Paracoccidioides brasilinsis-Induced Migration of Dendritic Cells and Subsequent T-Cell Activation in the Lung-Draining Lymph Nodes

Paracoccidioidomycosis is a mycotic disease caused by a dimorphic fungus, Paracoccidioides brasiliensis (Pb), that starts with inhalation of the fungus; thus, lung cells such as DC are part of the first line of defense against this microorganism. Migration of DC to the lymph nodes is the first step in initiating T cell responses. The mechanisms involved in resistance to Pb infection are poorly understood, but it is likely that DC play a pivotal role in the induction of effector T cells that control Pb infection. In this study, we showed that after Pb Infection, an important modification of lung DC receptor expression occurred. We observed an increased expression of CCR7 and CD103 on lung DC after infection, as well as MHC-II. After Pb infection, bone marrow-derived DC as well lung DC, migrate to lymph nodes. Migration of lung DC could represent an important mechanism of pathogenesis during PCM infection. In resume our data showed that Pb induced DC migration. Furthermore, we demonstrated that bone marrow-derived DC stimulated by Pb migrate to the lymph nodes and activate a T helper (Th) response. To the best of our knowledge, this is the first reported data showing that Pb induces migration of DC and activate a T helper (Th) response.

Dendritic Cells Transfected with scFv from Mab 7.B12 Mimicking Original Antigen gp43 Induces Protection against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), which primarily attacks lung tissue. Dendritic cells (DCs) are able to initiate a response in naive T cells, and they also participate in Th-cell education. Furthermore, these cells have been used for therapy in several disease models. Here we transfected DCs with a plasmid (pMAC/PS-scFv) encoding a single chain variable fragment (scFv) of an anti-Id antibody that is capable of mimicking gp43, the main antigenic component of P. brasiliensis. First, Balb/c mice were immunized subcutaneously with pMAC/PS-scFv and, after seven days, scFv protein was presented to the regional lymph nodes cells. Moreover, we showed that the DCs transfected with scFv were capable of efficiently activating proliferation of total lymph node cells and inducing a decrease in lung infection. Therefore, our results suggested that the use of scFv-transfected DCs may be a promising therapy in the paracoccidioidomycosis (PCM) model.

Plasmodium vivax: Induction of CD4(+)CD25(+)FoxP3(+) Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites

Circulation CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-beta) as well as pro-inflammatory (IFN-gamma, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4(+)CD25(+)FoxP3(+)GITR(+) or CD4(+)CD25(+)FoxP3(+)CTLA-4(+) and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.

Increased Endothelial Cell-Leukocyte Interaction in Murine Schistosomiasis: Possible Priming of Endothelial Cells by the Disease

Background and Aims: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. Methodology and Principal Findings: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. Conclusion/Significance: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.

Bronchus-Associated Lymphoid Tissue (BALT) and Survival in a Vaccine Mouse Model of Tularemia

Background: Francisella tularensis causes severe pulmonary disease, and nasal vaccination could be the ideal measure to effectively prevent it. Nevertheless, the efficacy of this type of vaccine is influenced by the lack of an effective mucosal adjuvant. Methodology/Principal Findings: Mice were immunized via the nasal route with lipopolysaccharide isolated from F. tularensis and neisserial recombinant PorB as an adjuvant candidate. Then, mice were challenged via the same route with the F. tularensis attenuated live vaccine strain (LVS). Mouse survival and analysis of a number of immune parameters were conducted following intranasal challenge. Vaccination induced a systemic antibody response and 70% of mice were protected from challenge as showed by their improved survival and weight regain. Lungs from mice recovering from infection presented prominent lymphoid aggregates in peribronchial and perivascular areas, consistent with the location of bronchus-associated lymphoid tissue (BALT). BALT areas contained proliferating B and T cells, germinal centers, T cell infiltrates, dendritic cells (DCs). We also observed local production of antibody generating cells and homeostatic chemokines in BALT areas. Conclusions: These data indicate that PorB might be an optimal adjuvant candidate for improving the protective effect of F. tularensis antigens. The presence of BALT induced after intranasal challenge in vaccinated mice might play a role in regulation of local immunity and long-term protection, but more work is needed to elucidate mechanisms that lead to its formation.

Sub-telomere directed gene expression during initiation of invasive aspergillosis

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility ( het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer ( HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated ""gene dumps'' and, perhaps, simultaneously, as "" gene factories''.

Cell-Free Antigens from Paracoccidioides brasiliensis Drive IL-4 Production and Increase the Severity of Paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. Methodology/Principal Findings: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. Conclusions: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.

New Genotype of Dengue Type 3 Virus Circulating in Brazil and Colombia Showed a Close Relationship to Old Asian Viruses

Dengue type 3 genotype V viruses have been recently detected in Brazil and Colombia. In this study, we described another Brazilian isolate belonging to this genotype. Phylogenetic analysis including dengue type 3 viruses isolated worldwide showed that Brazilian and Colombian viruses were closely related to viruses isolated in Asia more than two decades ago. The characteristic evolutionary pattern of dengue type 3 virus cannot explain the close similarity of new circulating viruses with old viruses. Further studies are needed to confirm the origin of the new dengue type III genotype circulating in Brazil and Colombia.

A New Baurusuchid (Crocodyliformes, Mesoeucrocodylia) from the Late Cretaceous of Brazil and the Phylogeny of Baurusuchidae

Background: Baurusuchidae is a group of extinct Crocodyliformes with peculiar, dog-faced skulls, hypertrophied canines, and terrestrial, cursorial limb morphologies. Their importance for crocodyliform evolution and biogeography is widely recognized, and many new taxa have been recently described. In most phylogenetic analyses of Mesoeucrocodylia, the entire clade is represented only by Baurusuchus pachecoi, and no work has attempted to study the internal relationships of the group or diagnose the clade and its members. Methodology/Principal Findings: Based on a nearly complete skull and a referred partial skull and lower jaw, we describe a new baurusuchid from the Vale do Rio do Peixe Formation (Bauru Group), Late Cretaceous of Brazil. The taxon is diagnosed by a suite of characters that include: four maxillary teeth, supratemporal fenestra with equally developed medial and anterior rims, four laterally visible quadrate fenestrae, lateral Eustachian foramina larger than medial Eustachian foramen, deep depression on the dorsal surface of pterygoid wing. The new taxon was compared to all other baurusuchids and their internal relationships were examined based on the maximum parsimony analysis of a discrete morphological data matrix. Conclusion: The monophyly of Baurusuchidae is supported by a large number of unique characters implying an equally large morphological gap between the clade and its immediate outgroups. A complex phylogeny of baurusuchids was recovered. The internal branch pattern suggests two main lineages, one with a relatively broad geographical range between Argentina and Brazil (Pissarrachampsinae), which includes the new taxon, and an endemic clade of the Bauru Group in Brazil (Baurusuchinae).

Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

Time Changes with the Embodiment of Another's Body Posture

The aim of the present study was to investigate whether the perception of presentation durations of pictures of different body postures was distorted as function of the embodied movement that originally produced these postures. Participants were presented with two pictures, one with a low-arousal body posture judged to require no movement and the other with a high-arousal body posture judged to require considerable movement. In a temporal bisection task with two ranges of standard durations (0.4/1.6 s and 2/8 s), the participants had to judge whether the presentation duration of each of the pictures was more similar to the short or to the long standard duration. The results showed that the duration was judged longer for the posture requiring more movement than for the posture requiring less movement. However the magnitude of this overestimation was relatively greater for the range of short durations than for that of longer durations. Further analyses suggest that this lengthening effect was mediated by an arousal effect of limited duration on the speed of the internal clock system.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Minor and Unsystematic Cortical Topographic Changes of Attention Correlates between Modalities

In this study we analyzed the topography of induced cortical oscillations in 20 healthy individuals performing simple attention tasks. We were interested in qualitatively replicating our recent findings on the localization of attention-induced beta bands during a visual task [1], and verifying whether significant topographic changes would follow the change of attention to the auditory modality. We computed corrected latency averaging of each induced frequency bands, and modeled their generators by current density reconstruction with Lp-norm minimization. We quantified topographic similarity between conditions by an analysis of correlations, whereas the inter-modality significant differences in attention correlates were illustrated in each individual case. We replicated the qualitative result of highly idiosyncratic topography of attention-related activity to individuals, manifested both in the beta bands, and previously studied slow potential distributions [2]. Visual inspection of both scalp potentials and distribution of cortical currents showed minor changes in attention-related bands with respect to modality, as compared to the theta and delta bands, known to be major contributors to the sensory-related potentials. Quantitative results agreed with visual inspection, supporting to the conclusion that attention-related activity does not change much between modalities, and whatever individual changes do occur, they are not systematic in cortical localization across subjects. We discuss our results, combined with results from other studies that present individual data, with respect to the function of cortical association areas.