Made available in DSpace on 2014-06-11T19:32:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-01-20Bitstream added on 2014-06-13T21:04:28Z : No. of bitstreams: 1 ramos_op_dr_arafcf.pdf: 948525 bytes, checksum: 0581866624e7c3f57ffffca838856184 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Yersinia pseudotuberculosis e Y. enterocolitica são patógenos que causam desordens gastrintestinais. Estudos utilizando infecção in vitro demonstraram que Y. enterocolitica pode ter como alvo as células dendríticas (DCs), afetando várias de suas funções, incluindo sua maturação e produção de citocinas, e, conseqüentemente, contribuindo para a diminuição da ativação de células T CD4+. O objetivo deste estudo foi investigar o papel das células dendríticas das placas de Peyer (PP) na determinação do padrão de resposta imune, Th1 e Th2, durante a infecção por via intragástrica de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6) com a amostra virulenta de Y. pseudotuberculosis (YpIII pIB1 – Yp+) ou seu par isogênico, curado do plasmídeo de virulência (YpIII – Yp-). As DCs das PP foram obtidas no 1°, 3° e 5° dia pós-infecção, quantificadas e analisadas quanto às suas subpopulações, expressões de moléculas de superfície e capacidade imunoestimulatória por citometria de fluxo, e quanto à secreção de citocinas (IL-4, IL-10, IL-12 e TNF-α) por ELISA. Os linfócitos das PP também foram obtidos no mesmo período e tiveram suas sub-populações e o padrão de citocinas intracelulares Th1/Th2 (IL-2, IL-4, IL-10 e IFN-γ) analisado por citometria de fluxo. A infecção por Yp+ reduziu o número de DCs no 1° dia pós-infecção e aumentou, no período inicial, a expressão de B7.1 e B7.2 nos camundongos BALB/c. Nos camundongos C57BL/6 reduziu o número de DCs durante todo o período analisado, aumentou a expressão de B7.1 e B7.2 no período inicial e a expressão de ICAM-1. A infecção por ambas as amostras provocou redução da sub-população CD8α+ e da expressão de MHC II nas duas linhagens de animais, aumentou a sub-população CD11b+ nos animais suscetíveis e diminuiu nos animais resistentes. Os animais estudados não apresentaram... / Yersinia pseudotuberculosis and Y. enterocolitica are pathogens that cause gastrointestinal disorders. Studies using in vitro infection demonstrated that Y. enterocolitica can have as a target dendritic cells (DCs), affecting several of its functions, including their maturation and production of cytokines, and, consequently, contributing to the diminished activation of the T CD4+ cells. The aim of this study was to investigate the role of dendritic cell from Peyer’s patches (PP) in determining of immune response pattern, Th1 and Th2, during infection by the intragastric route in susceptible (BALB/c) and resistant (C57BL/6) mice with a virulent sample of Yersinia pseudotuberculosis (YpIII pIB1 – Yp+) or its isogenic pair, cured of the virulence plasmid (YpIII – Yp-). The PP DCs were obtained on the 1st, 3rd and 5th days postinfection, quantified and analyzed as far as their subpopulations, expressions of surface molecules and immunostimulatory capacity by flow cytometry, and the cytokines secretion (IL-4, IL-10, IL-12 and TNF-α) by ELISA. The PP lymphocytes were also obtained in the same period, and had their subpopulations and the pattern of intracellular Th1/Th2 cytokines (IL-2, IL-4, IL-10 and IFN-γ) analysed by flow cytometry. The infection by Yp+ reduced the number of DCs on the 1st day post-infection and increased, in the initial period, the expression of B7.1 and B7.2 in BALB/c. In C57BL/6 mice reduced the number of DCs throughout the study period, increased the expression of B7.1 and B7.2 in the initial period and the expression of ICAM-1. The infection by both samples reduced CD8α+ subpopulation and expression of MHC II in both animals, increased CD11b+ sub-population in susceptible animals and reduced the same sub-population in resistant animals. The studied animals did not present important differences as far as secretion of cytokines by the DCs of PP and both... (Complete abstract click electronic access below)

Les dendrimères sont des entités chimiques "arborescentes" multi-ramifiées. Cette famille de polymères non linéaires comporte des applications dans différents domaines. Un dendrimère nommé ABP a montré des propriétés immuno-modulatrices sur le système immunitaire humain in vitro. Ce dendrimère comporte le profil d'un nouvel agent thérapeutique pour être utilisé dans le traitement de maladies inflammatoires chez l'homme. Parmi les cellules mononucléaires du sang humain, le dendrimère ABP cible principalement les monocytes et induit une activation anti-inflammatoire de ces cellules. Cette nouvelle étude a visé à comprendre l'interaction entre le dendrimère ABP et les monocytes humains. De manière intéressante, une interaction spécifique a été observée. En utilisant des expériences de modélisation, de physico-chimie et de biologie moléculaire, nous démontrons que le dendrimère ABP induit une activation anti-inflammatoire des monocytes humains par l'intermédiaire d'un ou de récepteur (s). / Dendrimers are multi-branched "tree-like" chemical entities. This family of non-linear polymers has already been applied in different fields. A dendrimer named ABP has shown some immuno-modulatory properties toward the Human immune system in vitro. This dendrimer is at the dawn to become a new therapeutic agent by itself for the treatment of inflammatory diseases in Human. Among human Peripheral Blood Mononuclear Cells (PBMC), the ABP dendrimer selectively targets human monocytes and induces an anti-inflammatory activation of these cells. This new study aims at understanding the interaction between the ABP dendrimer and human monocytes. Interestingly, a specific interaction was observed. Using, biological, physico-chemical and molecular modelling experiments we demonstrated that the ABP dendrimer triggers monocyte activation through receptor(s) recognition.

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Adenosine 5’ triphosphate (ATP) is a molecule central to life for its role as the cellular energy currency, and a purine nucleotide which serves as a building block of RNA. Thus, on the backdrop of an indispensible intracellular role of ATP, its identification as an extracellular signaling molecule in early 1970s came as a surprise. A novel doctrine, termed as ‘purinergic signaling’, was thus put forth. By definition, purinergic signaling consists of the signaling events triggered by binding of extracellular ATP- a purine nucleotide, and its breakdown products (viz., ADP, AMP, and adenosine) to their cognate receptors, which in turn are termed as ‘purinergic receptors’. Based on their ligand affinity, purinergic receptors are classified into two groups- P1 and P2 receptors. P2 receptors are further subclassified as P2X and P2Y receptors. Till date, four P1 receptors (viz. A1, A2a, A2b, and A3), seven P2X receptors (P2X1-7), and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and characterized. Conceptually, the first step of purinergic signaling is the release of ATP from an intact cell on encountering a stimulant or a modulator. The main mechanisms of such cellular ATP release include vesicular exocytosis and the release through conductive channels. ATP thus released, binds to its cognate receptors (i.e. P2X receptors, and certain P2Y receptors) and triggers the ‘purinergic signaling’ pathway that modulates the cellular response. In addition to purinergic receptors, cells also express ATP degrading enzymes on their surface, which break ATP down into ADP, AMP, and adenosine. ADP and adenosine, in turn, bind to their cognate receptors (certain P2Y receptors, and P1 receptors respectively) and further contribute to shaping the cellular response to a given cue. Thus, purinergic signaling is a highly dynamic process with pleiotropic downstream effects. First demonstrated in the context of neurotransmission, the phenomenon of purinergic signaling is now widely recognized and has been shown to play a role in regulating functional responses of cells of diverse origins, immune cells being one of them. Purinergic signaling in lymphocytes- an important subset of immune cells- is a common thread for the present research exercise, wherein we have addressed two sets of questions, one of academic curiosity and the other of clinical interest. In the former and the major part, we have examined whether purinergic signaling plays a role in functional aspects of ‘gamma delta (γδ) T cells’, which represent a unique subset of lymphocytes. Whereas, the latter part elaborates on the already identified involvement of purinergic signaling in T cell stimulatory action of ‘hypertonic saline (HS)’, which is used to treat trauma patients. The thesis, thus, is divided into five parts- the ‘Introduction’, ‘Aims and Scope of the study’, ‘Chapter 1’, ‘Chapter 2’, and ‘Summary of the work’. Understanding the questions posed in the present context, strategy designed to answer them, and eventually the experimental results answering these questions invoke basic knowledge of purinergic signaling, which has been attempted to be conferred through the ‘Introduction’ section. The discovery of purinergic signaling, its central theme, and individual molecular players involved in this signaling pathway are highlighted here. From the viewpoint of the present research endeavor, salient findings from the current literatureabout the involvement of purinergic signaling in the functional activities of various subsets of immune cells- are reviewed towards the end of this section. The ‘Introduction’ is followed by definition of the objectives for the present exercise, which are enlisted under ‘Aims and scope of the study’. Here, a brief overview of the background data that led us towards these objectives precedes the actual list of questions which we have approached. Purinergic signaling has been shown to play a role in the activation of ‘conventional αβ T’ cells. So we asked whether a similar purinergic signaling pathway also operates in unconventional γδ T cells. Thus, ‘Chapter 1’ is dedicated to answering the first set of questions about the role of purinergic signaling in γδ T cell activation. The chapter starts off by introducing γδ T cells. The topics such as discovery of γδ T cells, ontology, development, diversity, and distribution of these cells, and most importantly- their antigenic specificity and response are reviewed herein. The details of the experimental procedures employed to answer the defined objectives follow this introduction. We have carried out our experiments on γδ T cells in human circulation. For in vitro stimulation, we have used anti-CD3 + anti-CD28-coated beads (beads) or isopentenyl pyrophosphate (IPP), a γδ T cell specific stimulant. We observed that, circulating human γδ T cells rapidly release ATP on stimulation with beads or IPP. Pannexin-1 and connexin hemichannels, as well as vesicular exocytosis contribute to the ATP release. Real time RT-PCR data revealed that γδ T cells predominantly express purinergic receptors A2a, P2X1, P2X4, P2X7, and P2Y11. Of these, the inhibition of P2X4 receptors downregulated cytokine expression by γδ T cells post- in vitro stimulation, and also inhibited cytotoxic activity of γδ T cells towards Daudi cells. Selective translocation of P2X4 receptors to the immunological synapse was seen to be the underlying mechanism for these effects. Collectively, these data suggested that autocrine/paracrine purinergic signaling through P2X4 receptors indeed plays an important role in the functional aspects of circulating human γδ T cells. The experimental results are compiled in ‘Chapter 1’; which concludes with the ‘Discussion’ on the mentioned findings, and possible in vivo applications. ‘Chapter 2’ deals with the role of purinergic signaling in HS resuscitation. In addition to restoring the hemodynamic parameters, fluid replacement with small volumes of concentrated NaCl solution (HS) has been reported to reverse the suppression of T cells commonly found in the trauma subjects. Through an in vitro study using Jurkat cells as a model for primary human T cells, it has been shown earlier that, on HS exposure T cells release ATP- which binds to P2X7 receptors and promotes calcium influx. HS treatment also elicits phosphorylation of p38; and put together, Ca2+ influx and phosphorylated p38 synergize with TCR-induced stimulation resulting in the enhancement of transcriptional upregulation of IL-2. However, the mechanism of release of ATP on HS treatment and the possible involvement of P2X1 and P2X4 receptors expressed by T cells had not been addressed in this study. These very questions thus formed the objectives of the second part of present work. Experiments aimed to answer these questions showed that on HS treatment, Jurkat cells release ATP through pannexin-1 hemichannels. The released ATP binds to purinergic receptors P2X1, P2X4, and P2X7. This in turn triggers the downstream signaling cascade leading to phosphorylation of p38 and upregulation of IL-2 transcription, hence augmenting the T cell function. An overview of HS resuscitation, experimental protocols and results, and the discussion on the pathophysiological relevance of these findings comprise ‘Chapter 2’. Hence, we have found the answers to the questions we began with. The results are listed in a point-wise manner under the ‘Summary of the work’. Taken together, our data shows that: (i) Purinergic signaling does play a role in the functional aspects of circulating human γδ T cells. The release of ATP by γδ T cells post-stimulation, and autocrine/paracrine signaling through P2X4 receptors are the main components in this context. (ii) ATP release through pannexin-1 hemichannels, and autocrine/paracrine signaling through P2X1, P2X4, and P2X7 receptors underlie the mechanism of action of HS.

The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-induced proteins. The TCR proximal tyrosine kinase ITK simultaneously influences many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, I examined NFAT, NF-κB and MAP kinase pathways during early activation of individual naïve OT-I CD8+ T cells using peptide-loaded antigen presenting cells. Utilizing both measurement of transcription factor translocation in single T cell nuclei and conventional phospho-flow cytometry, I observed digital activation of Erk-MAPK and NFAT1 at all peptide doses and avidities. However, NF-κB activation showed a graded response to variation in TCR signal strength and was more sensitive to treatment with an ITK inhibitor. Inhibitor-treated cells showed poor induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis revealed genomic regions most sensitive to ITK inhibition are enriched for NF-κB and AP-1 motifs. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, I describe a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells.

Microtubules (MTs) are highly dynamic structures essential for the spatio-temporal intracellular organization and transport, signal propagation, cell differentiation, motility and division. To perform these roles, MTs create arrangements capable of fast and precise adaptation to various signals. MTs are under the control of many factors regulating MT nucleation, stability and dynamics. Bone marrow-derived mast cells (BMMCs) are important immune system cells, which can cause serious diseases if their functions are deregulated. Although MT reorganization during BMMC activation is well established, the molecular mechanisms that control their remodelling are largely unknown. In the presented thesis we functionally characterised GIT1/βPIX signalling proteins, PAK1 kinase, and Ca2+ signalling in the regulation of MT nucleation in BMMCs and other cell types. We also elucidated the function of miltefosine (hexadecylphosphocholine), a promising candidate for the treatment of mast cell-driven diseases. We found that GIT1/βPIX signalling proteins are γ-tubulin-interacting proteins associating with centrosomes in BMMCs. MT nucleation is positively regulated by GIT1 and Ca2+ , whereas βPIX is a negative regulator of MT nucleation in BMMCs. Cytosolic Ca2+ affects γ-tubulin properties and stimulates the...

Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS injury, and their activation is critical for maintaining homeostasis within the CNS. However, during inflammatory conditions, sustained microglial activation results in damage to surrounding neuronal cells. β-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that β-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate β-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of β-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, β-glucan suppressed TLR-mediated NF-κB activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that β-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions.

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The underlying cause of all vascular proliferative diseases is injury-induced activation of vascular endothelium and vascular smooth muscle cells (VSMC). Activated VSMC proliferate, than migrate from the arterial media to the intima, contributing to neointima formation. Activated immune cells, vascular cells, and their endogenous regulators mediate this complex process. One integral regulator of VSMC activation is allograft inflammatory factor-1 (AIF-1). AIF-1 is a cytoplasmic scaffold protein, expressed constitutively in lymphoid cells and induced in VSMC by injury. Stable over expression of AIF-1 increases VSMC proliferation and migration in vitro, causes increased injury-induced neointima formation, and increases Rac1 and p38 MAP Kinase activity. Recent studies show a correlation between VSMC expression of AIF-1 and atherosclerosis development. We hypothesize that VSMC over expression of AIF-1 contributes to atherosclerosis development by increasing activity of inflammatory signaling molecules, and that inhibiting VSMC AIF-1 expression will decrease injury-induced neointima formation. Rat carotid arteries transfected with AIF-1 si RNA adenovirus after balloon angioplasty developed significantly less neointima compared to controls. AIF-1 si RNA transfected VSMC proliferated significantly less than AIF-1 or GFP transfected VSMC, while AIF-1 si RNA transfection did not attenuate AIF-1-mediated migration. p38 inhibition showed that AIF-1-mediated proliferation is dependent on p38 activation while AIF-1-mediated migration is not. AIF-1 transgenic mice fed a high fat diet showed significantly more atherosclerotic lesions than WT littermates. Boyden Chamber assays showed OxLDL treatment increases VSMC migration but does not effect AIF-1-mediated migration. Expression of migration and inflammatory responsive genes in AIF-1 and XGal transfected VSMC after OxLDL treatment at various time points were examined. MMP-2 and -9 expression did not change. ICAM-1 and VCAM-1 expression increased in both groups. AIF-1 VSMC showed significantly higher ICAM-1 expression at baseline and early time points and elevated, but not significantly higher VCAM-1 expression at early time points. Western blots showed increased activation of NF-kB in AIF-1 transfected VSMC at baseline and 30 minutes after OxLDL stimulation compared to XGal transfected VSMC. Expression of the scavenger receptor receptors CD36 and SRA(I) expression increased after lipid treatment in AIF-1 and XGal transfected groups. AIF-1 VSMC showed sustained expression of both receptors after 16 hours of treatment compared to XGal VSMC, which showed decreased expression at that time point. CXCL16/PSOX expression increased with treatment, but differences in expression patterns were not seen between cell groups. Analysis showed significantly more OxLDL was taken up by AIF-1 VSMC compared to XGal VSMC. These data show that AIF-1 expression in VSMC is tightly linked to the vascular response to injury and development of vascular disease. Although AIF-1-mediated migration is not p38 dependent, AIF-1 may contribute to increased VSMC migration in part by upregulating NF- kB downstream effectors through increased NF-kB activity. AIF-1 may also speed the progression of atherosclerosis by increasing scavenger receptor expression and thereby increasing OxLDL uptake and foam cell formation. Although more study is required to fully elucidate the molecular mechanisms leading to AIF-1 mediated VSMC activation, these data have further established AIF-1 as an integral regulator of the VSMC response to injury. / Molecular and Cellular Physiology