A hidroquinona (HQ) é um composto fenólico de origem natural ou antropogênica, encontrada em grandes concentrações no cigarro, além de ser produto da biotransformação do benzeno. Nosso grupo de pesquisa tem demonstrado que a exposição à HQ compromete a resposta inflamatória in vivo. Dando continuidade a estas investigações, este trabalho visou investigar os efeitos da exposição in vivo à HQ sobre funções do tecido traqueal e atividades de macrófagos alveolares. Para tanto, camundongos Swiss machos foram expostos à HQ 25ppm (1,5mg/60mL/1h; 5 dias) ou veículo (solução salina etanol 1:20) por via sistêmica (nebulização). Concentrações de mediadores inflamatórios (interleucina (IL) 1β, IL-6, IL-10, IL-4, IL-12 fator de necrose tumoral-α (TNF-α) ou proteína quimiotáxica de monócitos (MCP-1); ensaio imunoenzimático (ELISA) e óxido nítrico (NO; reação de Griess) foram quantificados no lavado bronco-alveolar (LBA) em condições basais ou 3 horas após estímulo inflamatório (LPS in vivo; 100µL/mL; 10min) e no sobrenadante de macrófagos (MΦs) alveolares ou de cultura da traquéia obtidos dos animais e posteriormente estimulados in vitro (MΦs: 5µg/mL de LPS + 10ng/mL de IFN-γ; traquéia: 1µg/mL de LPS; 24h). Atividades fagocítica e fungicida (microscopia óptica) e a expressão de receptores envolvidos na fagocitose toll-like receptor (TLR, TLR-2, TLR4 e dectina-1; citometria de fluxo) foram determinadas após incubação in vitro de MΦs alveolares com o fungo Candida albicans e a expressão protéica de MyD88 foi realizada em MΦs alveolares (western blot). Quantificação do RNAm para MCP-1 (reação da transcriptase reversa em cadeia de polimerase, RT-PCR) foi realizada em tecido traqueal e células de linhagem monocítica humana THP-1 foram empregadas em ensaios de quimiotaxia in vitro (Câmara de Boyden) frente a diferentes concentrações de MCP-1. Reatividade do tecido traqueal foi quantificada frente à metacolina. Os resultados obtidos mostraram que a exposição in vivo à HQ reduziu a concentração de MCP-1 (54,98% vs. controle LPS) e IL-12 (51,45% vs. controle LPS) no LBA após inflamação; reduziu a secreção de MCP-1 por MΦs (basal: 87,96%; LPS+INF-γ: 61,20%) e tecido traqueal em cultura (79,77% vs. controle LPS). Neste último tecido, a diminuição foi dependente da menor expressão gênica. Concentrações de MCP-1 semelhantes à detectada no sobrenadante de cultura de traquéia de animais expostos à HQ induziram migração de células THP-1 menor (38,2%) que à provocada pela concentração de MCP-1 no sobrenadante da cultura traquéia de animais controles. Traquéias de animais expostos à HQ apresentaram hiperreatividade (193,48%), a qual foi revertida pela remoção do epitélio traqueal. Adicionalmente, cultura de MΦs obtidos de animais expostos à HQ apresentaram maior atividade fagocítica (porcentagem de fagocitose: 36,30%; índice de fagocitose: 83,97%) e fungicida (68,47%), que não foram dependentes de alterações nos receptores TLR2, TLR4 e dectina-1, mas podem ser decorrentes da menor expressão protéica de MyD88. Em conjunto, os dados obtidos apontam para alterações importantes resultantes da exposição in vivo à HQ sobre funções de MΦs alveolares e do tecido traqueal que, em conjunto, podem ser determinantes para a toxicidade observada nestes animais que culmina com prejuízo na defesa do organismo. / Hydroquinone (HQ) is a phenolic compound of natural or anthropogenic source, also found in high concentrations in cigarette, as well as benzene´s metabolite. Our research group has demonstrated that exposure to HQ impairs in vivo inflammatory response. Following these investigations, this work aimed to study the effects of in vivo exposure to HQ on tracheal tissue and alveolar macrophages (MΦs) activities. For this purpose, male Swiss mice were systemically (aerolised) exposed to 25ppm HQ (1.5 mg/60mL/1h; 5 days) or vehicle (saline ethanol solution, 1:20). Concentrations of inflammatory mediators (interleukin (IL) IL-1β, IL- 6, IL-10, IL-4, IL-12 tumor necrosis factor-α (TNF-α ) or monocyte chemoattractant protein (MCP-1); (enzyme immune assay, ELISA)) and nitric oxide (NO; Griess reaction) were quantified in bronchoalveolar lavage (BAL) at baseline or 3 hours after inflammatory stimulus (in vivo LPS, 100µL/mL; 10min); in the supernatant of cultured alveolar macrophages (MΦs) or trachea obtained from animals and subsequently in vitro stimulated (MΦs: 5µg/mL of LPS plus 10ng/mL IFN-γ; trachea: 1µg/mL LPS; 24 hours). Phagocytic and fungicidal activities (light microscopy) and expression of receptors involved in phagocytosis (toll-like receptor (TLR, TLR2, TLR4 and dectin-1, flow cytometry) were determined after in vitro incubation of alveolar MΦs with Candida albicans fungus and expression of MyD88 pathway was held in alveolar MΦs (western blot). Quantification of mRNA for MCP-1 (reaction of reverse transcriptase polymerase chain reaction, RT-PCR) was performed in tracheal tissue and cells human monocytic THP-1 were used in in vitro chemotaxis assays (Boyden chamber) using different concentrations of MCP-1. Tracheal reactivity was measured in response to methacholine. The results showed that in vivo HQ exposure reduced the concentration of MCP-1 (54.98% vs. control) and IL-12 (51.45% vs. control) in the BAL after inflammation; decreased secretion of MCP-1 by MΦs (basal: 87.96%, LPS+INF-γ: 61.20%) and in tracheal culture after LPS stimulation (79.77% vs. control). In the latter tissue, the MCP-1 protein content was dependent on impaired gene expression. Concentrations of MCP-1 similar to those detected in the supernatant of tracheal from HQ exposed rats induced smaller migration of THP-1 cells (38.2%) than that evoked by the MCP-1 concentration obtained in trachea supernatants collected from control animals. Tracheas from HQ exposed rats showed hyper reactivity (193.48%), which was reversed by removal of the tracheal epithelium. Additionally, culture of MΦs obtained from HQ exposed rats showed increased phagocytic (percentage of phagocytosis: 36.30%; phagocytosis index: 83.97%) and fungicide activity (68.47%), which were not dependent on changes in the receptors TLR2, TLR4 and dectin-1, but could be due to reduced MyD88 expression. Together, these data point out important alterations on MΦs and trachea after in vivo HQ exposure, which may be crucial for the toxicity observed in these animals that culminates with impaired host defense.

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Introduction: Available data suggest an association between presence of secondary (AA) amyloidosis and MCP-1 (monocyte chemoatracttant protein-1) and MIP-1alpha (macrophage inflammatory protein-1 alpha) genes polymorphisms. Some studies have also shown an impact of polymorphisms in exon 3 of SAA 1 (serum amyloid A 1) gene on the incidence of AA amyloidosis in different populations. Methods: The incidence of single genotypes MCP-1, MIP-1alpha and SAA 1 genes was investigated. Serum levels of SAA, MCP-1 and MIP-1alpha were measured and potential relation between serum levels and genotypes were analyzed. All examinations were performed in patients with AA amyloidosis (43), rheumatoid arthritis (RA) without amyloidosis and healthy control group (100). Results: Significantly more frequent occurrence of 1.1/1.1 genotype in SAA 1 was recorded in AA amyloidosis group compared to RA group as well as in control group (p<0,001). No statistically significant differences in distribution of another genotypes were found. Distribution of neither 1.1/1.1 genotype nor another ones did not vary among RA group and control group. No significant difference in distribution of another examined genotypes was recorded among all three groups. Serum concentrations of SAA were statistically significantly higher in AA amyloidosis group...

Introduction: Available data suggest an association between presence of secondary (AA) amyloidosis and MCP-1 (monocyte chemoatracttant protein-1) and MIP-1alpha (macrophage inflammatory protein-1 alpha) genes polymorphisms. Some studies have also shown an impact of polymorphisms in exon 3 of SAA 1 (serum amyloid A 1) gene on the incidence of AA amyloidosis in different populations. Methods: The incidence of single genotypes MCP-1, MIP-1alpha and SAA 1 genes was investigated. Serum levels of SAA, MCP-1 and MIP-1alpha were measured and potential relation between serum levels and genotypes were analyzed. All examinations were performed in patients with AA amyloidosis (43), rheumatoid arthritis (RA) without amyloidosis and healthy control group (100). Results: Significantly more frequent occurrence of 1.1/1.1 genotype in SAA 1 was recorded in AA amyloidosis group compared to RA group as well as in control group (p<0,001). No statistically significant differences in distribution of another genotypes were found. Distribution of neither 1.1/1.1 genotype nor another ones did not vary among RA group and control group. No significant difference in distribution of another examined genotypes was recorded among all three groups. Serum concentrations of SAA were statistically significantly higher in AA amyloidosis group...

One of the main effects of pro-inflammatory cytokines is the induction of chemokines and the expression of adhesive molecules that regulate the migration of immune cells to the center of the damage. Chemoattractant gradient also provides a physiological delivery of cells to tissues and lymphatic organs under normal circumstances. Chemokines are chemotactic cytokines that form a very large and diverse group of secreted proteins that have many functions both in processes that maintain homeostasis but also in inflammatory states. Production of some chemokines also has a major effect on graft rejection. Further understanding of the mechanisms involved in the acute rejection chemokine could contribute to improving treatment steps in transplantology. In this diploma thesis, serum chemokine levels were monitored in renal transplant patients, but these measurements did not show significant dynamics. Furthermore, the effect of pro-inflammatory cytokines on the release of chemokines from renal epithelial cells and monocytes was studied. Experiments were performed to monitor the levels of individual chemokines such as ENA-78, IL-8, MCP-1, MIP-1 β, RANTES, GRO alpha, THP-1 (monocyte/macrophage cell line), RPTEC (renal epithelial cells of proximal tubules) and RA (renal cell tumor lines). TNF-α (tumor necrosis...

One of the main effects of pro-inflammatory cytokines is the induction of chemokines and the expression of adhesive molecules that regulate the migration of immune cells to the center of the damage. Chemoattractant gradient also provides a physiological delivery of cells to tissues and lymphatic organs under normal circumstances. Chemokines are chemotactic cytokines that form a very large and diverse group of secreted proteins that have many functions both in processes that maintain homeostasis but also in inflammatory states. Production of some chemokines also has a major effect on graft rejection. Further understanding of the mechanisms involved in the acute rejection chemokine could contribute to improving treatment steps in transplantology. In this diploma thesis, serum chemokine levels were monitored in renal transplant patients, but these measurements did not show significant dynamics. Furthermore, the effect of pro- inflammatory cytokines on the release of chemokines from renal epithelial cells and monocytes was studied. Experiments were performed to monitor the levels of individual chemokines such as ENA-78, IL-8, MCP-1, MIP-1β, RANTES, GRO-α, THP-1 (monocyte/macrophage cell line), RPTEC (renal epithelial cells of proximal tubules) and RA (renal cell tumor lines). TNF-α had an effect on the...

Previous studies demonstrated that intravenous administration of human umbilical cord blood (HUCB) cells could improve behavioral and neurological recovery of stroked animals following middle cerebral artery occlusion (MCAO). In addition, HUCB cell recipients had less of an inflammatory response with less leukocyte infiltration. In these studies we explored how HUCB cells change the inflammatory response of neurons, astrocytes, and microglia to hypoxia/ischemia. Initiation of the inflammatory response occurs with the expression of chemokines. We determined that monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1alph (MIP-1a), which are upregulated in the brain early after a stroke, induce migration of HUCB cells to the site of injury. Neutralizing these chemokines with antibodies prevented migration in an in vitro migration assay. We next explored the interaction of the whole HUCB mononuclear cell fraction, as well as subpopulations from within the mononuclear fraction (T cell alone, B cell alone, and monocytes/macrophage alone) with cultures of enriched neurons, astrocytes or microglia exposed to hypoxia in an oxygen, glucose deprivation paradigm. We showed that HUCB cells increased the cell viability of neurons and astrocytes, while decreasing cell viability of microglia. There was also a change in the cytokine secretion profile from the cells exposed to HUCB cells under hypoxic conditions. These results suggested that chemokines, MCP-1 and MIP-1a increased in stroked brain, and they played an important role in recruitment of HUCB into the CNS after intravenous administration. Once inside the brain, HUCB could suppress the immune response by promoting microglial death and modulating the function of astrocytes. In addition, HUCB cells provide neuron protection against the injury caused by stroke. However, it is unlikely to contribute the effect of HUCB to a single population of HUCB.

Bacillary angiomatosis (BA), one of the clinical manifestations resulting from infection with the facultative intracellular bacterium Bartonella henselae, is characterized by angiogenic lesions. Endothelial cells have been identified as host cells for this pathogen and are presumed important for pathogenesis as lesions contain bacteria in direct contact with the endothelium. Lesions also contain infiltrating macrophages, which contribute to the angiogenic process during B. henselae infection by secreting vascular endothelial growth factor (VEGF). While virulence factors have been characterized, and the role for macrophages in B. henselae infection has been established, endothelial cell mediators of angiogenesis have not been well defined. We investigated three potentially important pathways that are triggered by the bacterial interactions with endothelial cells. We examined the ability of endothelial cells to upregulate the chemokines monocyte-macrophage chemoattracta nt protein-1 (MCP-1) and CXCL8 and the mechanism by which B. henselae secreted proteins (BHSP) induce endothelial cell proliferation. We determined that MCP-1 production is upregulated in response to B. henselae infection, which very likely contributes to angiogenic lesion formation by recruiting the VEGF-secreting macrophage. The chemokine CXCL8 is an important mediator of angiogenesis which can cause endothelial cell survival, proliferation, and capillary tube formation. We determined that CXCL8 is secreted from B. henselae-infected cells and contributes to B. henselae-induced angiogenesis in an autocrine manner. We also investigated the role of Ca2+ signaling during B. henselae infection. We determined that BHSP induce a robust intracellular Ca2+ response in HUVEC which originates from intracellular Ca2+ pools. Additionally, endothelial cell proliferation in response to BHSP required Ca2+ activity, indicating a role for intracellular Ca2+ pools during B. henselae-induced angio genesis. Endothelial cell proliferation during B. henselae infection possibly indicates a mechanism by which a pathogen induces proliferation of its host cell in order to propagate its own survival. Numerous factors culminate in the vascular lesions that are characteristic of BA. B. henselae infection represents an important and unique model for pathogen-triggered angiogenesis, and studies into the specific mechanisms of this process may elucidate host cell-pathogen interactions as well as pathways of pathogenic angiogenesis.

La nostra proposta ha profunditzat per primera vegada en l’estudi de les lipoproteïnes d’alta densitat (HDL) en la infecció pel virus de la immunodeficiència humana (VIH), i hem determinat la seva implicació en el curs de la infecció i en el desenvolupament de l’arteriosclerosi amb la finalitat de dissenyar noves estratègies terapèutiques. Els nostres resultats mostren com l’estat inflamatori i oxidant persistent associat a la infecció té un paper central en l’aparició d’alteracions metabòliques i el desenvolupament d’aterosclerosi en aquests pacients. Concretament, utilitzant tècniques cromatogràfiques i proteòmiques, mostrem com la presència anòmala de gammaglobulines, molècules pro-inflamatories i reactants de fase aguda en el plasma d’aquests pacients indueixen la modificació del metabolisme, composició i distribució de les partícules HDL, suggerint que la monitorització de les partícules HDL en aquests pacients no ha d’estar basada únicament en la determinació de la concentració del contingut de colesterol, sinó que ha d’incloure la determinació d’altres paràmetres associats a la qualitat, i per tant, a la funcionalitat, de les partícules HDL. / Our proposal has explored for the first time the effect of human immunodeficiency virus (HIV)- infection on high-density lipoprotein (HDL) particles, and we determined their involvement in the course of infection itself and the development of atherosclerosis in order to design novel therapeutic strategies. Our results show how the chronic oxidant and inflammatory status associated with infection plays a central role in the onset of metabolic disorders and the development of subclinical atherosclerosis in these patients. Concretely, using both, chromatographic and proteomic techniques, we observed how the increased levels of gammaglobulin, pro-inflammatory molecules and acute phase reactants in the plasma of these patients induced metabolism alterations and modifications on the distribution and composition of HDL particles, suggesting that the monitoring of HDL particles in these patients should not be only based on the determination of the concentration of cholesterol content, but must include the determination of other parameters associated with particle quality, and therefore the functionality, of HDL particles

G-CSF is routinely used to treat neutropenia/leukopenia or to increase hematopoietic stem cell generation in bone marrow donors. G-CSF and its receptor, G-CSFR, are produced by various cell types both in the peripheral circulation and within brain. As a consequence, exogenous administration of G-CSF results in a broad spectrum of effects involving hematopoietic, immune and central nervous systems. G-CSF administration in a mouse model of Alzheimer's disease (AD) has revealed both cognitive benefits and disease modifying effects: a) decreased Aβ plaque burden, b) increased microgliosis, c) increased neurogenesis and d) improved performance in radial arm water maze (RAWM). In clinical studies, G-CSF plasma levels were found to be lower in patients with early AD in comparison to healthy age matched controls. A course of G-CSF administration in humans is known to increase levels of circulating hematopoietic stem cells (CD34 cells), monocytes and neutrophils in patients with neutropenia and when administered to patients with AD, there is also a similar increase in absolute monocyte count, CD34 cells and total neutrophils. The extent to which the beneficial effects of G-CSF in AD depend on monocyte infiltration into CNS, compared to direct neurotrophic actions of G-CSF on the CNS, is not known. The overall goal of this study was to investigate and understand the effects of G-CSF in an AD mouse model, but more specifically to distinguish the actions of G-CSF that affect the peripheral monocyte population from the direct actions on CNS. The first approach was to examine in vitro effects of G-CSF within a monocytic cell line (THP-1) and a neuronal cell line (SH-SY5Y). The second approach was to study effects of G-CSF on infiltration of bone marrow-derived cells into the brain by utilizing a chimeric GFP+ APP/PS1 AD mouse model. The third approach was to assess the effects of G-CSF on hippocampal neurogenesis in both a wild-type and AD mouse model. Comparison of the monocytic and neuronal cell lines showed a) G-CSF interacts with its cognate receptor with different binding kinetics and with a greater affinity for the monocyte G-CSFR, b) the number of G-CSF receptors in neurons is greater than in monocytes, and c) the anti-apoptotic response in neurons occurs at lower concentrations of G-CSF than in monocytes. Various concentrations of G-CSF increased proliferation of both the monocytic and neuronal cell line in vitro. G-CSF did not improve migratory properties of the monocytic cell line, either adhesiveness or migration through a membrane. In vivo G-CSF treatment (250μg/kg s.c. qod for 2 ½ weeks) in both the AD chimeric and non-chimeric AD mice resulted in increased microgliosis and decreased amyloid plaque burden in the hippocampus. In the chimeric AD mice, G-CSF treatment did not increase infiltration of GFP+ bone marrow derived cells (BMDC) into brain parenchyma and did not increase adhesion to microvasculature. In the non-chimeric AD mice there was improvement of neurogenesis to non-transgenic levels after G-CSF treatment and an increase in synaptogenesis in the CA1 region of the hippocampus. The effects of G-CSF on the endogeneous microglial population are most likely responsible for the increase in microgliosis, as no significant increase of BMDC infiltration into the brain parenchyma was found in vivo. The enhanced proliferation and improved viability of the neuronal cell line after G-CSF treatment may explain the improvement in neurogenesis and significant increase in synaptogenesis seen in the AD mouse model. The actions of G-CSF on neural stem/progenitor cells to stimulate hippocampal neurogenesis and to enhance resident microglial capacity to decrease amyloid burden are the most likely mechanisms responsible for the behavioral improvement seen in the AD mouse model.