Dengue Virus Directly Stimulates Polyclonal B Cell Activation.

Dengue infection is associated to vigorous inflammatory response, to a high frequency of activated B cells, and to increased levels of circulating cross-reactive antibodies. We investigated whether direct infection of B cells would promote activation by culturing primary human B lymphocytes from healthy donors with DENV in vitro. B cells were susceptible, but poorly permissive to infection. Even though, primary B cells cultured with DENV induced substantial IgM secretion, which is a hallmark of polyclonal B cell activation. Notably, DENV induced the activation of B cells obtained from either DENV immune or DENV naïve donors, suggesting that it was not dependent on DENV-specific secondary/memory response. B cell stimulation was dependent on activation of MAPK and CD81. B cells cultured with DENV also secreted IL-6 and presented increased expression of CD86 and HLA-DR, which might contribute to B lymphocyte co-stimulatory function. Indeed, PBMCs, but not isolated B cells, secreted high amounts of IgG upon DENV culture, suggesting that interaction with other cell types in vivo might promote Ig isotype switching and IgG secretion from different B cell clones. These findings suggest that activation signaling pathways triggered by DENV interaction with non-specific receptors on B cells might contribute to the exacerbated response observed in dengue patients.

Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

Immunobiologic and antiinflammatory properties of a bark extract from Ampelozizyphus amazonicus Ducke.

Ampelozizyphus amazonicus is used in the treatment and prevention of malaria. The effect of an aqueous extract from this plant (SART) on the immune response was investigated by measuring immunoglobulin production induced by immunization with the antigen TNP-Ficoll in Plasmodium chabaudi-infected mice. SART treatment increased antigen-specific IgM and IgG levels in TNP-Ficoll-immunized mice. The B cell response during malarial infection was also modified by SART. There was an increase in total serum IgM and IgG and a decrease in the percentage of splenic plasma cells (CD138+ cells) in P. chabaudi-infected, SART-treated animals. SART (1, 3 or 10 mg/kg, p.o.) and the reference drug dexamethasone (5 mg/kg) were also tested in carrageenan-induced leukocyte migration to the subcutaneous air pouch (SAP). All SART doses significantly reduced leukocyte migration into the SAP. The protein concentration resulting from extravasation into the peritoneum was also significantly reduced. Our data indicate that SART possesses immunomodulatory properties, inducing an in vivo modification of the B lymphocyte response and anti-inflammatory properties, which are partly due to a reduction in cell migration and are most likely due to an inhibition of the production of inflammatory mediators. Preliminary HPLC-ESI-MS/MS analysis of SART shows a complex saponin profile with deprotonated molecule [M-H](-) ions in the range of m/z 800-1000.

Bradykinin enhances Sindbis virus infection in human brain microvascular endothelial cells.

Sindbis virus (SINV) induces inflammatory and vasoactive responses that are associated with rash and arthritis in human infections. The mechanisms underlying infection-associated microvasculopathy are still unknown. We investigated whether endothelial cells infected by SINV are differentially responsive to bradykinin (BK), a potent inducer of inflammatory edema in a broad range of infectious diseases. Human endothelial cells (HBMECs) infected with SINV presented an upregulation of bradykinin B2 receptors (BK2R) expression. Also, BK reduced SINV-induced apoptosis and enhanced virus replication in HBMECs in a way dependent on BK2R, PI3 kinase and ERK signaling. Strikingly, intracerebral infection of mice in the presence of a BK2R antagonist reduced the local viral load. Our data suggest that SINV infection renders human endothelial cells hypersensitive to BK, which increases host cell survival and viral replication. Ongoing studies may clarify if the deregulation of the kinin pathway contributes to infection-associated vasculopathies in life-threatening arbovirus infections.