Intracellular Hmgb1 inhibits inflammatory nucleosome release and limits acute pancreatitis in mice

BACKGROUND & AIMS: High mobility group box 1 (HMGB1) is an abundant protein that regulates chromosome architecture and also functions as a damage-associated molecular pattern molecule. Little is known about its intracellular roles in response to tissue injury or during subsequent local and systemic inflammatory responses. We investigated the function of Hmgb1 in mice after induction of acute pancreatitis. METHODS: We utilized a Cre/LoxP system to create mice with pancreas-specific disruption in Hmbg1 (Pdx1-Cre; HMGB1(flox/flox) mice). Acute pancreatitis was induced in these mice (HMGB1(flox/flox) mice served as controls) after injection of l-arginine or cerulein. Pancreatic tissues and acinar cells were collected and analyzed by histologic, immunoblot, and immunohistochemical analyses. RESULTS: After injection of l-arginine or cerulein, Pdx1-Cre; HMGB1(flox/flox) mice developed acute pancreatitis more rapidly than controls, with increased mortality. Pancreatic tissues of these mice also had higher levels of serum amylase, acinar cell death, leukocyte infiltration, and interstitial edema than controls. Pancreatic tissues and acinar cells collected from the Pdx1-Cre; HMGB1(flox/flox) mice after l-arginine or cerulein injection demonstrated nuclear catastrophe with greater nucleosome release when compared with controls, along with increased phosphorylation/activation of RELA nuclear factor kappaB, degradation of inhibitor of kappaB, and phosphorylation of mitogen-activated protein kinase. Inhibitors of reactive oxygen species (N-acetyl-l-cysteine) blocked l-arginine-induced DNA damage, necrosis, apoptosis, release of nucleosomes, and activation of nuclear factor kappaB in pancreatic tissues and acinar cells from Pdx1-Cre; HMGB1(flox/flox) and control mice. Exogenous genomic DNA and recombinant histone H3 proteins significantly induced release of HMGB1 from mouse macrophages; administration of antibodies against H3 to mice reduced serum levels of HMGB1 and increased survival after l-arginine injection. CONCLUSIONS: In 2 mouse models of acute pancreatitis, intracellular HMGB1 appeared to prevent nuclear catastrophe and release of inflammatory nucleosomes to block inflammation. These findings indicate a role for the innate immune response in tissue damage

High Pro-Inflammatory Cytokine Secretion and Loss of High Avidity Cross-Reactive Cytotoxic T-Cells during the Course of Secondary Dengue Virus Infection

BACKGROUND: Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype. METHODS AND FINDINGS: Virus-specific cytotoxic T lymphocytes (CTL) showing high level cross reactivity between dengue serotypes could be expanded from blood samples taken during the acute phase of secondary dengue infection. These could not be detected in convalescence when only CTL populations demonstrating significant serotype specificity were identified. Dengue cross-reactive CTL clones derived from these patients were of higher avidity than serotype-specific clones and produced much higher levels of both type 1 and certain type 2 cytokines, many previously implicated in dengue pathogenesis. CONCLUSION: Dengue serotype cross-reactive CTL clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. That such cells cannot be expanded from convalescent samples suggests that they may be depleted, perhaps as a consequence of activation-induced cell death. Such high avidity cross-reactive memory CTL may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. These cells appear to be subsequently deleted leaving a more serotype-specific memory CTL pool. Further studies are needed to relate these cellular observations to disease phenotype in a large group of patients. If confirmed they have significant implications for understanding the role of virus-specific CTL in pathogenesis of dengue disease

Transduction of Human T Cells with a Novel T-Cell Receptor Confers Anti-HCV Reactivity

Hepatitis C Virus (HCV) is a major public health concern, with no effective vaccines currently available and 3% of the world's population being infected. Despite the existence of both B- and T-cell immunity in HCV-infected patients, chronic viral infection and HCV-related malignancies progress. Here we report the identification of a novel HCV TCR from an HLA-A2-restricted, HCV NS3:1073–1081-reactive CTL clone isolated from a patient with chronic HCV infection. We characterized this HCV TCR by expressing it in human T cells and analyzed the function of the resulting HCV TCR-transduced cells. Our results indicate that both the HCV TCR-transduced CD4+ and CD8+ T cells recognized the HCV NS3:1073–1081 peptide-loaded targets and HCV+ hepatocellular carcinoma cells (HCC) in a polyfunctional manner with cytokine (IFN-γ, IL-2, and TNF-α) production as well as cytotoxicity. Tumor cell recognition by HCV TCR transduced CD8− Jurkat cells and CD4+ PBL-derived T cells indicated this TCR was CD8-independent, a property consistent with other high affinity TCRs. HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections

O potencial pedagógico da Hora do Conto para o desenvolvimento da Comunicação Oral

O presente relatório é produto de um projeto de investigação que pretendeu caracterizar a adequação e dinamização da hora do conto com a finalidade de promover o desenvolvimento da comunicação oral, implementada numa sala de 3/4 anos e a adequação didática de um processo de intervenção educativa de estratégias para a promoção do desenvolvimento da Oralidade (incidindo na dinamização da hora conto) implementado numa turma de 3.º ano do 1.º Ciclo do Ensino Básico. Tendo consciência da desvalorização que é atribuída à temática em análise, acreditamos que os profissionais de educação se tornam responsáveis pelo desenvolvimento e aperfeiçoamento das capacidades de comunicação das crianças/alunos. Neste sentido, consideramos que foi extremamente importante investir neste domínio e nas competências a ele associado, nas vertentes de compreensão e expressão orais. Para atingir os objetivos delineamos, planificamos, construímos, aplicamos e avaliamos em sala de aula, um conjunto de intervenções educativas adotando como estratégia principal a dinamização da hora do conto. Assim sendo, podemos verificar através dos resultados obtidos, que efetivamente, a hora do conto e a utilização de diferentes estratégias para a dinamização da mesma, contribuíram positivamente para o desenvolvimento das competências orais dos intervenientes, potenciando melhorias, a vários níveis, nomeadamente na vertente de compressão e de expressão oral dos dois grupos de aplicação.This report is the product of a research project that aimed to characterize the adequacy and dynamization of the story time with the purpose of promoting the development of oral communication, implemented in a 3/4 year classroom and the didactic adequacy of a process of educational intervention strategies to promote the development of Orality (focusing on the dynamization of the story time) implemented in a 3rd year class of the 1st Cycle of Basic Education. Being aware of the devaluation that is attributed to the theme under analysis, we believe that education professionals become responsible for the development and improvement of communication skills of children/students. In this sense, we believe that it was extremely important to invest in this field and in the skills associated with it, in the areas of understanding and oral expression. In order to achieve the objectives, we delineate, plan, build, apply and evaluate in the classroom, a set of educational interventions adopting as the main strategy the dynamization of the story time. Therefore, we can verify through the results obtained, that effectively, the story time and the use of different strategies for the dynamization of the tale, contributed positively to the development of the oral skills of the participants, enhancing improvements, at various levels, particularly in the compression and oral expression of the two groups of application. Keywords: Oral Communication; Compression and Oral Expression; Story Time; Pre-School; 1st Cycle of Basic Education

PBMC are as good a source of tumor-reactive T lymphocytes as TIL after selection by Melan-A/A2 multimer immunomagnetic sorting.: Immunomagnetic sort of Melan-A specific T cells from TIL and PBMC

International audienceChoosing a reliable source of tumor-specific T lymphocytes and an efficient method to isolate these cells still remains a critical issue in adoptive cellular therapy (ACT). In this study, we assessed the capacity of MHC/peptide based immunomagnetic sorting followed by polyclonal T cell expansion to derive pure polyclonal and tumor-reactive Melan-A specific T cell populations from melanoma patient's PBMC and TIL. We first demonstrated that this approach was extremely efficient and reproducible. We then used this procedure to compare PBMC and TIL-derived cells from three melanoma patients in terms of avidity for Melan-A A27L analog, Melan-A(26-35)and Melan-A(27-35), tumor reactivity (lysis and cytokine production) and repertoire. Regardless of their origin, i.e., fresh PBMC, peptide stimulated PBMC or TIL, all sorted populations (from the three patients) were cytotoxic against HLA-A2+ melanoma cell lines expressing Melan-A. Although some variability in peptide avidity, lytic activity and cytokine production was observed between populations of different origins in a given patient, it differed from one patient to another and thus no correlation could be drawn between T cell source and reactivity. Analysis of Vbeta usage within the sorted populations showed the recurrence of Vbeta3 and Vbeta14 subfamilies in the three patients but differences in the rest of the Melan-A repertoire. In addition, in two patients, we observed major repertoire differences between populations sorted from the three sources. We especially documented that in vitro peptide stimulation of PBMC, used to facilitate the sort by enriching in specific T lymphocytes, could significantly alter their repertoire and reactivity towards tumor cells. We conclude that PBMC which are easily obtained from all melanoma patients, can be as good a source as TIL to derive high amounts of tumor-reactive Melan-A specific T cells, with this selection/amplification procedure. However, the conditions of peptide stimulation should be improved to prevent a possible loss of reactive clonotypes