P2 purinoceptors signaling in fibroblasts of rat subcutaneous tissue

Mestrado em Biologia Molecular e CelularO tecido conjuntivo parece estar envolvido na génese de diversas condições patológicas. O aumento da rigidez do tecido conjuntivo, resultante da fibrose, pode constituir um factor importante no mecanismo patogénico da dor crónica resistente a fármacos (Langevin & Sherman, 2007). Por outro lado, os nucleótidos extracelulares parecem estar envolvidos na fisiopatologia da dor crónica (Burnstock, 2001). Assim, este estudo teve como objectivo averiguar o efeito dos nucleótidos de adenina e uridina na proliferação e síntese de colagénio tipo I de fibroblastos do tecido subcutâneo de rato em cultura. Os resultados obtidos mostram que a incubação com UTP (0.3-100 M, n=5) induz um aumento da proliferação e da produção de colagénio tipo I, o qual é dependente da concentração. Contrariamente, o agonista selectivo dos receptores P2Y2, o MRS 2768 (10 μM, n=3), não teve qualquer efeito no que se refere à proliferação, mas diminuiu significativamente (P<0.05) a síntese de colagénio tipo I. Uma vez que o aumento da produção de colagénio induzida pelo UTP (100 μM) foi proporcional ao aumento do número de células (proliferação celular),podemos especular que este aumento se deve ao aumento do número de células per si do que a uma maior actividade sintética de cada célula. Assim, ao normalizar os valores do colagénio tipo I em relação aos valores obtidos do MTT para os mesmos momentos/dias, deixamos de observar diferenças estatisticamente significativas entre o controlo e as células expostas ao UTP. Uma vez que os receptores P2Y2 não parecem estar envolvidos nesta resposta do UTP (100 μM), esta poderá estar a ser mediada pela activação dos receptores P2Y4 e/ou P2Y6. Considerando que o RB-2 (10 μM, n=5), um antagonista não selectivo que actua preferencialmente no subtipo de receptores P2Y4, não foi capaz de modificar a resposta induzida pelo UTP (100 μM), os receptores P2Y4 parecem também não estar envolvidos. Por outro lado, o MRS 2578 (100 nM), um antagonista selectivo dos receptores P2Y6, atenuou de forma significativa o aumento induzido pelo UTP (100 μM). A corroborar os nossos resultados, uma análise imunocitoquímica mostrou uma imunorreactividade positiva contra os receptores P2Y2 e P2Y6, mostrando um padrão de marcação citoplasmático/membranar, o qual é típico para este tipo de receptores, ao contrário do padrão nuclear exibido pelo anticorpo contra os receptores P2Y4. Relativamente ao envolvimento dos receptores sensíveis ao ADP, os resultados obtidos mostraram que o ADPβS (10-100 μM, n=3-6), um análogo estável do ADP, não parece induzir efeitos significativamente diferentes (P>0.05) na proliferação celular. Contudo, a sua incubação continuada aumentou a produção de colagénio tipo I de forma dependente da concentração (P<0.05). De modo a identificar os receptores purinérgicos envolvidos neste efeito, testamos o ADPβS (100 μM) na presença do MRS 2179 (0.3 μM), do AR-C 66096 (0.1 μM), e do MRS 2211 (10 μM), os quais antagonizam selectivamente os receptores P2Y1, P2Y12 e P2Y13, respectivamente. O efeito facilitatório induzido pelo ADPβS (100 μM) foi atenuado de forma significativa na presença do antagonista dos receptores P2Y1, o MRS 2179 (0.3 μM, n=3), sem ser afectado pelo antagonista dos receptores P2Y12, o AR- C 66096 (0.1 μM, n=3). Pelo contrário, o MRS 2211 (10 μM, n=2) potenciou o aumento da produção de colagénio induzida pelo ADPβS (100 μM), indicando assim que a síntese de colagénio tipo I induzida pelo receptor P2Y1 pode estar a ser parcialmente influenciada por uma activação síncrona do receptor inibitório P2Y13. Por último, uma análise por imunocitoquímica mostrou que estas células apresentam imunorreactividade positiva para os receptores P2Y1 e P2Y13, exibindo um padrão citoplasmático/membranar, contrariamente ao padrão nuclear dos receptores ostentado pelo anticorpo contra os receptores P2Y12. Concluindo, a remodelação da fáscia superficial induzida pelos fibroblastos parece ser regulada por um balanço entre a activação dos receptores P2Y2 e P2Y6, assim como dos receptores P2Y13 e P2Y1. Clarificar as vias que conduzem ao processo de fibrose pode representar uma oportunidade para esclarecer o seu envolvimento na patogénese da dor crónica musculo-esquelética, bem como ser útil no desenvolvimento de novas estratégias terapêuticas.Connective tissue may be involved in the pathogenesis of a wide variety of disease conditions. Increased connective tissue stiffness due to fibrosis may be an important link to the pathogenic mechanism leading to drug-resistant chronic pain (Langevin & Sherman, 2007). In addition, extracellular nucleotides seem to be involved in the pathophysiology of chronic pain (Burnstock, 2001). Therefore, we aimed at investigating the effect of adenine and uridine nucleotides on the proliferation and synthesis of type I collagen by rat fibroblasts from subcutaneous connective tissue. The results showed that continuous incubation of UTP (0.3-100 M, n=5) concentration-dependently increased fibroblasts proliferation, as also increased the synthesis of type I collagen above the control levels. Conversely, the selective P2Y2 agonist, MRS 2768 (10 μM, n=3), was devoid of effect in what concerns proliferation, but significantly (P<0.05) decreased type I collagen synthesis. Since the increase in type I collagen synthesis induced by UTP (100 μM) was proportional to the increase in the amount of cells in the culture (fibroblasts proliferation), we speculated that such an increase could be related to the increase in the cell number rather than a higher synthetic activity. Thus, we performed a more detailed data analysis, in which we normalized type I collagen production taking into consideration the MTT values obtained at the same time points, and we observed no longer significant differences between control and UTP-exposed cells. Discounting the contribution of MRS 2768-sensitive P2Y2 receptors, UTP (100 μM)-induced increase in cells proliferation could be due to P2Y4 and/or P2Y6 receptor activation. Since RB-2 (10 μM, n=5), a non-selective antagonist that acts preferentially on the P2Y4 subtype, did not modify the effect of UTP (100 μM), P2Y4 does not seem to be involved. In turn, MRS 2578 (100 nM), which is a selective P2Y6 antagonist, significantly attenuated UTP (100 μM)-induced increase. To corroborate our results, an immunocytochemistry analysis showed a positive immunoreactivity against the P2Y2 and P2Y6 receptors exhibiting a cytoplasmic/membrane labeling pattern, which is typical for those receptors in many different cells, conversely to the nuclear labeling pattern exhibited by the antibody against the P2Y4. To investigate the involvement of ADP-sensitive P2 receptors on cell proliferation and extracellular matrix production, fibroblast cultures were continuously incubated with the stable ADP analogue, ADPβS (10-100 μM). Results obtained with ADPβS (10-100 μM, n=3-6) showed no significant (P>0.05) differences in fibroblast cells proliferation. However, a continuous incubation with ADPβS (10-100 μM, n=2-5) concentration-dependently increased type I collagen production by fibroblasts (P<0.05). In order to identify which purinoceptor(s) that could be mediating this effect, we tested ADPβS (100 μM) in the presence of MRS 2179 (0.3 μM), AR-C 66096 (0.1 μM), and MRS 2211 (10 μM), which antagonize selectively ADP-sensitive P2Y1, P2Y12 and P2Y13 receptors, respectively. The facilitatory effect of ADPβS (100 μM) was significantly attenuated in the presence of the P2Y1 antagonist, MRS 2179 (0.3 μM, n=3), without being affected by the P2Y12 antagonist, AR- C 66096 (0.1 μM, n=3). In contrast, MRS 2211 (10 μM, n=2) potentiated the effect of ADPβS (100 μM) on type I collagen synthesis, thus indicating that the P2Y1-receptor-induction of type I collagen synthesis may be partially counteracted by synchronous activation of the inhibitory P2Y13 receptor. Finally, an immunocytochemistry analysis showed that these cells exhibit immunoreactivity to P2Y1 and P2Y13 receptors with a cytoplasmic/membrane staining pattern, conversely to the nuclear pattern of P2Y12. Concluding, a delicate balance between the activation of P2Y2 and P2Y6, as well as P2Y13 and P2Y1 purinoceptors, might regulate fibroblast’s induced superficial fascia remodeling. Targeting the pathways leading to fibrosis may represent an opportunity to clarify its involvement in the pathogenesis of musculoskeletal chronic pain and it may be useful for designing novel therapeutic strategies to overcome this disease

Reducing prostaglandin E2 production to raise cancer immunogenicity

Cyclooxygenases (COX), commonly upregulated in numerous cancers, generate prostaglandin E2 (PGE2), which has been implicated in key aspects of malignant growth including proliferation, invasion and angiogenesis. Recently, we showed that production of PGE2 by cancer cells dominantly enables progressive tumor growth via immune escape and that cyclooxygenase inhibitors synergize with immunotherapy to enhance tumor eradication

Cytosolic Sensing of Viruses

Cells are equipped with mechanisms that allow them to rapidly detect and respond to viruses. These defense mechanisms rely partly on receptors that monitor the cytosol for the presence of atypical nucleic acids associated with virus infection. RIG-I-like receptors detect RNA molecules that are absent from the uninfected host. DNA receptors alert the cell to the abnormal presence of that nucleic acid in the cytosol. Signaling by RNA and DNA receptors results in the induction of restriction factors that prevent virus replication and establish cell-intrinsic antiviral immunity. In light of these formidable obstacles, viruses have evolved mechanisms of evasion, masking nucleic acid structures recognized by the host, sequestering themselves away from the cytosol or targeting host sensors, and signaling adaptors for deactivation or degradation. Here, we detail recent advances in the molecular understanding of cytosolic nucleic acid detection and its evasion by viruses

Sensing infection and tissue damage

Innate and adaptive immunity work concertedly in vertebrates to restore homoeostasis following pathogen invasion or other insults. Like all homoeostatic circuits, immunity relies on an integrated system of sensors, transducers and effectors that can be analysed in cellular or molecular terms. At the cellular level, T and B lymphocytes act as an effector arm of immunity that is mobilised in response to signals transduced by innate immune cells that detect a given insult. These innate cells are spread around the body and include dendritic cells (DCs), the chief immune sensors of pathogen invasion and tumour growth. At the molecular level, DCs possess receptors that directly sense pathogen presence and tissue damage and that signal via transduction pathways to control antigen presentation or regulate a plethora of genes encoding effector proteins that regulate immunity. Notably, molecular circuits for pathogen detection are not confined to DCs or even to immune cells. All cells express sensors and transducers that monitor invasion by viruses and bacteria and elicit suitable effector barriers to pathogen propagation. Here, I discuss work from my laboratory that has contributed to our understanding of these issues over the years

Economic Impact of Prosthetic Joint Infection - an Evaluation Within the Portuguese National Health System

Introduction: Prosthetic infection is a devastating complication of arthroplasty and carries significant economic burden. The objective of this study was to analyze the economic impact of prosthetic hip and knee infection in Portuguese National Health System. Material and Methods: Case-control study carried out from January 2014 to December 2015. The mean costs of primary arthroplasties and prosthetic revision surgeries for non-infectious reasons were compared with the costs of prosthetic infections treated with debridement and preservation of the prosthesis or with two-stage exchange arthroplasty.The reimbursement for these cases was also evaluated and compared with its real costs. Results: A total of 715 primary arthroplasties, 35 aseptic revisions, 16 surgical debridements and 15 revisions for infectious reasons were evaluated. The cost of primary arthroplasties was 3,230€ in the hips and 3,618€ in the knees. The cost of aseptic revision was 6,089€ in the hips and 7,985€ in the knees. In the cases treated with debridement and implant retention the cost was 5,528€ in the hips and 4,009€ in the knees. In cases of infections treated with a two-stage revision the cost was 11,415€ and 13,793€ for hips and knees, respectively. Conclusion: As far as we know this is the first study that analyzes the economic impact of prosthetic infection in the Portuguese context. Although direct compensation for treating infected cases is much lower than calculated costs, infected cases push the overall hospital case-mix-index upwards thus increasing financial compensation for the entire cohort of treated patients. This knowledge will allow for more informed decisions about health policies in the future.info:eu-repo/semantics/publishedVersio

Panorama do uso do BIM 4D e 5D no planejamento e gerenciamento de obras na construção civil

Trabalho de Conclusão de Curso (Graduação)O governo brasileiro instituiu metas para implementação da indústria 4.0 para alavancar os avanços na indústria da construção civil. Segundo a Agenda Brasileira para a Indústria 4.0, atualmente o setor da AEC (Arquitetura, Engenharia e Construção) ocupa o ranking de 69º lugar no índice global de inovação, e de 2010 a 2016 teve uma queda na produção de 7%, enquanto países da Europa, como a Alemanha e Itália ocupam respectivamente a primeira e segunda posição. A introdução de novas tecnologias que consigam minimizar a geração de resíduos, organizar e integrar as partes interessadas reduzindo conflitos e gerenciar o tempo e custo se faz necessária. A metodologia BIM (Building Information Modeling) fornece a interação das informações de um projeto, unindo a modelagem 3D, com o gerenciamento do tempo (BIM 4D) e a gestão de custos (BIM 5D). O interesse com relação à adoção dessa metodologia por parte das empresas vem crescendo, porém, a falta de informação, de suporte, de conhecimento, de documentos para consulta, além de profissionais qualificados e experientes na área, são fatores que geram a desistência das instituições. Este artigo visou mapear sistematicamente referências que fazem o uso do BIM 4D e 5D no planejamento e gerenciamento de obras, com o objetivo de apresentar um panorama da atualidade e as lacunas a serem preenchidas, no Brasil e no mundo. Dentre os principais tópicos identificados, a falta de expertise da mão de obra em relação ao BIM e a estagnação da indústria AEC em antigos métodos foram os mais levantados pelos estudos aderidos a este artigo, logo em seguida, a falta de padronização da metodologia por meio da criação de normas e regulamentos

Molecular mechanism of influenza A NS1-mediated TRIM25 recognition and inhibition

RIG-I is a viral RNA sensor that induces the production of type I interferon (IFN) in response to infection with a variety of viruses. Modification of RIG-I with K63-linked poly-ubiquitin chains, synthesised by TRIM25, is crucial for activation of the RIG-I/MAVS signalling pathway. TRIM25 activity is targeted by influenza A virus non-structural protein 1 (NS1) to suppress IFN production and prevent an efficient host immune response. Here we present structures of the human TRIM25 coiled-coil-PRYSPRY module and of complexes between the TRIM25 coiled-coil domain and NS1. These structures show that binding of NS1 interferes with the correct positioning of the PRYSPRY domain of TRIM25 required for substrate ubiquitination and provide a mechanistic explanation for how NS1 suppresses RIG-I ubiquitination and hence downstream signalling. In contrast, the formation of unanchored K63-linked poly-ubiquitin chains is unchanged by NS1 binding, indicating that RING dimerisation of TRIM25 is not affected by NS1

Dendritic cell quiescence during systemic inflammation driven by LPS stimulation of radioresistant cells in vivo

Dendritic cell (DC) activation is a prerequisite for T cell priming. During infection, activation can ensue from signaling via pattern-recognition receptors after contact with pathogens or infected cells. Alternatively, it has been proposed that DCs can be activated indirectly by signals produced by infected tissues. To address the contribution of tissue-derived signals, we measured DC activation in a model in which radioresistant cells can or cannot respond to lipopolysaccharide (LPS). We report that recognition of LPS by the radioresistant compartment is sufficient to induce local and systemic inflammation characterized by high circulating levels of tumor necrosis factor (TNF) α, interleukin (IL) 1β, IL-6, and CC chemokine ligand 2. However, this is not sufficient to activate DCs, whether measured by migration, gene expression, phenotypic, or functional criteria, or to render DC refractory to subsequent stimulation with CpG-containing DNA. Similarly, acute or chronic exposure to proinflammatory cytokines such as TNF-α ± interferon α/β has marginal effects on DC phenotype in vivo when compared with LPS. In addition, DC activation and migration induced by LPS is unimpaired when radioresistant cells cannot respond to the stimulus. Thus, inflammatory mediators originating from nonhematopoietic tissues and from radioresistant hematopoietic cells are neither sufficient nor required for DC activation in vivo

Dendritic cells in remodeling of lymph nodes during immune responses

A critical hallmark of adaptive immune responses is the rapid and extensive expansion of lymph nodes. During this process, the complex internal structure of the organs is maintained revealing the existence of mechanisms able to balance lymph node integrity with structural flexibility. This article reviews the extensive architectural remodeling that occurs within lymph nodes during adaptive immune responses and how it is regulated by dendritic cells (DCs). In particular we focus on previously unappreciated functions of DCs in coordinating remodeling of lymph node vasculature, expansion of the fibroblastic reticular network and maintenance of lymphoid stromal phenotypes. Our increased understanding of these processes indicates that DCs need to be viewed not only as key antigen-presenting cells for lymphocytes but also as broad-acting immune sentinels that convey signals to lymphoid organ stroma and thereby facilitate immune response initiation at multiple levels

Platelet-rich blood derivatives for tendon regeneration

Tendon injuries constitute a significant healthcare problem with variable clinical outcomes. The complex interplay of tissue homeostasis, degeneration, repair, and regeneration makes the development of successful delivery therapeutic strategies challenging. Platelet-rich hemoderivatives, a source of supra-physiologic concentrations of human therapeutic factors, are a promising application to treat tendon injuries from the perspective of tendon tissue engineering, although the outcomes remain controversial.The authors acknowledge the finan-cial support from the ERC GrantCoG MagTendon nr 772817, theProject NORTE-01-0145-FEDER-000021, the Project FOOD4CELLS(Pedro Babo post-doc grant)(PTDC/CTM-BIO/4706/2014-POCI-01-0145-FEDER 016716), andHORIZON 2020 under the TEAM-ING Grant agreement no 739572—The Discoveries CTR