Specific features of endothelial cells in primary tumors

RESUMO: As células endoteliais definem e delineiam todo o sistema vascular...Nesta tese procurámos explorar o papel que o ambiente tumoral exerce sobre as células endoteliais. ... Avaliamos também a capacidade anti-angiogénica de alguns derivados do estrogénio... Em suma os nossos resultados mostram a importância de um controlo rigoroso da regulação transcricional...

Immunophenotyping of circulating T cells in a mucosal leishmaniasis patient coinfected with HIV

HIV coinfection modifies the clinical course of leishmaniasis by promoting a Th2 pattern of cytokine production. However, little information is available regarding the lymphocytic response in untreated coinfected patients. This work presents the immunophenotyping of Leishmania-stimulated T cells from a treatment-naÏve HIV+ patient with ML. Leishmania braziliensis antigens induced CD69 expression on CD3+CD4+ and CD3+CD8+ cells. It also increased IL-4 intracellular staining on CD3+CD4+GATA3- population and decreased the percentage of CD3+CD4+IL-17+ cells. This suggests that modulations in the IL-4R/STAT6 pathway and the Th17 population may serve as parasitic evasion mechanisms in HIV/ML. Further studies are required to confirm these results.

Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

Glutathione levels in and total antioxidant capacity of Candida sp. cells exposed to oxidative stress caused by hydrogen peroxide

INTRODUCTION: The capacity to overcome the oxidative stress imposed by phagocytes seems to be critical for Candida species to cause invasive candidiasis. METHODS: To better characterize the oxidative stress response (OSR) of 8 clinically relevant Candida sp., glutathione, a vital component of the intracellular redox balance, was measured using the 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB)-glutathione disulfide (GSSG) reductase reconversion method; the total antioxidant capacity (TAC) was measured using a modified method based on the decolorization of the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic) acid radical cation (ABTS*+). Both methods were used with cellular Candida sp. extracts treated or not with hydrogen peroxide (0.5 mM). RESULTS: Oxidative stress induced by hydrogen peroxide clearly reduced intracellular glutathione levels. This depletion was stronger in Candida albicans and the levels of glutathione in untreated cells were also higher in this species. The TAC demonstrated intra-specific variation. CONCLUSIONS: Glutathione levels did not correlate with the measured TAC values, despite this being the most important non-enzymatic intracellular antioxidant molecule. The results indicate that the isolated measurement of TAC does not give a clear picture of the ability of a given Candida sp. to respond to oxidative stress.

The influence of carbohydrates in the interaction of Paracoccidioides brasiliensis with CCL-6 cells in vitro

INTRODUCTION: Little is known about the early events in the interaction between Paracoccidioides brasiliensis and its host. To understand the effect of carbohydrates in the interaction between the fungus and epithelial cell in culture, we analyzed the influence of different carbohydrate solutions on the adhesion of P. brasiliensis yeast cells to CCL-6 cells in culture. METHODS: Fungal cells were cultivated with the epithelial cell line, and different concentrations of D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine, D-galactosamine, sorbitol and fructose were added at the beginning of the experiment. Six hours after the treatment, the cells were fixed and observed by light microscopy. The number of P. brasiliensis cells that were adhered to the CCL-6 monolayer was estimated. RESULTS: The number of adhesion events was diminished following treatments with D-fucose, N-acetyl-glucosamine, D-mannose, D-glucosamine and D-galactosamine as compared to the untreated controls. Sorbitol and fructose-treated cells had the same adhesion behavior as the observed in the control. P. brasiliensis propagules were treated with fluorescent lectins. The FITC-labeled lectins WGA and Con-A bound to P. brasiliensis yeast cells, while SBA and PNA did not. CONCLUSIONS: The perceptual of adhesion between P. brasiliensis and CCL-6 cells decreased with the use of D-mannose, N-acetyl-glucosamine and D-glucosamine. The assay using FITC-labeled lectins suggests the presence of N-acetyl-glucosamine, α-mannose and α-glucose on the P. brasiliensis cell surface. An enhanced knowledge of the mediators of adhesion on P. brasiliensis could be useful in the future for the development of more efficient and less harmful methods for disease treatment and control.

Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis

Visceral leishmaniasis (VL) or kala-azar, a disseminated infection of the lymphoreticular system of the body, is marked by severe defect in immune system of the host. Successful cure of VL depends on the immune status of the host in combination with the effects of the antileishmanial drugs. The rationale approach towards eradication of this disease would be to potentiate the immune functioning of the host in addition to parasite killing. This review deals with different aspects of adaptive and innate immune responses and explores their role in protection or pathogenesis of VL. IL-10 has emerged as the principal cytokine responsible for disease pathogenesis, although evidences regarding its source during active VL remain inconclusive. On the other hand, IFNγ, under the influence of IL-12, is mostly correlated with healing of the disease. Chemokines are important in mounting cell-mediated immune response as they can prevent parasite invasion in association with cytokines. Different types of T cells like CD4, CD8 and NK T cells also contribute to the immunology of this disease. In spite of conflicting reports, the role of regulatory T cells in VL pathogenesis is important. Recently discovered Th17 subset and its different members have been reported to perform diverse functions in the course of VL and leishmaniasis as a whole. Innate immune responses, depending on the cell types, are essential in early parasite detection and subsequent development of an efficient NK cell response. Immunotherapy targeting IL-10 could be looked upon as an interesting option for the treatment of VL.

Analysis of metabolic flux distributions in relation to the extracellular environment in Avian cells

Continuous cell lines that proliferate in chemically defined and simple media have been highly regarded as suitable alternatives for vaccine production. One such cell line is the AG1.CR.pIX avian cell line developed by PROBIOGEN. This cell line can be cultivated in a fully scalable suspension culture and adapted to grow in chemically defined, calf serum free, medium [1]–[5]. The medium composition and cultivation strategy are important factors for reaching high virus titers. In this project, a series of computational methods was used to simulate the cell’s response to different environments. The study is based on the metabolic model of the central metabolism proposed in [1]. In a first step, Metabolic Flux Analysis (MFA) was used along with measured uptake and secretion fluxes to estimate intracellular flux values. The network and data were found to be consistent. In a second step, Flux Balance Analysis (FBA) was performed to access the cell’s biological objective. The objective that resulted in the best predicted results fit to the experimental data was the minimization of oxidative phosphorylation. Employing this objective, in the next step Flux Variability Analysis (FVA) was used to characterize the flux solution space. Furthermore, various scenarios, where a reaction deletion (elimination of the compound from the media) was simulated, were performed and the flux solution space for each scenario was calculated. Growth restrictions caused by essential and non-essential amino acids were accurately predicted. Fluxes related to the essential amino acids uptake and catabolism, the lipid synthesis and ATP production via TCA were found to be essential to exponential growth. Finally, the data gathered during the previous steps were analyzed using principal component analysis (PCA), in order to assess potential changes in the physiological state of the cell. Three metabolic states were found, which correspond to zero, partial and maximum biomass growth rate. Elimination of non-essential amino acids or pyruvate from the media showed no impact on the cell’s assumed normal metabolic state.

CD8+ T cells in situ in different clinical forms of human cutaneous leishmaniasis

Introduction Leishmania braziliensis infection induces a large spectrum of lesions that clinically manifest as nodules or papules that progress to ulcers. Although it is already known that T helper cells predominate in the lesions, cytotoxic T cells have also been reported to be present, and their role in leishmaniasis immunopathogenesis is not well known. This study investigated the amounts of CD8+ and granzyme B+ cells in different clinical forms of human cutaneous leishmaniasis (CL). Methods Forty tissue fragments from early (E-CL) and late CL (L-CL) lesions and from disseminated leishmaniasis (DL) - papules and ulcers - were characterized. The inflamed area per fragment was calculated, and the CD8 and granzyme B expression levels in the infiltrates were quantified by counting positive cells in 15 fields. The localization of CD8 and granzyme B was graded subjectively. Results Inflammation was higher in L-CL and DL ulcers. CD8 expression was increased in late ulcerated lesions compared to recent lesions. The increase in CD8+ cells also correlated with the duration of the lesion. Papules had a higher frequency of granzyme B+ cells than E-CL lesions, although the frequency was similar to those for late and DL ulcers. CD8+ cells were mostly found in the papillary dermis. Conclusions CD8+ T and granzyme B+ cells are present in the inflammatory infiltrates of CL and DL and may participate in the immunopathogenesis of Leishmania infection.

Unravelling the role of alpha 2,6 sialic acid on mouse dentritic cells functions

RESUMO: As células dendríticas (CDs) são fundamentais na imunomodulação e iniciação de respostas imunes adaptativas, enquanto os ácidos siálicos (Sias) são potenciais imunomoduladores. Estas células expressam níveis elevados da sialiltransferase ST6Gal-1, que transfere Sias para a posição terminal de oligossacáridos. De facto, a maturação de CDs está associada a uma diminuição da sialilação na sua superfície celular. Apesar de ter função biológica desconhecida, a forma solúvel, extracelular de ST6Gal-1 aumenta em cancros e inflamação. Ainda assim, esta foi recentemente identificada como moduladora da hematopoiese. Considerando o importante papel das CDs na iniciação de respostas anticancerígenas, uma ligação entre a sialilação extrínseca induzida por ST6Gal-1 extracelular e o seu papel na modulação de CDs deve ser identificada. Neste trabalho hipotetizou-se que a sialilação α2,6 extrínseca de CDs diminui o seu perfil de maturação mediante ativação por lipopolissacarídeo (LPS). O objetivo principal foi sialilar extrinsecamente em α2,6 CDs da medula óssea de murganhos, avaliando os seus perfis de maturação e de libertação de citocinas, após estimulação com LPS (por Citometria de Fluxo e ELISA, respetivamente). Ao contrário da hipótese, o perfil celular não foi modulado, usando várias abordagens. Por outro lado, a consequência da falta de α2,6 Sias na maturação de CDs foi avaliada analisando: 1) CDs da medula óssea de murganhos tratadas com sialidase, 2) CDs da medula óssea e 3) CDs das vias aéreas, ambas de murganhos deficientes em ST6Gal-1, comparando com a estirpe selvagem. Estes resultados sugerem que a perta total de α2,6 Sias se relaciona com o aumento da expressão do complexo de histocompatibilidade principal de classe II. Apesar de controverso, é provável existirem mecanismos inerentes à ativação por LPS, reduzindo a eficácia de ST6Gal-1 extracelular. Por outro lado, a modificação no perfil de CDs de murganhos deficientes em ST6Gal-1 poderá relacionar-se com uma predisposição para um estado inflamatório severo. Com isto, o trabalho desenvolvido abriu futuras linhas de investigação, nomeadamente explorar outros fatores envolvidos na (de)sialilação α2,6 de CDs, podendo ter impacto em imunoterapia com uso de CDs.--------------------------ABSTRACT: Dendritic cells (DCs) are vital for immunomodulation and the initiation of adaptive immune responses, whereas sialic acids (Sias) are potential immunomodulators. These cells express high levels of sialyltransferase ST6Gal-1, responsible for transferring Sias to the terminal position of oligosaccharide chains. Indeed, DCs’ maturation is associated with decreased cell surface sialylation. Although its biological significance is unknown, the soluble, extracellular form of ST6Gal-1 increases in cancers and inflammation. However, extracellular ST6Gal-1 was recently identified as modulator of hematopoiesis. Considering that DCs play a crucial role in the initiation of a productive anti-cancer immune response, a link between extrinsic sialylation by the extracellular ST6Gal-1 on DC function needs to be investigated. We hypothesize that extrinsic α2,6 sialylation of DCs diminishes their maturation features upon lipopolysaccharide (LPS) stimulation. The main goal was to extrinsically α2,6 sialylate mice bone marrow derived DCs (BMDCs) and to evaluate their maturation and cytokine profiles upon LPS stimulation (by Flow Cytometry and ELISA, respectively). Unlike the hypothesis, we observed that BMDCs’ profile is not modulated, even using several approaches. In contrast, the consequence of lacking cell surface α2,6 Sias in DC maturation was assessed by analysing: 1) sialidase treated BMDCs, 2) BMDCs from mice lacking ST6Gal-1 and 3) DCs from mice airways, comparing wild type with ST6Gal-1 knockout mice. These results suggest that overall lack in α2,6 Sias is related with increased expression of major histocompatibility class II (MHC-II). Although appearing to be controversial findings, other intracellular mechanisms might be occurring upon LPS-induced BMDC activation, probably reducing extracellular ST6Gal-1 effect. In opposite, the modification observed in DC profile of ST6Gal-1 knockout mice might be related to its predisposition to a more severe inflammatory status. With this, the developed work opened future lines of investigation, namely exploring other factors involved in α2,6 (de)sialylation of DC, which might have influence in immunotherapy using DCs.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells

IntroductionPurpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro.MethodsSpores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37°C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy.ResultsAfter 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinumconidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells.ConclusionsP. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

Light harvesting in solar cells using natural pigments from red fruits adsorbed to mesoporous TiO2

Nature has developed strategies to present us with a wide variety of colours, from the green of leaves to the bright colours seen in flowers. Anthocyanins are between these natural pigments that are responsible for the great diversity of colours seen in flowers and fruits. Anthocyanins have been used to sensitize titanium dioxide (TiO2) in Dye-Sensitized Solar Cells (DSSCs). DSSCs have become one of the most popular research topic in photovoltaic cells due to their low production costs when compared to other alternatives. DSSCs are inspired in what happens in nature during photosynthesis. A primary charge separation is achieved by means of a photoexcited dye capable of performing the electron injection into the conduction band of a wide band-gap semiconductor, usually TiO2. With this work we aimed to synthesize a novel mesoporous TiO2 structure as the semiconductor in order to increase the dye loading. We used natural occurring dyes such as anthocyanins and their synthetic flavylium relatives, as an alternative to the widely used metal complexes of Ru(II) which are expensive and are environmentally unsafe. This offers not only the chance to use safer dyes for DSSCs, but also to take profit of waste biological products, such as wine and olive oil production residues that are heavily loaded with anthocyanin dyes. We also performed a photodegradation study using TiO2 as the catalyst to degrade dye contaminants, such as those from the wine production waste, by photo-irradiation of the system in the visible region of the light spectrum. We were able to succeed in the synthesis of mesoporous TiO2 both powder and thin film, with a high capacity to load a large amount of dye. We proved the concept of photodegradation using TiO2 as catalyst. And finally, we show that wine production waste is a possible dye source to DSSCs application.

Development of process control strategies exploiting knowledge from systems biology: application to MDCK suspension cells

Madine Darby Canine Kidney (MDCK) cell lines have been extensively evaluated for their potential as host cells for influenza vaccine production. Recent studies allowed the cultivation of these cells in a fully defined medium and in suspension. However, reaching high cell densities in animal cell cultures still remains a challenge. To address this shortcoming, a combined methodology allied with knowledge from systems biology was reported to study the impact of the cell environment on the flux distribution. An optimization of the medium composition was proposed for both a batch and a continuous system in order to reach higher cell densities. To obtain insight into the metabolic activity of these cells, a detailed metabolic model previously developed by Wahl A. et. al was used. The experimental data of four cultivations of MDCK suspension cells, grown under different conditions and used in this work came from the Max Planck Institute, Magdeburg, Germany. Classical metabolic flux analysis (MFA) was used to estimate the intracellular flux distribution of each cultivation and then combined with partial least squares (PLS) method to establish a link between the estimated metabolic state and the cell environment. The validation of the MFA model was made and its consistency checked. The resulted PLS model explained almost 70% of the variance present in the flux distribution. The medium optimization for the continuous system and for the batch system resulted in higher biomass growth rates than the ones obtained experimentally, 0.034 h-1 and 0.030 h-1, respectively, thus reducing in almost 10 hours the duplication time. Additionally, the optimal medium obtained for the continuous system almost did not consider pyruvate. Overall the proposed methodology seems to be effective and both proposed medium optimizations seem to be promising to reach high cell densities.

Optimization of production of anti-cancer vaccines based on dendritic cells

Cancer remains as one of the top killing diseases in first world countries. It’s not a single, but a set of various diseases for which different treatment approaches have been taken over the years. Cancer immunotherapy comes as a “new” breath on cancer treatment, taking use of the patients’ immune system to induce anti-cancer responses. Dendritic Cell (DC) vaccines use the extraordinary capacity of DCs’ antigen presentation so that specific T cell responses may be generated against cancer. In this work, we report the ex vivo generation of DCs from precursors isolated from clinical-grade cryopreserved umbilical cord blood (UCB) samples. After the thawing protocol for cryopreserved samples was optimized, the generation of DCs from CD14+ monocytes, i.e., moDCs, or CD34+ hematopoietic stem cells (HSCs), i.e, CD34-derived DCs, was followed and their phenotype and function evaluated. Functional testing included the ability to respond to maturation stimuli (including enzymatic removal of surface sialic acids), Ovalbumin-FITC endocytic capacity, cytokine secretion and T cell priming ability. In order to evaluate the feasibility of using DCs derived from UCB precursors to induce immune responses, they were compared to peripheral blood (PB) moDCs. We observed an increased endocytosis capacity after moDCs were differentiated from monocyte precursors, but almost 10-fold lower than that of PB moDCs. Maturation markers were absent, low levels of inflammatory cytokines were seen and T cell stimulatory capacity was reduced. Sialidase enzymatic treatment was able to mature these cells, diminishing endocytosis and promoting higher T cell stimulation. CD34-derived DCs showed higher capacity for both maturation and endocytic capacity than moDCs. Although much more information was acquired from moDCs than from CD34-derived DCs, we conclude the last as probably the best suited for generating an immune response against cancer, but of course much more research has to be performed.

Activity of natural killer cells during HIV-1 infection in Brazilian patients

Natural killer cells are increasingly being considered an important component of innate resistance to viruses, but their role in HIV infection is controversial. Some investigators have found that natural killer cells do not confer a protective effect during the progression of HIV disease, whereas others have shown that natural killer cells may be protective and retard the progression of the disease, either through their lytic activity or by a chemokine-related suppression of HIV replication. In this study, we analyzed functional alterations in the activity of natural killer cells during HIV-1 infection using a natural killer cells activity assay with K562 cells as targets. RESULTS: Our results show that the activity of natural killer cells decreases only in the advanced phase of HIV infection and when high (40:1) effector cell-target cell ratios were used. The depression at this stage of the disease may be related to increased levels of some viral factors, such as gp120 or gag, that interfere with the binding capacity of natural killer cells, or to the decreased production of natural killer cells -activity-stimulating cytokines, such as IFN-a and IL-12, by monocytes, a subset of cells that are also affected in the late stage of HIV infection. The data suggest that decreased natural killer cells cell activity may contribute to the severe impairment of the immune system of patients in the late stages of HIV infection.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor β-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to “in-cell” Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.