Baculovirus surface display of a chimeric E-NS1 protein of YFV protects against YFV infection.

Yellow fever (YF) is a disease caused by the homonymous flavivirus that can be prevented by a vaccine containing attenuated viruses. Since some individuals cannot receive this vaccine, the development of alternatives is desirable. Here, we developed a recombinant baculovirus (rBV) surface display platform utilizing a chimeric E-NS1 protein as a vaccine candidate. A pBacPAK9 vector containing the baculoviral GP64 signal peptide, the YFV prM, E, NS1 and the ectodomain of VSV-G sequences was synthesized. This transfer plasmid and the bAcGOZA bacmid were cotransfected into Sf9 cells, and an rBV-E-NS1 was obtained, which was characterized by PCR, WB, IFI and FACS analysis. Mice immunized with rBV-E-NS1 elicited a specific humoral and cellular immune response and were protected after YFV infection. In summary, we have developed an rBV that expresses YFV major antigen proteins on its surface, which opens new alternatives that can be tested in a mouse model.

Phagocyte Escape of Leptospira: The Role of TLRs and NLRs.

The spirochetal bacteria Leptospira spp. are causative agents of leptospirosis, a globally neglected and reemerging zoonotic disease. Infection with these pathogens may lead to an acute and potentially fatal disease but also to chronic asymptomatic renal colonization. Both forms of disease demonstrate the ability of leptospires to evade the immune response of their hosts. In this review, we aim first to recapitulate the knowledge and explore the controversial data about the opsonization, recognition, intracellular survival, and killing of leptospires by scavenger cells, including platelets, neutrophils, macrophages, and dendritic cells. Second, we will summarize the known specificities of the recognition or escape of leptospire components (the so-called microbial-associated molecular patterns; MAMPs) by the pattern recognition receptors (PRRs) of the Toll-like and NOD-like families. These PRRs are expressed by phagocytes, and their stimulation by MAMPs triggers pro-inflammatory cytokine and chemokine production and bactericidal responses, such as antimicrobial peptide secretion and reactive oxygen species production. Finally, we will highlight recent studies suggesting that boosting or restoring phagocytic functions by treatments using agonists of the Toll-like or NOD receptors represents a novel prophylactic strategy and describe other potential therapeutic or vaccine strategies to combat leptospirosis.

Bovine macrophages responses to the infection with virulent and attenuated Leptospira interrogans serovar Pomona.

Leptospirosis is a zoonosis, caused by pathogenic spirochetes of the genus Leptospira. Although cattle are usually the maintenance hosts of serovar Hardjo, Pomona is the most frequent serovar circulating in Argentina. The understanding of bovine innate immune response and the virulence of this serovar is important for future control measures. This work compares infection of bovine macrophages with the virulent L. interrogans sv Pomona strain AKRFB (P1) and its attenuated counterpart (P19). First, we confirmed attenuation in the hamster model. Mortality and lung hemorrhages occurred after P1 inoculation, while the survival rate was 100% in P19-infected animals. Cells infected with both strains showed statistically upregulated gene expression of pro-inflammatory cytokines, IL-1ß, IL-6 and TNFα. The level of expression of anti-inflammatory cytokine IL-10 was statistically different between strains. Increased expression of IL-10 was observed only in P1-infected cells. For the first time, we describe macrophages extracellular traps induced by infection of bovine macrophages (bMETs) with both, the virulent and attenuated Leptospira interrogans Pomona strains. P1 was found higher internalized when the phagocytosis was inhibited, suggesting a cell entrance of this strain also by an independent-phagocytosis pathway. Furthermore, P1 was higher colocalized with acidic and late endosomal compartments compared with P19. This data emphasizes the importance to deepen in Leptospira bovine macrophages particular invasion mechanisms and, furthermore, underline the value of studying the main hosts.

Role of neutrophils in CVB3 infection and viral myocarditis.

Coxsackievirus B3 (CVB3) is a globally prevalent enterovirus of the Picornaviridae family that is frequently associated with viral myocarditis (VM). Neutrophils, as first responders, may be key cells in determining viral disease outcomes; however, neutrophils have been poorly studied with respect to viral infection. Although neutrophils have been ascribed a relevant role in early cardiac inflammation, their precise role in CVB3 infection has not yet been evaluated. In this study, we aimed to determine if the interaction between human neutrophils and CVB3 could lead to viral replication and/or modulation of neutrophil survival and biological functions, and whether neutrophil depletion in a murine model has a beneficial or harmful effect on CVB3 infection. Our results show that CVB3 interacted with but did not replicate in human neutrophils. Neutrophils recognized CVB3 mainly through endosomal TLR-8, and infection triggered NFκB activation. Virus internalization resulted in increased cell survival, up-regulation of CD11b, enhanced adhesion to fibrinogen and fibronectin, and the secretion of IL-6, IL-1ß, TNF-α, and IL-8. Supernatants from infected neutrophils exerted chemotactic activity partly mediated by IL-8. The infected neutrophils released myeloperoxidase and triggered neutrophil extracellular trap formation in the presence of TNF-α. In mice infected with CVB3, viral RNA was detected in neutrophils as well as in mononuclear cells. After neutrophil depletion, mice showed reduced VM reflected by a reduction in viral titers, cell exudates, and CCL-2 mRNA levels, as well as the abrogation of reactive cardiomyocyte hypertrophy. Our results indicate that neutrophils have relevant direct and indirect roles in the pathogenesis of CVB3-induced VM.

Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets.

In addition to being key elements in hemostasis and thrombosis, platelets amplify neutrophil function. We aimed to gain further insight into the stimuli, mediators, molecular pathways, and regulation of neutrophil extracellular trap formation mediated by human platelets. Platelets stimulated by lipopolysaccharide, a wall component of gram-negative bacteria, Pam3-cysteine-serine-lysine 4, a mimetic of lipopeptide from gram-positive bacteria, Escherichia coli, Staphylococcus aureus, or physiologic platelet agonists promoting neutrophil extracellular trap formation and myeloperoxidase-associated DNA activity under static and flow conditions. Although P-selectin or glycoprotein IIb/IIIa were not involved, platelet glycoprotein Ib, neutrophil cluster of differentiation 18, and the release of von Willebrand factor and platelet factor 4 seemed to be critical for the formation of neutrophil extracellular traps. The secretion of these molecules depended on thromboxane A(2) production triggered by lipopolysaccharide or Pam3-cysteine-serine-lysine 4 but not on high concentrations of thrombin. Accordingly, aspirin selectively inhibited platelet-mediated neutrophil extracellular trap generation. Signaling through extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Src kinases, but not p38 or reduced nicotinamide adenine dinucleotide phosphate oxidase, was involved in platelet-triggered neutrophil extracellular trap release. Platelet-mediated neutrophil extracellular trap formation was inhibited by prostacyclin. Our results support a role for stimulated platelets in promoting neutrophil extracellular trap formation, reveal that an endothelium-derived molecule contributes to limiting neutrophil extracellular trap formation, and highlight platelet inhibition as a potential target for controlling neutrophil extracellular trap cell death.

Activation of cyclic AMP pathway prevents CD34(+) cell apoptosis.

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.

Aspirin and salicylate suppress polymorphonuclear apoptosis delay mediated by proinflammatory stimuli.

During inflammation, polymorphonuclear leukocyte (PMN) apoptosis can be delayed by different proinflammatory mediators. Classically, it has been accepted that the widely used anti-inflammatory drug acetyl salicylic acid (ASA) exerts its action through inhibition of cyclooxygenases and subsequent prostaglandin synthesis. We hypothesized that another anti-inflammatory action of ASA could be the shortening of PMN survival. We found that at therapeutic concentrations (1-3 mM), ASA and its metabolite salicylate (NaSal), but not indomethacin or ibuprofen, counteracted the prolonged PMN survival mediated by lipopolysaccharide (LPS) through inhibition of nuclear factor-kappaB (NF-kappaB) activation. Both salicylates also inhibited interleukin (IL)-1alpha or acidic conditions antiapoptotic activity. Higher concentrations of both drugs had a direct apoptotic effect. Salicylates were not effective when PMN apoptosis delay was induced by granulocyte macrophage-colony-stimulating factor (GM-CSF), a NF-kappaB-independent cytokine. Promotion of PMN survival by the combination of IL-1alpha and LPS was also reversed by salicylates, but higher concentrations were required. ASA concentrations that did not trigger PMN death increase the zymosan- or tumor necrosis factor-alpha-mediated proapoptotic effect. The LPS- and IL-1alpha- but not GM-CSF-mediated antiapoptotic effect was markedly reduced in PMNs from donors who had ingested ASA. Using a thioglycolate-induced peritonitis model, we showed that in ASA- or NaSal-treated mice there was not only a decrease in the number of cells recruited but also an increase in the percentage of apoptotic PMNs as well as an enhancement of phagocytosis compared with controls. Our findings demonstrate that acceleration of PMN apoptosis by turning off the NF-kappaB-mediated survival signals elicited by proinflammatory stimuli is another anti-inflammatory action of ASA and NaSal.

Endothelial cell function alteration after Junin virus infection.

Hematologic involvement is the main feature of Argentine hemorrhagic fever (AHF), an endemo-epidemic disease caused by Junin virus (JV). Since endothelial dysfunction could play a role in AHF-altered hemostasis, we studied human umbilical vein endothelial cell (HUVEC) infection with a virulent (JVv) and a non-virulent (JVa) JV strain. Cells were infected by the two JV variants with no detectable apoptosis or cytopathic effect. Both viral variants up-regulated ICAM-1 and VCAM-1 levels, while von Willebrand factor (VWF) production was decreased. Prostacyclin (PGI2) release and decay accelerating factor (DAF) expression were greater in JVv- than in JVa-infected or control cells. Furthermore, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression was only raised in JVv-infected supernatants. Significant NO and PGI2 values were also detected in AHF patient sera. These data demonstrate that endothelial cell responses are triggered subsequently by JV infection, suggesting that such alterations play a major role in the pathogenesis of AHF and perhaps in other viral-induced hemorrhagic diseases.