Soluble flagellin coimmunization attenuates Th1 priming to Salmonella and clearance by modulating dendritic cell activation and cytokine production.

Soluble flagellin (sFliC) from Salmonella Typhimurium (STm) can induce a Th2 response to itself and coadministered antigens through ligation of TLR5. These properties suggest that sFliC could potentially modulate responses to Th1 antigens like live STm if both antigens are given concurrently. After coimmunization of mice with sFliC and STm there was a reduction in Th1 T cells (T-bet(+) IFN-γ(+) CD4 T cells) compared to STm alone and there was impaired clearance of STm. In contrast, there was no significant defect in the early extrafollicular B-cell response to STm. These effects are dependent upon TLR5 and flagellin expression by STm. The mechanism for these effects is not related to IL-4 induced to sFliC but rather to the effects of sFliC coimmunization on DCs. After coimmunization with STm and sFliC, splenic DCs had a lower expression of costimulatory molecules and profoundly altered kinetics of IL-12 and TNFα expression. Ex vivo experiments using in vivo conditioned DCs confirmed the effects of sFliC were due to altered DC function during a critical window in the coordinated interplay between DCs and naïve T cells. This has marked implications for understanding how limits in Th1 priming can be achieved during infection-induced, Th1-mediated inflammation.

Differential activation of dendritic cells by Mycobacterium tuberculosis Beijing genotype.

Mycobacterium tuberculosis (Mtb) inhibits dendritric cells (DC) function in order to delay T cell response. Furthermore, there is increasing evidence that genetic diversity of Mtb strains can affect their interaction with the immune system. Beijing genotype has attracted attention because of its high prevalence and multi-drug resistance. Although it is known that this genotype is hypervirulent and differentially activates macrophages when compared to other genotypes, little is known about its interaction with DC. In order to address this issue, murine bone marrow derived DC (BMDC) were stimulated with soluble extracts (SE) from BCG, H37Rv, Canetti and Beijing genotypes. We observed that unlike other mycobacteria strains, SE-Beijing was unable to induce maturation of DC as assessed by cell surface MHC-II expression. DC stimulated with SE-Beijing failed to produce IL-12 and TNF-α, but did secrete IL-10. Interestingly, SE-Beijing induced CCR7 and PDL-1 on BMDC, but did not induce the expression of CD86. When BMDC stimulated with SE-Beijing were used to activate CD4+ cells they were unable to induce a Th1 response when compared with less virulent genotypes. These results indicate that Beijing is able to modulate DC activation and function, which may be related to the pathogenesis induced by this genotype.

Activation of regulatory dendritic cells by Mertk coincides with a temporal wave of apoptosis in neonatal lungs.

Multiple mechanisms restrain inflammation in neonates, most likely to prevent tissue damage caused by overly robust immune responses against newly encountered pathogens. Here, we identify a population of pulmonary dendritic cells (DCs) that express intermediate levels of CD103 (CD103int) and appear in the lungs and lung-draining lymph nodes of mice between birth and 2 weeks of age. CD103int DCs express XCR1 and CD205 and require expression of the transcription factor BATF3 for development, suggesting that they belong to the cDC1 lineage. In addition, CD103int DCs express CCR7 constitutively and spontaneously migrate to the lung-draining lymph node, where they promote stromal cell maturation and lymph node expansion. CD103int DCs mature independently of microbial exposure and TRIF- or MyD88-dependent signaling and are transcriptionally related to efferocytic and tolerogenic DCs as well as mature, regulatory DCs. Correlating with this, CD103int DCs show limited ability to stimulate proliferation and IFN-γ production by CD8+ T cells. Moreover, CD103int DCs acquire apoptotic cells efficiently, in a process that is dependent on the expression of the TAM receptor, Mertk, which drives their homeostatic maturation. The appearance of CD103int DCs coincides with a temporal wave of apoptosis in developing lungs and explains, in part, dampened pulmonary immunity in neonatal mice. Together, these data suggest a mechanism by which DCs sense apoptotic cells at sites of noninflammatory tissue remodeling, such as tumors or the developing lungs, and limit local T cell responses.

Elevated levels of enteric IgA in an unimmunised mouse model of Hyper IgM syndrome derived from gut-associated secondary lymph organs even in the absence of germinal centres.

Introduction: Patients with Human Hyper IgM syndromes (HIGM) developed pulmonary and gastrointestinal infections since infancy and most patients have mutations in the CD40 ligand (CD40L) gene. Most HIGM patients compared to healthy subjects have higher/similar IgM and lower IgG, and IgA serum concentrations but gut antibody concentrations are unknown. CD40L on activated T-cells interacts with CD40 on B-cells, essential for the formation of germinal centres (GCs) inside secondary lymphoid organs (SLOs), where high-affinity antibodies, long-lived antibody-secreting plasma cells, and memory B-cells, are produced. C57BL6-CD40 ligand deficient mice (C57BL6-cd40l -/-), are a model of HIGM, because serum immunoglobulin concentrations parallel levels observed in HIGM patients and have higher faecal IgA concentrations. In mice, TGFß and other cytokines induce IgA production. Aims: To compare and evaluate B-cell populations and IgA-producing plasma cells in peritoneal lavage, non-gut-associated SLOs, spleen/inguinal lymph nodes (ILN), and gut-associated SLOs, mesenteric lymph nodes (MLN)/Peyer´s patches (PP) of unimmunised C57BL6-cd40l -/- and C57BL6-wild-type (WT) mice. Material and methods: Peritoneal lavages, spleens, ILN, MLN, and PP from 8-10 weeks old C57BL6-cd40l -/- and WT mice, were obtained. Organ cryosections were analysed by immunofluorescence and B-cell populations and IgA-positive plasma cell suspensions by flow cytometry. Results: In unimmunised WT mice, GCs were only observed in the gut-associated SLOs, but GCs were absent in all C57BL6-cd40l -/- SLOs. PP and MLN of C57BL6-cd40l -/- mice exhibited a significantly higher number of IgA-producing cells than WT mice. In the spleen and ILN of C57BL6-cd40l- /- mice IgA-producing cells significantly decreased, while IgM-positive plasma cells increased. C57BL6-cd40l -/- B-1 cells were more abundant in all analysed SLOs, whereas in WT mice most B-1 cells were contained within the peritoneal cavity. C57BL6-cd40l -/- B-cells in MLN expressed a higher TGFß receptor-1 than WT mice. Mouse strains small intestine microvilli (MV), have a similar frequency of IgA-positive cells. Discussion: Together our results confirm the role of PP and MLN as gut inductive sites, whose characteristic features are to initiate an IgA preferential immune response production in these anatomical sites even in the absence of GCs. IgA antibodies play a pivotal role in neutralising, eliminating, and regulating potential pathogens and microorganisms in the gut.

Neonate antigen presenting cells within murine intestinal muscular layer.

The intestinal mucosa is exposed to a vast antigenic contact. Several antigen presenting cell (APCs) have been described within the gut associated lymphoid tissue (GALT) (Peyer's patches, lamina propria, mesenteric lymph nodes, muscular layer); however, this has been done almost exclusively in adult organisms. As there is no characterization of intestinal muscular layer's APCs during early neonate development we adapted the conventional technique used in adults, to the neonate intestine. We obtained the intestinal muscular layer from early neonates (days 0-3 upon birth) and from young mice (2 and 3 weeks after birth). A planar network of CD45(+), MHC-II(+), DEC-205(+) cells with irregular, some with prominent dendritic morphology was found at birth under basal physiological conditions, whereas Langerin(+) DCs appeared after two weeks. The variations seen in CD45(+), MHC-II(+) and DEC-205(+) cells along the early neonatal development, could be related to the new challenges by intestinal antigen exposure from the newborn diet (breast milk, solid food), and to important environmental changes (start walking, exploring the surroundings, etc). Our study reveals the presence of APCs in intestinal muscular layer at birth, and their subsequent changes in physiological, non-induced conditions, contributing basic information about these cells in the neonate intestinal immune system.

Reduced in vivo cytotoxicity and increased Mycobacterial burden are associated with virulent Mycobacterium tuberculosis strains during lung infection.

Cytotoxic cellular responses are crucial for clearing intracellular pathogens and generating host resistance. Experimental pulmonary tuberculosis is associated with an early delay in T cell responses and with elevated lung bacterial burden during chronic infection. In this study we quantified the in vivo cytotoxicity and the mycobacterial burden from two pertinent tissues in groups of mice infected each with a mycobacterial strain of different virulence. None of the strains induced cytotoxic responses during early (day 14) infection. Interestingly, at 21 and 60 days post-infection, Mycobacterium canettii (lowest virulence) triggered the strongest in vivo cytotoxicity both in lungs and mediastinal lymph nodes. In contrast, Mycobacterium tuberculosis H37Rv (intermediate virulence) and Beijing strains (highest virulence) induced lower cytotoxic responses, and exhibited high bacterial growth, especially in lungs. These in vivo data suggest that virulence of Mycobacterium strains are somehow associated with subverting cytotoxic responses, thus contributing to early bacterial replication and subsequent persistence in the lungs.

A subpopulation of green turtle suprabasal epidermal cells are Langerin+ and migrate under in vitro stimulation of the chemokine CCL21.

Dendritic cells form the link between the innate and adaptative immune response, particularly on mucosal and epidermal surfaces. The Langerhans, an epidermal dendritic cell subpopulation, play a key role in the skin immune response across several species. Scarse immune cell subpopulations, including Langerhans-like cells, have been identified in endangered green turtles thereby complicating the understanding of the pathogenesis of diseases such as fibropapillomatosis, which induces skin tumours in this species worldwide. In biopsies from green turtle skin, we demonstrated that the polyclonal anti-human Langerin antibodies strongly stained a Langerin+ cell population in epidermal sheets, the suprabasal layer of the epidermis in cryosections and in cells from cytospin preparation of migration assays. The morphology of these cells was round to amoeboid in normal skin; however, in skin with ulcerative dermatitis, Langerin+ cells aggregated around ulcers and adopted a more pleomorphic morphology. To our knowledge, this is the first identification of Langerin+ cells with a molecular marker in a reptile species.

Rotavirus infection activates dendritic cells from Peyer's patches in adult mice.

This study used an in vivo mouse model to analyze the response of dendritic cells (DCs) in Peyer's patches (PPs) within the first 48 h of infection with the wild-type murine rotavirus EDIM (EDIM(wt)). After the infection, the absolute number of DCs was increased by 2-fold in the PPs without a modification of their relative percentage of the total cell number. Also, the DCs from PPs of infected mice showed a time-dependent migration to the subepithelial dome (SED) and an increase of the surface activation markers CD40, CD80, and CD86. This response was more evident at 48 h postinfection (p.i.) and depended on viral replication, since DCs from PPs of mice inoculated with UV-treated virus did not show this phenotype. As a result of the activation, the DCs showed an increase in the expression of mRNA for the proinflammatory cytokines interleukin-12/23p40 (IL-12/23p40), tumor necrosis factor alpha (TNF-alpha), and beta interferon (IFN-beta), as well as for the regulatory cytokine IL-10. These results suggest that, a short time after rotavirus infection, the DCs from PPs play a critical role in controlling the infection and, at the same time, avoiding an excessive inflammatory immune response.

Robust Plasma Cell Response to Skin-Inoculated Dengue Virus in Mice.

Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG+ PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM+ PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67+) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model.

Multiparameter flow cytometry analysis of leukocyte markers for diagnosis in preterm neonatal sepsis.

PURPOSE: Neonatal sepsis is an important public health concern worldwide due to its immediate lethality and long-term morbidity rates, Clinical evaluation and laboratory analyses are indispensable for diagnosis of neonatal sepsis. However, assessing multiple biomarkers in neonates is difficult due to limited blood availability. The aim is to investigate if the neonatal sepsis in preterm could be identified by multiparameter analysis with flow cytometry. MATERIALS AND METHODS: The expression of activation-related molecules was evaluated by flow cytometry in newborn with or without risk factors for sepsis. RESULTS: Our analysis revealed that several markers could be useful for sepsis diagnosis, such as CD45RA, CD45RO, or CD71 on T cells; HLA-DR on NKT or classic monocytes, and TREM-1 on non-classic monocytes or neutrophils. However, ROC analysis shows that the expression of CD45RO on T lymphocytes is the only useful biomarker for diagnosis of neonatal late-onset sepsis. Also, decision tree analyses showed that CD45RO plus CD27 could help differentiate the preterm septic neonates from those with risk factors. CONCLUSIONS: Our study shows a complementary and practical strategy for biomarker assessment in neonatal sepsis.

Germinal Center Cells Turning to the Dark Side: Neoplasms of B Cells, Follicular Helper T Cells, and Follicular Dendritic Cells.

Gaining knowledge of the neoplastic side of the three main cells-B cells, Follicular Helper T (Tfh) cells, and follicular dendritic cells (FDCs) -involved in the germinal center (GC) reaction can shed light toward further understanding the microuniverse that is the GC, opening the possibility of better treatments. This paper gives a review of the more complex underlying mechanisms involved in the malignant transformations that take place in the GC. Whilst our understanding of the biology of the GC-related B cell lymphomas has increased-this is not reviewed in detail here-the dark side involving neoplasms of Tfh cells and FDCs are poorly studied, in great part, due to their low incidence. The aggressive behavior of Tfh lymphomas and the metastatic potential of FDCs sarcomas make them clinically relevant, merit further attention and are the main focus of this review. Tfh cells and FDCs malignancies can often be misdiagnosed. The better understanding of these entities linked to their molecular and genetic characterization will lead to prediction of high-risk patients, better diagnosis, prognosis, and treatments based on molecular profiles.

Cutaneous Dengue Virus Inoculation Triggers Strong B Cell Reactions but Contrastingly Poor T Cell Responses.

Dengue is a global health problem without current specific treatment nor safe vaccines available. While severe dengue is related to pre-existing non-neutralizing dengue virus (DENV) antibodies, the role of T cells in protection or pathology is unclear. Using cutaneous DENV infection in immunocompetent mice we previously showed the generation of PNA+ germinal centers (GCs), now we assessed the activation and proliferation of B and T cells in draining lymph nodes (DLNs). We found a drastic remodelling of DLN compartments from 7 to 14 days post-infection (dpi) with greatly enlarged B cell follicles, occupying almost half of the DLN area compared to ~24% in naïve conditions. Enormous clusters of proliferating (Ki-67+) cells inside B follicles were found 14 dpi, representing ~33% of B cells in DLNs but only ~2% in non-infected mice. Inside GCs, we noticed an important recruitment of tingle body macrophages removing apoptotic cells. In contrast, the percentage of paracortex area and total T cells decreased by 14-16 dpi, compared to controls. Scattered randomly distributed Ki-67+ T cells were found, similar to non-infected mice. CD69 expression by CD4+ and CD8+ T cells was minor, while it was remarkable in B cells, representing 1764.7% of change from basal levels 3 dpi. The apparent lack of T cell responses cannot be attributed to apoptosis since no significant differences were observed compared to non-infected mice. This study shows massive B cell activation and proliferation in DLNs upon DENV infection. In contrast, we found very poor, almost absent CD4+ and CD8+ T cell responses.

Dendritic cells and Brucella spp. interaction: the sentinel host and the stealthy pathogen.

As dendritic cells (DCs) are among the first cells to encounter antigens, these cells trigger both innate and T cell responses, and are the most potent antigen-presenting cells. Brucella spp., which is an intracellular facultative and stealthy pathogen, is able to evade the bactericidal activities of professional phagocytes. Several studies have demonstrated that Brucella can survive and replicate intracellularly, thereby provoking impaired maturation of DCs. Therefore, the interaction between DCs and Brucella becomes an interesting model to study the immune response. In this review, we first will describe the most common techniques for DCs differentiation in vitro as well as general features of brucellosis. Then, the interaction of DCs and Brucella, including pathogen recognition, molecular mechanisms of bacterial pathogenesis, and intracellular trafficking of Brucella to subvert innate response, will be reviewed. Finally, we will debate diversity in immunological DC response and the controversial role of DC activation against Brucella infection.

Outer Membrane Vesicles From Brucella melitensis Modulate Immune Response and Induce Cytoskeleton Rearrangement in Peripheral Blood Mononuclear Cells.

Similar to what has been described in other Gram-negative bacteria, Brucella melitensis releases outer membrane vesicles (OMVs). OMVs from B. melitensis 16M and the rough-mutant B. melitensis VTRM1 were able to induce a protective immune response against virulent B. melitensis in mice models. The presence of some proteins which had previously been reported to induce protection against Brucella were found in the proteome of OMVs from B. melitensis 16M. However, the proteome of OMVs from B. melitensis VTRM1 had not previously been determined. In order to be better understand the role of OMVs in host-cell interactions, the aim of this work was to compare the proteomes of OMVs from B. melitensis 16M and the derived rough-mutant B. melitensis VTRM1, as well as to characterize the immune response induced by vesicles on host cells. Additionally, the effect of SDS and proteinase K on the stability of OMVs was analyzed. OMVs from B. melitensis 16M (smooth strain) and the B. melitensis VTRM1 rough mutant (lacking the O-polysaccharide side chain) were analyzed through liquid chromatography-mass spectrometry (LC-MS/MS). OMVs were treated with proteinase K, sodium deoxycholate, and SDS, and then their protein profile was determined using SDS-PAGE. Furthermore, PBMCs were treated with OMVs in order to measure their effect on cytoskeleton, surface molecules, apoptosis, DNA damage, proliferation, and cytokine-induction. A total of 131 proteins were identified in OMVs from B. melitensis16M, and 43 in OMVs from B. melitensis VTRM1. Proteome comparison showed that 22 orthologous proteins were common in vesicles from both strains, and their core proteome contained Omp31, Omp25, GroL, and Omp16. After a subsequent detergent and enzyme treatment, OMVs from B. melitensis VTRM1 exhibited higher sensitive compared to OMVs from the B. melitensis 16M strain. Neither OMVs induced IL-17, proliferation, apoptosis or DNA damage. Nonetheless, OMVs from the smooth and rough strains induced overproduction of TNFα and IL-6, as well as actin and tubulin rearrangements in the cytoskeleton. Moreover, OMVs from both strains inhibited PD-L1 expression in T-cells. These data revealed significant differences in OMVs derived from the rough and smooth Brucella strains, among which, the presence or absence of complete LPS appeared to be crucial to protect proteins contained within vesicles and to drive the immune response.

Crosstalk Between Dermal Fibroblasts and Dendritic Cells During Dengue Virus Infection.

Dengue virus infection (DENV-2) is transmitted by infected mosquitoes via the skin, where many dermal and epidermal cells are potentially susceptible to infection. Most of the cells in an area of infection will establish an antiviral microenvironment to control viral replication. Although cumulative studies report permissive DENV-2 infection in dendritic cells, keratinocytes, and fibroblasts, among other cells also infected, little information is available regarding cell-to-cell crosstalk and the effect of this on the outcome of the infection. Therefore, our study focused on understanding the contribution of fibroblast and dendritic cell crosstalk to the control or promotion of dengue. Our results suggest that dendritic cells promote an antiviral state over fibroblasts by enhancing the production of type I interferon, but not proinflammatory cytokines. Infected and non-infected fibroblasts promoted partial dendritic cell maturation, and the fibroblast-matured cells were less permissive to infection and showed enhanced type I interferon production. We also observed that the soluble mediators produced by non-infected or Poly (I:C) transfected fibroblasts induced allogenic T cell proliferation, but mediators produced by DENV-2 infected fibroblasts inhibited this phenomenon. Additionally, the effects of fibroblast soluble mediators on CD14+ monocytes were analyzed to assess whether they affected the differentiation of monocyte derived dendritic cells (moDC). Our data showed that mediators produced by infected fibroblasts induced variable levels of monocyte differentiation into dendritic cells, even in the presence of recombinant GM-CSF and IL-4. Cells with dendritic cell-like morphology appeared in the culture; however, flow cytometry analysis showed that the mediators did not fully downregulate CD14 nor did they upregulate CD1a. Our data revealed that fibroblast-dendritic cell crosstalk promoted an antiviral response mediated manly by type I interferons over fibroblasts. Furthermore, the maturation of dendritic cells and T cell proliferation were promoted, which was inhibited by DENV-2-induced mediators. Together, our results suggest that activation of the adaptive immune response is influenced by the crosstalk of skin resident cells and the intensity of innate immune responses established in the microenvironment of the infected skin.

Langerhans Cells From Mice at Birth Express Endocytic- and Pattern Recognition-Receptors, Migrate to Draining Lymph Nodes Ferrying Antigen and Activate Neonatal T Cells in vivo.

Antigen capturing at the periphery is one of the earliest, crucial functions of antigen-presenting cells (APCs) to initiate immune responses. Langerhans cells (LCs), the epidermal APCs migrate to draining lymph nodes (DLNs) upon acquiring antigens. An arsenal of endocytic molecules is available to this end, including lectins and pathogen recognition receptors (PRRs). However, cutaneous LCs are poorly defined in the early neonatal period. We assessed endocytic molecules expression in situ: Mannose (CD206)-, Scavenger (SRA/CD204)-, Complement (CD2l, CDllb)-, and Fc-Receptors (CD16/32, CD23) as well as CD1d, CD14, CD205, Langerin (CD207), MHCII, and TLR4 in unperturbed epidermal LCs from both adult and early neonatal mice. As most of these markers were negative at birth (day 0), LC presence was revealed with the conspicuous, epidermal LC-restricted ADPase (and confirmed with CD45) staining detecting that they were as numerous as adult ones. Unexpectedly, most LCs at day 0 expressed CD14 and CD204 while very few were MHCII+ and TLR4+. In contrast, adult LCs lacked all these markers except Langerin, CD205, CD11b, MHCII and TLR4. Intriguingly, the CD204+ and CD14+ LCs predominant at day 0, apparently disappeared by day 4. Upon cutaneous FITC application, LCs were reduced in the skin and a CD204+MHCII+FITC+ population with high levels of CD86 subsequently appeared in DLNs, with a concomitant increased percentage of CD3+CD69+ T cells, strongly suggesting that neonatal LCs were able both to ferry the cutaneous antigen into DLNs and to activate neonatal T cells in vivo. Cell cycle analysis indicated that neonatal T cells in DLNs responded with proliferation. Our study reveals that epidermal LCs are present at birth, but their repertoire of endocytic molecules and PRRs differs to that of adult ones. We believe this to be the first description of CDl4, CD204 and TLR4 in neonatal epidermal LCs in situ. Newborns' LCs express molecules to detect antigens during early postnatal periods, are able to take up local antigens and to ferry them into DLNs conveying the information to responsive neonatal T cells.

Proteomic Analysis of Membrane Blebs of Brucella abortus 2308 and RB51 and Their Evaluation as an Acellular Vaccine.

Membrane blebs are released from Gram-negative bacteria, however, little is known about Brucella blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and in silico analysis. The second aim was to evaluate the use of membrane blebs of Brucella abortus 2308 and B. abortus RB51 as an acellular vaccine in vivo and in vitro. To achieve these aims, membrane blebs from B. abortus 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. Brucella membrane blebs were used as a "vaccine" to induce an immune response in BALB/c mice, using the strain B. abortus RB51 as a positive vaccine control. After subsequent challenge with B. abortus 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known Brucella immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth B. abortus induced similar protective immune responses as well as the vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308 (P < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or B. abortus RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19+CD69+) in vitro. Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected.

Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus.

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long-lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen-associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long-lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules on dendritic cells and macrophages, and long-lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen-presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long-lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Characterization of langerhans cells in epidermal sheets along the body of Armadillo (Dasypus novemcinctus).

Armadillos are apparently important reservoirs of Mycobacterium leprae and an animal model for human leprosy, whose immune system has been poorly studied. We aimed at characterizing the armadillo's langerhans cells (LC) using epidermal sheets instead of tissue sections, since the latter restrict analysis only to cut-traversed cells. Epidermal sheets by providing an en face view, are particularly convenient to evaluate dendritic morphology (cells are complete), spatial distribution (regular vs. clustered), and frequency (cell number/tissue area). Lack of anti-armadillo antibodies was overcome using LC-restricted ATPase staining, allowing assessment of cell frequency, cell size, and dendrites extension. Average LC frequency in four animals was 528 LC/mm(2), showing a rather uniform non-clustered distribution, which increased towards the animal's head, while cell size increased towards the tail; without overt differences between sexes. The screening of antibodies to human DC (MHC-II, CD 1a, langerin, CD86) in armadillo epidermal sheets, revealed positive cells with prominent dendritic morphology only with MHC-II and CD86. This allowed us to test DC mobilization from epidermis into dermis under topical oxazolone stimulation, a finding that was corroborated using whole skin conventional sections. We hope that the characterization of armadillo's LC will incite studies of leprosy and immunity in this animal model.

Competitive suppression of dengue virus replication occurs in chikungunya and dengue co-infected Mexican infants.

BACKGROUND: Co-circulation of dengue virus (DENV) and chikungunya virus (CHIKV) is increasing worldwide but information on the viral dynamics and immune response to DENV-CHIKV co-infection, particularly in young infants, is scant. METHODS: Blood samples were collected from 24 patients, aged 2 months to 82 years, during a CHIKV outbreak in Mexico. DENV and CHIKV were identified by RT-PCR; ELISA was used to detect IgM and IgG antibodies. CHIKV PCR products were cloned, sequenced and subjected to BLAST analysis. To address serological findings, HMEC-1 and Vero cells were inoculated with DENV-1, DENV-2 and CHIKV alone and in combination (DENV-2-CHIKV and DENV-1-CHIKV); viral titers were measured at 24, 48 and 72 h. RESULTS: Nine patients (38%) presented co-infection, of who eight were children. None of the patients presented severe illness. Sequence analysis showed that the circulating CHIKV virus belonged to the Asian lineage. Seroconversion to both viruses was only observed in the four patients five years or older, while the five infants under two years of age only seroconverted to CHIKV. Viral titers in the CHIKV mono-infected cells were greater than in the DENV-1 and DENV-2 mono-infected cells. Furthermore, we observed significantly increased CHIKV progeny and reduction of DENV progeny in the co-infected cells. CONCLUSIONS: In our population, DENV-CHIKV co-infection was not associated with increased clinical severity. Our in vitro assay findings strongly suggest that the lack of DENV IgG conversion in the co-infected infants is due to suppression of DENV replication by the Asian lineage CHIKV. The presence of maternal antibody and immature immune responses in the young infants may also play a role.