Human T Cell and Antibody-Mediated Responses to the Mycobacterium tuberculosis Recombinant 85A, 85B, and ESAT-6 Antigens

Tuberculosis remains a major health problem throughout the world causing large number of deaths. Effective disease control and eradication programs require the identification of major antigens recognized by the protective responses against M. tuberculosis. In this study, we have investigated humoral and cellular immune responses to M. tuberculosis-specific Ag85A, Ag85B, and ESAT-6 antigens in Brazilian patients with pulmonary (P, n = 13) or extrapulmonary (EP, n = 12) tuberculosis, patients undergoing chemotherapy (PT, n = 23), and noninfected healthy individuals (NI, n = 7). Compared to NI, we observed increased levels of IgG1 responses to Ag85B and ESAT-6 in P and PT groups. Regarding cellular immunity, Ag85A and ESAT-6 were able to discriminate P, PT, and EP patients from healthy individuals by IFN-γ production and P and PT groups from EP individuals by production of TNF-α. In summary, these findings demonstrate the ability of Ag85A, Ag85B, and ESAT-6 to differentiate TB patients from controls by IgG1, IFN-γ and TNF-α production

Contemporary Centralities in São Paulo. Study on the construction of the Berrini - Chucri Zaidan axis

This article investigates the morphological and functional changes of Chucri Zaidan avenue and its relationship with the new axis of urban centrality of São Paulo, also composed of Berrini Avenue. The study seeks to reveal the construction logics and the process that has been transforming the area into a financial center and condenser axis of a mixture of uses, including leisure activities, services, commerce and culture that enrich the social life of the area. To study it, we will seek to identify the main urban transformations that have occurred throughout history, drawing the changes that have occurred at the ground level, the building typology, the uses and contours of public and collective spaces. The analyses will be based on official documents, drawings drawn from registration maps of the city, on data collected on site, and on the theoretical discussion about the way as a powerful element of condensation of collective spaces and their centrality and urbanity. As a result, it is verified that the axis is constructed as a linear centrality, through the implementation of mixed-use buildings, with predominance of office buildings, implanted in partially open blocks configured with paths and permeable free areas, with a wide offer of green areas. On the other hand, it lacks social mixing. Keywords: Linear Centrality, Collective Spaces, Metropolis of São Paulo, Urban life.Este artigo investiga as mudanças morfológicas e funcionais da avenida Chucri Zaidan e sua relação com o novo eixo de centralidade urbana de São Paulo, composto também pela avenida Berrini. O estudo busca revelar as lógicas de construção e os processos que vem transformando a área em centro financeiro e eixo condensador de atividades de lazer, serviços, comércio e cultura que enriquecem a vida social da cidade. Para estudá-la, buscaremos identificar as principais transformações urbanas que ocorreram ao longo da história, desenhando as mudanças transcorridas na tipologia edificada, nos usos e contornos dos espaços públicos e coletivos. As análises serão embasadas por documentos oficiais, desenhos elaborados a partir de mapas cadastrais da prefeitura, dados coletados in loco, bem como na discussão teórica sobre a via como elemento estruturador dos espaços coletivos e de sua centralidade e urbanidade. Como resultado, verifica-se que este eixo se constrói como uma centralidade linear, mediante a implantação de edifícios de uso misto, com predominância de edifícios de escritórios, implantados em quadras parcialmente abertas configuradas com caminhos e áreas livres permeáveis, com ampla oferta de áreas verdes. Por outro lado, é carente de mistura social. Palavras-chave: Centralidade Linear, Espaços Coletivos, Metrópole de São Paulo, Vida urbana.Peer Reviewe

I contratti di credito immobiliare fra diritto europeo e attuazione nazionale. Strumenti di prevenzione del sovraindebitamento del consumatore

Il volume affronta, in prospettiva anche comparatistica, le principali problematiche attinenti alla concessione del credito immobiliare ipotecario nei rapporti con i consumatori. Partendo da una disamina della Mortage Credit Directive (MCD) il volume ne evidenzia le criticità in relazione agli obblighi precontrattuali di informazione e annesso accesso alle banche dati, alla verifica del merito creditizio, ai casi di estinzione anticipata dei mutui, alle pratiche di pratiche di commercializzazione, alla gestione dell’inadempimento

Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as a Model to Study Trypanosoma cruzi Infection

Submitted by Nuzia Santos ([email protected]) on 2019-08-21T14:03:04Z No. of bitstreams: 1 Using Human Induced Pluripotent Stem .pdf: 1908077 bytes, checksum: 178f6efbded1fadb06946707daced99a (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2019-08-21T14:07:21Z (GMT) No. of bitstreams: 1 Using Human Induced Pluripotent Stem .pdf: 1908077 bytes, checksum: 178f6efbded1fadb06946707daced99a (MD5)Made available in DSpace on 2019-08-21T14:07:21Z (GMT). No. of bitstreams: 1 Using Human Induced Pluripotent Stem .pdf: 1908077 bytes, checksum: 178f6efbded1fadb06946707daced99a (MD5) Previous issue date: 2019Stanford Cardiovascular Institute. Stanford, CA, USA/Fundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, BrazilStanford Cardiovascular Institute. Stanford, CA, USA/Department of Medicine. Division of Cardiovascular Medicine. Stanford University. School of Medicine., Stanford, CA, USA/Department of Radiology. Stanford University. School of Medicine. Stanford, CA, USAStanford Cardiovascular Institute. Stanford, CA, USA/Department of Medicine. Division of Cardiovascular Medicine. Stanford University. School of Medicine., Stanford, CA, USA/Department of Radiology. Stanford University. School of Medicine. Stanford, CA, USADivision of Infectious Diseases and Geographic Medicine. Stanford University. School of Medicine, Stanford, CA, USA/California Institute for Medical Research. San Jose, CA, USAStanford Cardiovascular Institute. Stanford, CA, USA/Department of Medicine. Division of Cardiovascular Medicine. Stanford University. School of Medicine., Stanford, CA, USA/Department of Radiology. Stanford University. School of Medicine. Stanford, CA, USADivision of Infectious Diseases and Geographic Medicine. Stanford University. School of Medicine, Stanford, CA, USA/California Institute for Medical Research. San Jose, CA, USAFundação Oswaldo Cruz. Instituto René Rachou. Belo Horizonte, MG, BrazilDivision of Infectious Diseases and Geographic Medicine. Stanford University. School of Medicine, Stanford, CA, USA/California Institute for Medical Research. San Jose, CA, USAStanford Cardiovascular Institute. Stanford, CA, USA/Department of Medicine. Division of Cardiovascular Medicine. Stanford University. School of Medicine., Stanford, CA, USA/Department of Radiology. Stanford University. School of Medicine. Stanford, CA, USAChagas disease (ChD) is one of the most neglected tropical diseases, with cardiomyopathy being the main cause of death in Trypanosoma cruzi-infected patients. As the parasite actively replicates in cardiomyocytes (CMs), the heart remains a key target organ in the pathogenesis of ChD. Here we modeled ChD using human induced pluripotent stem cell-derived CMs (iPSC-CMs) to understand the complex interplay between the parasite and host cells. We showed that iPSC-CMs can get infected with the T. cruzi Y strain and that all parasite cycle stages can be identified in our model system. Importantly, characterization of T. cruzi-infected iPSC-CMs showed significant changes in their gene expression profile, cell contractility, and distribution of key cardiac markers. Moreover, these infected iPSC-CMs exhibited a pro-inflammatory profile as indicated by significantly elevated cytokine levels and cell-trafficking regulators. We believe our iPSC-CM model is a valuable platform to explore new treatment strategies for ChD

The levels of IL-17A and of the cytokines involved in Th17 cell commitment are increased in patients with chronic immune thrombocytopenia

Th17 cells have been associated with immune-mediated diseases in humans but it has still not been determined whether they play a role in immune thrombocytopenia. We evaluated representative cytokines of the Th17, Th1, Th2 and Treg cell commitment in the serum of patients with chronic immune thrombocytopenia, as well as the cell source of IL-17A. Higher levels of IL-17A and Th17-related cytokines, and an increased percentage of IL-17A producing CD4+ and neutrophils were observed in patients. The levels of cytokines involved in Th1 cell commitment IFN-γ, IL-2, IL12-p70 and the percentages of Th1 cells were also increased, but IL-4 was not detected. Although the concentrations of IL-10 were higher, the levels of TGF-β were similar in both groups. In conclusion, our results point to a putative role for Th-17 cells/IL-17A cytokine in the pathogenesis of chronic immune thrombocytopenia

New Developments of RNAi in <i>Paracoccidioides brasiliensis</i>: Prospects for High-Throughput, Genome-Wide, Functional Genomics

<div><p>Background</p><p>The Fungal Genome Initiative of the Broad Institute, in partnership with the <i>Paracoccidioides</i> research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for <i>P. brasiliensis</i>.</p><p>Methodology/Principal Findings</p><p>In this paper, we report <i>Agrobacterium tumefaciens</i> mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×10⁶ viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the <i>Streptoalloteichus hindustanus</i> bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in <i>P. brasiliensis</i>. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach.</p><p>Conclusions/Significance</p><p>We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future <i>Paracoccidioides</i> spp. functional genomics research.</p></div

ATMT of <i>P. brasiliensis</i> to certify the Zeocin/ZeoR positive selection system and the terminal GP43 gene transcription region.

<p>(<b>A</b>). Four independent transformation experiments were performed by co-cultivating 1×10⁶ viable <i>Pb18</i> yeast cells with <i>A. tumefaciens</i> at different ratios. <i>A. tumefaciens</i> carried the “Prm_CBP1::<i>Shble</i>::Ttr_GP43” cassette to express the ZeoR selection marker (*, P = 0.0002; **, P = 0.001). (<b>B</b>). Three independent transformation experiments were performed by co-cultivating 1×10⁶ viable <i>Pb18</i> yeast cells with <i>A. tumefaciens</i> at a ratio of 1∶100 and recovering the yeast cells using two different selective media (*, P = 0.02).</p

RNA interference of <i>Pb18</i> GP43 gene expression triggered artificially with intron-containing dsRNA.

<p>(<b>A</b>). GP43 gene expression levels were investigated in six cAlf-GP43 transformants in relation to cAlfG-control by qRT-PCR after 8 months of subculturing in non-selective media. Results were normalized against the internal control L34. (<b>B–C</b>) Epifluorescence microscopy analysis of transformant C4 (C), in which the GP43 gene expression was nearly abolished, in comparison to cAlfG-control (<b>B</b>). Scale bar: 10 µm.</p

Cassettes assembled in the present work.

<p>(<b>A</b>). Cassette for the expression of the ZeoR marker under the transcriptional regulation of Prm_CBP1 and Ttr_GP43,which was used in preliminary ATMT of <i>P. brasiliensis</i>. (<b>B–D</b>). Cassettes assembled to validate Prm_Act and Prm_GP43against Prm_CBP1 through the expression of the bifunctional selective marker “Shble::mCherry” in yeast cells. (<b>E</b>). Purpose-built cAlfG, adapted for the Gateway Technology, intended for the expression of intron_GP43-containing hairpin RNA to trigger RNAi in yeast cells. Abbreviations: Ttr, transcription termination region of <i>Pb18</i> GP43 gene; Prm_GP43, promoter region of <i>Pb18</i> GP43 gene; Prm_Act, promoter region of <i>Pb18</i> actin gene; Prm_CBP1, promoter region of <i>H. capsulatum</i> CBP1 gene; Rfc, gateway reading frame cassette; Intr, first intron of the <i>Pb18</i> GP43 gene.</p