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Summary: Objectives: The study of a predicted outer membrane leptospiral protein encoded by the gene LIC12690 in mediating the adhesion process. Methods: The gene was cloned and expressed in Escherichia coli BL21 (SI) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and used to assess its ability to activate human umbilical vein endothelial cells (HUVECs). Results: The recombinant leptospiral protein of 95kDa, named Lp95, activated E-selectin in a dose-dependent fashion but not the intercellular adhesion molecule 1 (ICAM-1). In addition, we show that pathogenic and non-pathogenic Leptospira are both capable to stimulate endothelium E-selectin and ICAM-1, but the pathogenic L. interrogans serovar Copenhageni strain promotes a statistically significant higher activation than the non-pathogenic L. biflexa serovar Patoc (P < 0.01). The Lp95 was identified in vivo in the renal tubules of animal during experimental infection with L. interrogans. The whole Lp95 as well as its fragments, the C-terminal containing the domain of unknown function (DUF), the N-terminal and the central overlap regions bind laminin and fibronectin ECM molecules, being the binding stronger with the DUF containing fragment. Conclusion: This is the first leptospiral protein capable to mediate the adhesion to ECM components and the activation of HUVECS, thus suggesting its participation in the pathogenesis of Leptospira. [Copyright &y& Elsevier]

Accidental skin contact with the Lonomia caterpillar bristles causes a severe hemorrhagic syndrome. While fibrinolytic activation is considered to be the main cause of hemorrhage in Lonomia achelous envenomation, a consumptive coagulopathy was found to be a major component involved in the bleeding complications observed in patients envenomed by contact with Lonomia obliqua. Although we have previously observed that in L. obliqua envenomations, fibrinolysis activation appeared to be secondary to coagulation system activation, there are no reports regarding the ability of L. obliqua venom to activate directly fibrinolytic pathways. We examined the action of L. obliqua crude bristles extract (LOCBE) on several fibrinolytic system components. We demonstrated that LOCBE degraded the A-alpha fibrinogen chain only at high concentrations and after long incubation times. Under these conditions, LOCBE also induced prolongation of the fibrinogen clotting time, but no clot lysis was observed before 24 h. LOCBE did not contain t-PA- or u-PA-like activities. Gel filtration and SDS-PAGE showed that LOCBE did not induce FXIII digestion. In addition, no FXIII activity inhibition was detected by dansylcadaverin method. FXIII levels remained unchanged when FXIII was measured in fibrinogen-depleted LOCBE-treated rat plasma, suggesting that the observed 50% FXIII reduction in rats was related to consumption. In conclusion, our results clearly demonstrated that LOCBE did not display either FXIII inhibition or degradation nor fibrinolytic activity. Furthermore, although proteolytic activity on Aα fibrinogen chain was observed, cross-linked fibrin was not affected by LOCBE. [Copyright &y& Elsevier]

We investigated the contribution of human platelets to macrophage effector properties in the presence of lipopolysaccharide (LPS), as well as the beneficial effects and time frame for platelet transfusion in septic animals. Our results show that platelets sequester both pro-(TNF-α/IL-6) and anti-(IL-10) inflammatory cytokines released by monocytes. Low LPS concentrations (0.01 ng/mL) induced M2 macrophage polarization by decreasing CD64 and augmenting CD206 and CD163 expression; yet, the presence of platelets skewed monocytes toward type 1 macrophage (M1) phenotype in a cell-contact-dependent manner by the glycoprotein Ib (GPIb)-CD11b axis. Accordingly, platelet-licensed macrophages showed increased TNF-α levels, bacterial phagocytic activity, and a reduced healing capability. Platelet transfusion increased inducible nitric oxide synthase (iNOS)+ macrophages, improving bacterial clearance and survival rates in septic mice up to 6 h post-infection, an effect that was abolished by CD11b and GPIb blockade. Our results demonstrate that platelets orchestrate macrophage effector responses, improving the clinical outcome of sepsis in a narrow but relevant time frame. • Platelets sequester pro- and anti-inflammatory cytokines released by monocytes • In the presence of LPS, platelets skew monocytes toward a pro-inflammatory phenotype • During sepsis, platelet transfusion increases iNOS+ macrophages and bacterial clearance • Platelet transfusion increases septic mice survival in a narrow time frame Carestia et al. describe the beneficial role of platelet transfusion for animal survival during sepsis by reprogramming macrophages and fostering antimicrobial functions. The cross-talk between platelets and monocytes that promotes pro-inflammatory macrophages depends on the intimal cellular contact between platelet GPIb and CD11b in the monocyte surface. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Platelets express Toll-like receptors (TLRs) that recognise molecular components of pathogens and, in nucleated cells, elicit immune responses through nuclear factor-kappaB (NF-κB) activation. We have shown that NF-κB mediates platelet activation in response to classical agonists, suggesting that this transcription factor exerts non-genomic functions in platelets. The aim of this study was to determine whether NF-κB activation is a downstream signal involved in TLR2 and 4-mediated platelet responses. Material and methods: Aggregation and ATP release were measured with a Lumi-aggregometer. Fibrinogen binding, P-selectin and CD40 ligand (CD40L) levels and platelet-neutrophil aggregates were measured by cytometry. I kappa B alpha (IκBα) degradation and p65 phosphorylation were determined by Western blot and von Willebrand factor (vWF) by ELISA. Results: Platelet stimulation with Pam3CSK4 or LPS resulted in IκBα degradation and p65 phosphorylation. These responses were suppressed by TLR2 and 4 blocking and synergised by thrombin. Aggregation, fibrinogen binding and ATP and vWF release were triggered by Pam3CSK4. LPS did not induce platelet responses per se, except for vWF release, but it did potentiate thrombin-induced aggregation, fibrinogen binding and ATP secretion. Pam3CSK4, but not LPS, induced P-selectin and CD40L expression and mixed aggregate formation. All of these responses, except for CD40L expression, were inhibited in platelets treated with the NF-κB inhibitors BAY 11-7082 or Ro 106-9920. Conclusion: TLR2 and 4 agonists trigger platelet activation responses through NF-κB. These data show another non-genomic function of NF-κB in platelets and highlight this molecule as a potential target to prevent platelet activation in inflammatory or infectious diseases. [Copyright &y& Elsevier]

Abstract: Argentine haemorrhagic fever (AHF) is a systemic febrile syndrome characterized by several haematological and neurological alterations caused by Junín virus (JUNV), a member of the Arenaviridae family. Newborn mice are highly susceptible to JUNV and the course of infection has been associated with the viral strain used. Galectin-3 (Gal-3) is an animal lectin that has been proposed to play an important role in some central nervous system (CNS) diseases. In this study, we analysed Gal-3 expression at the transcriptional and translational expression levels during JUNV-induced CNS disease. We found that Candid 1 strain induced, with relatively low mortality, a subacute/chronic CNS disease with significant glia activation and upregulation of Gal-3 in microglia cells as well as in reactive astrocytes that correlated with viral levels. Our results suggest an important role for Gal-3 in viral-induced CNS disease. [Copyright &y& Elsevier]

Objective: Megakaryo/thrombopoiesis is a complex process regulated by multiple signals provided by the bone marrow microenvironment. Because macrophages are relevant components of the bone marrow stroma and their activation induces an upregulation of molecules that can regulate hematopoiesis, we analyzed the impact of these cells on the control of megakaryocyte development and platelet biogenesis. Materials and Methods: The different stages of megakaryo/thrombopoiesis were analyzed by flow cytometry using an in vitro model of human cord blood CD34+ cells stimulated with thrombopoietin in either a transwell system or conditioned media from monocyte-derived macrophages isolated from peripheral blood. Cytokines secreted from macrophages were characterized by protein array and enzyme-linked immunosorbent assay. Results: Resting macrophages released soluble factors that promoted megakaryocyte growth, cell ploidy, a size increase, proplatelet production, and platelet release. Lipopolysaccharide stimulation triggered the secretion of cytokines that exerted opposite effects together with a dramatic switch of CD34+ commitment to the megakaryocytic lineage toward the myeloid lineage. Neutralization of interleukin-8 released by stimulated macrophages partially reversed the inhibition of megakaryocyte growth. Activation of nuclear factor κB had a major role in the synthesis of molecules involved in the megakaryocyte inhibition mediated by lipopolysaccharide-stimulated macrophages. Conclusions: Our study extends our understanding about the role of the bone marrow microenvironment in the regulation of megakaryo/thrombopoiesis by showing that soluble factors derived from macrophages positively or negatively control megakaryocyte growth, differentiation, maturation, and their ability to produce platelets. [Copyright &y& Elsevier]

Abstract: Junín virus of the Arenaviridae family is the etiological agent of Argentine hemorrhagic fever, a febrile syndrome causing hematological and neurological symptoms. We review historical perspectives of current knowledge on the disease, and update information related to the virion and its potential pathogenic mechanisms. [Copyright &y& Elsevier]

Abstract: In a previous study of experimental murine encephalitis induced by Junín virus (JV), an arenavirus, we showed increased expression of iNOS by unidentified cells, concomitant with the astrocyte reaction. The specific inhibition of iNOS was associated with greater mortality but lower astrocytosis, suggesting that the protective role of nitric oxide (NO) synthesized by iNOS was related to enhanced astrocyte activation, representing a beneficial cellular response to virus-induced central nervous system damage. In the present work, cultured astrocytes were used to study whether JV infection could trigger iNOS expression and assess its eventual relationship with viral replication, glial fibrilary acidic protein (GFAP) expression levels and the presence of apoptosis. We found that JV infection of astrocytes did not induce apoptosis but produced both increased iNOS synthesis, detected by immunocytochemistry and fluorescence activated cell sorting (FACS) analysis, and increased NO, which was indirectly measured by nitrite/nitrate levels. These changes occurred early relative to the increases in GFAP expression, as detected by immunocytochemistry, FACS analysis and RT-PCR. The fact that iNOS inhibition abolished enhanced GFAP expression in infected monolayers suggests that NO was directly involved. In addition, iNOS inhibition enhanced virus replication. Together with data from confocal microscopy, these results suggest that JV induces iNOS expression in infected astrocytes and that the resulting NO has an important role both in reducing viral replication and in enhancing subsequent astrocyte activation. [Copyright &y& Elsevier]

Objective: Although cAMP is involved in a number of physiologic functions, its role in hematopoietic cell fate decision remains poorly understood. We have recently demonstrated that in CD34+-derived megakaryocytes, cAMP-related agents prevent apoptosis. In this study we addressed the question of whether cAMP also regulates survival of their precursors, CD34+ cells. Methods: Apoptosis was evaluated by fluorescence microscopy, and detection of hypodiploid or annexin V+ cells by flow cytometry. Mitochondrial membrane potential and bcl-xL or caspase-3 expression were assessed by flow cytometry. Colony-forming units were studied by clonogenic assays in methylcellulose. Results: We found that two different cAMP analogs such as Dibutiril-cAMP and sp-5,6-DCl-BIMPS (BIMPS) promoted survival of human umbilical cord–derived CD34+ cells by suppressing apoptosis induced by either nitric oxide (NO) or serum deprivation. Involvement of PKA and PI3K pathway was demonstrated by the ability of their specific inhibitors Rp-cAMP and Wortmannin or LY294002 respectively to reverse the antiapoptotic effect of BIMPS. Treatment of CD34+ cell with BIMPS not only restrained the bcl-xL downregulation but also suppressed the loss of mitochondrial membrane potential and caspase-3 activation induced by serum starvation. While thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF) were not able to increase cAMP levels, the antiapoptotic activity exerted by these growth factors was blocked by inhibition of the adenylate cyclase and synergized by BIMPS. Cyclic AMP analogs suppressed the decreased colony formation in cells exposed to NO or serum deprivation. Conclusion: Altogether, our results strongly suggest that cAMP appears to be not only a key pathway controlling CD34+ survival, but also a mediator of the TPO-, G-CSF- and SCF-mediated cytoprotection. [Copyright &y& Elsevier]