratories around the world to better compare and discuss data regarding different features of ZIKV biology and disease, contributing to more efficient collaborations and progression in ZIKV research.
Pereira JC, Ramos TD, Silva JD, de Mello MF, Pratti JES, da Fonseca-Martins AM, Firmino-Cruz L, Kitoko JZ, Chaves SP, Gomes DCO, Diaz BL, Rocco PRM, de Matos Guedes HL. Effects of Bone Marrow Mesenchymal Stromal Cell Therapy in Experimental Cutaneous Leishmaniasis in BALB/c Mice Induced by Leishmania amazonensis. Front Immunol. 2017 Aug 10;8:893.
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Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis. In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106 Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously administered. As a conclusion, in the current murine model of L. amazonensis-induced cutaneous disease, MSCs did not control the damage of cutaneous disease and, depending on the administration route, it could result in deleterious effects.
Mendes MA, de Carvalho DS, Amadeu TP, Silva BJA, Prata RBDS, da Silva CO, Ferreira H, Hacker MA, Nery JAC, Pinheiro RO, Sampaio EP, Sarno EN, Schmitz V. Elevated Pentraxin-3 Concentrations in Patients With Leprosy: Potential Biomarker of Erythema Nodosum Leprosum. J Infect Dis. 2017 Dec 19;216(12):1635-1643.
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Leprosy, the leading infectious cause of disability worldwide, remains a major public health challenge in the most severely affected countries despite the sharp decline in new cases in recent years. The search for biomarkers is essential to achieve a better understanding of the molecular and cellular mechanisms underlying the disease.
Travassos LH, Vasconcellos LR, Bozza MT, Carneiro LA. Heme and iron induce protein aggregation. Autophagy. 2017 Mar 4;13(3):625-626.
DOI: 10.1080/15548627.2016.1271515
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Heme is an essential molecule expressed in many tissues where it plays key roles as the prosthetic group of several proteins involved in vital physiological and metabolic processes such as gas and electron transport. Structurally, heme is a tetrapyrrole ring containing an atom of iron (Fe) in its center. When released into the extracellular milieu, heme exerts several deleterious effects, which make it an important player in infectious and noninfectious hemolytic diseases where large amounts of free heme are observed such as malaria, dengue fever, β-thalassemia, sickle cell disease and ischemia-reperfusion. Our recent work has uncovered an unappreciated cellular response triggered by heme or Fe, one of its degradation products, on macrophages, which is the formation of protein aggregates known as aggresome-like induced structres (ALIS). This response was shown to be fully dependent on ROS production and the activation of the transcription factor NFE2L2/NRF2. In addition, we have demonstrated that heme degradation by HMOX1/HO-1 (heme oxygenase 1) is required and that Fe is essential for the formation of ALIS, as heme analogs lacking the central atom of Fe are not able to induce these structures. ALIS formation is also observed in vivo, in a model of phenylhydrazine (PHZ)-induced hemolysis, indicating that it is an integral part of the host response to excessive free heme and that it may play a role in cellular homeostasis.
Babdor J, Descamps D, Adiko AC, Tohmé M, Maschalidi S, Evnouchidou I, Vasconcellos LR, De Luca M, Mauvais FX, Garfa-Traore M, Brinkmann MM, Chignard M, Manoury B, Saveanu L. IRAP+ endosomes restrict TLR9 activation and signaling. Nat Immunol. 2017 May;18(5):509-518.
DOI: 10.1038/ni.3711
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The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.
de Castro LL, Xisto DG, Kitoko JZ, Cruz FF, Olsen PC, Redondo PAG, Ferreira TPT, Weiss DJ, Martins MA, Morales MM, Rocco PRM. Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma. Stem Cell Res Ther. 2017 Jun 24;8(1):151.
DOI: 10.1186/s13287-017-0600-8
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Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma.
Nascimento CR, Andrade D, Carvalho-Pinto CE, Serra RR, Vellasco L, Brasil G, Ramos-Junior ES, da Mota JB, Almeida LN, Andrade MV, Correia Soeiro MN, Juliano L, Alvarenga PH, Oliveira AC, Sicuro FL, de Carvalho ACC, Svensjö E, Scharfstein J. Mast Cell Coupling to the Kallikrein-Kinin System Fuels Intracardiac Parasitism and Worsens Heart Pathology in Experimental Chagas Disease. Front Immunol. 2017 Aug 2;8:840.
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During the course of Chagas disease, infectious forms of Trypanosoma cruzi are occasionally liberated from parasitized heart cells. Studies performed with tissue culture trypomastigotes (TCTs, Dm28c strain) demonstrated that these parasites evoke neutrophil/CXCR2-dependent microvascular leakage by activating innate sentinel cells via toll-like receptor 2 (TLR2). Upon plasma extravasation, proteolytically derived kinins and C5a stimulate immunoprotective Th1 responses via cross-talk between bradykinin B2 receptors (B2Rs) and C5aR. Awareness that TCTs invade cardiovascular cells in vitro via interdependent activation of B2R and endothelin receptors [endothelin A receptor (ETAR)/endothelin B receptor (ETBR)] led us to hypothesize that T. cruzi might reciprocally benefit from the formation of infection-associated edema via activation of kallikrein–kinin system (KKS). Using intravital microscopy, here we first examined the functional interplay between mast cells (MCs) and the KKS by topically exposing the hamster cheek pouch (HCP) tissues to dextran sulfate (DXS), a potent “contact” activator of the KKS. Surprisingly, although DXS was inert for at least 30 min, a subtle MC-driven leakage resulted in factor XII (FXII)-dependent activation of the KKS, which then amplified inflammation via generation of bradykinin (BK). Guided by this mechanistic insight, we next exposed TCTs to “leaky” HCP—forged by low dose histamine application—and found that the proinflammatory phenotype of TCTs was boosted by BK generated via the MC/KKS pathway. Measurements of footpad edema in MC-deficient mice linked TCT-evoked inflammation to MC degranulation (upstream) and FXII-mediated generation of BK (downstream). We then inoculated TCTs intracardiacally in mice and found a striking decrease of parasite DNA (quantitative polymerase chain reaction; 3 d.p.i.) in the heart of MC-deficient mutant mice. Moreover, the intracardiac parasite load was significantly reduced in WT mice pretreated with (i) cromoglycate (MC stabilizer) (ii) infestin-4, a specific inhibitor of FXIIa (iii) HOE-140 (specific antagonist of B2R), and (iv) bosentan, a non-selective antagonist of ETAR/ETBR. Notably, histopathology of heart tissues from mice pretreated with these G protein-coupled receptors blockers revealed that myocarditis and heart fibrosis (30 d.p.i.) was markedly and redundantly attenuated. Collectively, our study suggests that inflammatory edema propagated via activation of the MC/KKS pathway fuels intracardiac parasitism by generating infection-stimulatory peptides (BK and endothelins) in the edematous heart tissues.
