T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production

T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived F(AB), G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., "TCR-like" specificity. These chimeric receptors comprised the FcepsilonRI-gamma signaling element. We analyzed whether linking the CD28 costimulation structure to it (gamma + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8(POS) T lymphocytes comprising the gamma + CD28 vs the gamma signaling element alone produced substantially more IL-2, TNF-alpha, and IFN-gamma in response to HLA-A1/MAGE-A1(POS) melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8(POS) T cells, equipped with gamma + CD28 vs the gamma-chain alone was observed

Selection of Intracellular Single-Domain Antibodies Targeting the HIV-1 Vpr Protein by Cytoplasmic Yeast Two-Hybrid System

International audienceThe targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system

A Universidade Corporativa Como Fator Determinante da Geração do Conhecimento nas Organizações: o Caso da Embratel.

Esse artigo propõe uma análise da Universidade Corporativa como uma das ferramentas estratégicas do conhecimento organizacional, em seus aspectos essenciais. Contextualiza-a de acordo com a evolução da Administração de Recursos Humanos, abordando o processo de desenvolvimento de pessoas. Os procedimentos metodológicos adotados consistem em uma pesquisa de natureza descritiva, qualitativa e teórica aplicada. Apresenta o estudo de caso realizado na Universidade Corporativa Embratel (UCE), como uma experiência prática de sucesso

Virtual screening for inhibitors of the human TSLP:TSLPR interaction

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathophysiology of various allergy disorders that are mediated by type 2 helper T cell (Th2) responses, such as asthma and atopic dermatitis. TSLP forms a ternary complex with the TSLP receptor (TSLPR) and the interleukin-7-receptor subunit alpha (IL-7Ra), thereby activating a signaling cascade that culminates in the release of pro-inflammatory mediators. In this study, we conducted an in silico characterization of the TSLP: TSLPR complex to investigate the drugability of this complex. Two commercially available fragment libraries were screened computationally for possible inhibitors and a selection of fragments was subsequently tested in vitro. The screening setup consisted of two orthogonal assays measuring TSLP binding to TSLPR: a BLI-based assay and a biochemical assay based on a TSLP: alkaline phosphatase fusion protein. Four fragments pertaining to diverse chemical classes were identified to reduce TSLP: TSLPR complex formation to less than 75% in millimolar concentrations. We have used unbiased molecular dynamics simulations to develop a Markov state model that characterized the binding pathway of the most interesting compound. This work provides a proof-ofprinciple for use of fragments in the inhibition of TSLP: TSLPR complexation

Adaptation of HIV-1 Envelope Glycoprotein gp120 to Humoral Immunity over the Course of the Epidemic

Since 2009, a large panel of broad and potent monoclonal neutralizing antibodies (MoNAbs) against HIV-1 have been isolated. These MoNAbs can protect from lllV-1 infection and suppress established infection in animal models. Because their efficacy should be evaluated in human clinical trials, it is of importance to define the sensitivity of the most contemporary transmitted variants to these MoNAbs. We, and others previously, reported that HIV-1 has become more resistant to neutralization over the course of the epidemic (Bunnik et al., Nature Med 2010, Bouvin-Pley et al., PloS Pathog 2013). Methods: Here we extended the analyses to the most potent MoNAbs described since then, either more recently isolated or improved by structure-based gene modifications. Results: We fully confirmed the first observations showing an increasing resistance of HIV-1 clade B over time to MoNAbs targeting the major gp l20 epitopes but not to MoNAbs targeting the gp41 MPER. Despite this evolution, some MoNAbs still were able to neutralize efficiently the most recently transmitted HIV-1 variants (2006-2010). The most potent MoNAbs were the bi-specific PG9- and PG16-iMab that alone were able to neutralize an variants at less than 0.4 mg/mL. The sensitivity to iMAb remained similar over time, suggesting that the trend of increasing resistance to PG9-/PG16-iMAb may be attributed only to die antigen binding domain of PG9/PG16. NIH45-46m2 (and -m7), 10-1074 and 10E8 were also highly potent and, if combined, reached the potency of PG9-/PG16-iMAb. We also observed that 3BNC 117 was almost as potent as the modified NIH45-46 antibodies, and that the lama-derived JM4IgG2b was the most potent Ab among those that do not target the major gp 120 neutralizing epitopes. Conclusions: These data clearly suggest a continuous drift of the env gene of HIV-1 elude B over the epidemic, and that not a single epitope is concerned but the entire gp120 as a whole. The consequences of this adaptation on the envelope functionality are being explored

Tissue-specific cytokine release from human extra-placental membranes stimulated by lipopolysaccharide in a two-compartment tissue culture system

<p>Abstract</p> <p>Background</p> <p>The extra-placental gestational membranes secrete cytokines in response to bacteria and other infectious agents, with potentially adverse consequences for pregnancy. The present study used lipopolysaccharide (LPS) as a prototype endotoxin to investigate the pattern of stimulated cytokine release from the amniotic and choriodecidual sides of full-thickness human gestational membranes in a two-compartment tissue culture system.</p> <p>Methods</p> <p>Gestational membranes were collected from healthy non-laboring caesarean deliveries at term. Full-thickness membranes from each placenta were cut into pieces, mounted on Transwell frames, and placed in culture wells to create a two-compartment culture with the gestational membranes serving as the barrier between compartments. The LPS (100 ng/ml) was added to the amniotic, choriodecidual or both chambers of the culture, and cytokines were assayed in the medium of the amniotic and choriodecidual chambers after 8 h of LPS exposure. Cytokine concentrations were analyzed by two-way analysis of variance for effects of treatment and side specificity of cytokine release from the membranes.</p> <p>Results</p> <p>LPS exposure on the choriodecidual side of the membranes significantly increased TNF-alpha, IL-6, IL-10 and IL-8 in the choriodecidual compartment, whereas TNF-alpha was the only cytokine observed to increase in the amniotic compartment. When LPS treatment was to the amniotic side of the membranes, there were significant increases in TNF-alpha and IL-6 in the amniotic compartment as well as increased concentrations of TNF-alpha, IL-6 and IL-8 in the choriodecidual compartment; however, there were no statistically significant differences for IL-10 in either compartment. No statistically significant differences were observed for IL-1beta, TGF-beta or IL-4 concentrations in response to LPS, regardless of the exposure modality.</p> <p>Conclusion</p> <p>The amnion and choriodecidua exhibited distinct patterns of response to LPS with evidence of inflammatory signaling across the layers of the gestational membranes. These results suggest a complicated network of signaling within the gestational membranes, in which cytokine- and tissue-specific responses to inflammatory stimulation may have important implications for maintaining pregnancy in the challenge of microbial invasion of the uterine compartment.</p

Projeto de rebranding da marca de vestuário SRI Clothing

Este projeto tem como objetivo o redesenho da identidade visual da marca SRI Clothing. A SRI é uma marca de vestuário do Rio de Janeiro, criada em 2012, que nunca passou por um processo de branding completo. Por isso, a proposta deste rebranding é criar um sistema de identidade visual coeso que comunique a essência da marca. Para isso foram utilizados como base metodológica o livro Design de Identidade de Marca , de Alina Wheeler (2008), que guia o processo de construção de marca, e o livro A Moda Imita a Vida , de André Carvalhal, (2020), que oferece uma visão mais nichada do processo de branding, voltada para o mercado da moda

Analysis of the binding loops configuration and surface adaptation of different crystallized single‐domain antibodies in response to various antigens

Monoclonal antibodies have revolutionized the biomedical field through their ubiquitous utilization in different diagnostics and therapeutic applications. Despite this widespread use, their large size and structural complexity have limited their versatility in specific applications. The antibody variable region that is responsible for binding antigen is embodied within domains that can be rescued individually as single-domain antibody (sdAb) fragments. Because of the unique characteristics of sdAbs, such as low molecular weight, high physicochemical stability, and the ability to bind antigens inaccessible to conventional antibodies, they represent a viable alternative to full-length antibodies. Consequently, 149 crystal structures of sdAbs, originating from human (VH), camelids (VHH), or sharks (VNAR), were retrieved from the Protein Data Bank, and their structures were compared. The 3 types of sdAbs displayed complementarity determining regions (CDRs) with different lengths and configurations. CDR3 of the VHH and VNAR domains were dominated by pleated and extended orientations, respectively. Although VNAR showed the smallest average molecular weight and molecular surface area compared with VHH and VH antibodies. However, the solvent accessible surface area measurements of the 3 tested sdAbs types were very similar. All the antihapten VHH antibodies showed pleated CDR3, which were sufficient to create a binding pocket to accommodate haptens (methotrexate and azo dyes) in terms of shape and electrostatic potential. The sdAbs that recognized lysozyme showed more diversity in their CDR3 orientation to enable them to recognize various topographies of lysozyme. Subsequently, the three sdAb classes were different in size and surface area and have shown distinguishable ability to optimize their CDR length and orientation to recognize different antigen classes

Tomada de decisão: uma abordagem utilizando a linguagem corporal da dança e a gestão do conhecimento

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia e Gestão do Conhecimento, Florianópolis, 2010O campo de atuação do gestor, executivo ou dirigente organizacional, tornou-se complexa, à medida que as transformações organizacionais determinaram formas de produção eficazes e, conseqüentemente eficientes maneiras de os profissionais exercerem suas atividades, interferindo diretamente nos processos decisórios. Assim, ao se tomar a decisão de argumentar, agir, avaliar e tornar a argumentar, deve-se levar em consideração não somente conhecimentos técnico-administrativos, mas o conhecimento sensível presente na expressão, intuição, percepção, imaginação. Articular o conhecimento dos aspectos administrativos com o sensível por meio da linguagem corporal da dança faz a diferença na flexibilização de ações que buscam no movimento vivido uma conexão, uma harmonização entre a inteligência, as sensações, as percepções, contribuindo para a construção de seres humanos conscientes de suas capacidades. O objetivo do trabalho foi demonstrar como técnicas da linguagem corporal da dança contribuíram para auxiliar gestores, executivos ou dirigentes corporativos em sua tomada de decisões, por meio da criação de um ferramental dessa atividade corporal. Para os fins a que se propôs este estudo, os procedimentos metodológicos seguiram a pesquisa qualitativa, por meio das técnicas de pesquisa descritiva e exploratória. Na pesquisa de campo, o universo foram os "gestores de unidade" de uma organização de economia privada, denominada Banco X S/A. Para a análise de dados utilizou-se a análise de conteúdo e a triangulação de dados. Para permitir a validação, realizou-se também uma entrevista semi-estruturada, por meio da técnica de triangulação de especialistas. Constatou-se que a utilização das técnicas da dança pautadas principalmente na improvisação e em processos coreográficos permitiu além de novas experiências corporais, o estímulo à criatividade e à ousadia. Essas elaborações permitiram que, a todo o momento, os participantes tomassem decisões numa perspectiva de entender que movimento selecionar e conectar para a criação dessas figuras, exercitando de forma individual a construção do coletivo. No caso, a aquisição de conhecimentos tornou-se um processo interminável de atualização constante, com a contribuição das experiências práticas que ampliaram a capacidade criadora, contribuindo significativamente para a construção de uma Gestão do Conhecimento, destacando-se, também, que as pessoas são fundamentais à consecução dos objetivos organizacionais relacionados à Gestão do Conhecimento. Concluiu-se que a criação do ferramental utilizando-se técnicas da linguagem corporal da dança, contribuiu para o processo de tomada de decisões, pois além de potencializar as atividades dos participantes em questão, permitindo uma maior flexibilidade em suas decisões, favoreceu o desenvolvimento de vertentes artísticas, culturais, éticas, estéticas, sociais, vislumbrando-se uma dança mais democrática, rompendo com a idéia de que é necessária uma técnica específica para realizá-la. Espera-se que estas reflexões levem a conexões, idéias e discussões, sobretudo ao aprofundamento das atividades de dança, numa oportunidade de investimento inovador, contemplando, também, a valorização da potencialidade humana