Semantics bias in cross-national comparative analyses : Is it good or bad to have "fair" health?

The Health Behavior in School-aged Children is a cross-national study collecting data on social and health indicators on adolescents in 43 countries. The study provides comparable data on health behaviors and health outcomes through the use of a common protocol, which have been a back bone of the study sine its initiation in 1983. Recent years, researchers within the study have noticed a questionable comparability on the widely used item on self-rated health. One of the four response categories to the item "Would you say your health is….?" showed particular variation, as the response category "Fair" varied from 20 % in Latvia and Moldova to 3-4 % in Bulgaria and Macedonia. A qualitative mini-survey of the back-translations showed that the response category "Fair" had a negative slant in 25 countries, a positive slant in 10 countries and was considered neutral in 9 countries. This finding indicates that there are what may be called semantic issues affecting comparability in international studies, since the same original word (in an English original) is interpreted differently across countries and cultures. The paper test and discuss a few possible explanations to this, however, only leaving to future studies to hold a cautious approach to international comparisons if working with the self-rated health item with four response categories.

Impact of oral health on health-related quality of life : A cross-sectional study

Background: Despite the consensus regarding the existence of a relationship between “impacts on oral health” and “health-related quality of life”, this relationship, considering the latent nature of these variables, is still poorly investigated. Thus, we performed this study in order to determine the magnitude of the impacts of oral health, demographic and symptom/clinical variables on the health-related quality of life in a Brazilian sample of dental patients. Methods: A total of 1,007 adult subjects enrolled in the School of Dentistry of São Paulo State University (UNESP) - Araraquara Campus for dentistry care between September/2012 and April/2013, participated. 72.4 % were female. The mean age was 45.7 (SD = 12.5) years. The Oral Health Impact Profile (OHIP-14) and the Short Form Health Survey (SF-36) were used. The demographic and symptom/clinical variables collected were gender, age, economic status, presence of pain and chronic disease. The impact of studied variables on health-related quality of life were evaluated with a structural equation model, considering the factor “Health” as the central construct. The fit of the model was first analyzed by the evaluation of the goodness of fit indices (χ 2 /df ≤ 2.0, CFI and TLI ≥ 0.90 and RMSEA < 0.10) and the evaluation of the variables’ impact over health-related quality of life was based on the statistical significance of causal paths (β), evaluated by z tests, for a significance level of 5 %. Results: We observed adequate fit of the model to the data (χ 2 /df = 3.55; CFI = 0.95; TLI = 0.94; RMSEA = 0.05). The impacts on oral health explained 28.0 % of the variability of the health-related quality of life construct, while the total variance explained of the model was 39.0 %. For the demographic and symptom/clinical variables, only age, presence of pain and chronic disease showed significant impacts (p < 0.05). Conclusion: The oral health, age, presence of pain and chronic disease of individuals had significant influence on health-related quality of life.

Frequency of the toxic shock syndrome toxin-1 gene in methicillin-susceptible and -resistant Staphylococcus aureus isolates from teaching hospitals in Shiraz, Iran

INTRODUCTION: Staphylococcus aureus produces a range of virulence factors such as toxic shock syndrome toxin-1. METHODS: In this cross-sectional study of 345 clinical S. aureus isolates, the presence of the tst gene was assessed by polymerase chain reaction (PCR). RESULTS: The study revealed 53/345 (15.4%) isolates were positive for the tst gene. The tst gene was present in 18.1% of methicillin-susceptible S. aureus (MSSA) isolates and 11.6% of methicillin-resistant S. aureus (MRSA) isolates (p = 0.136). CONCLUSIONS: These results reveal the remarkable risk of S. aureus infections in hospitals, regardless of methicillin-resistance status.

The determinants of mutual fund performance: A cross-country study

We use a new data set to study the determinants of the performance of open–end actively managed equity mutual funds in 27 countries. We find that mutual funds underperform the market overall. The results show important differences in the determinants of fund performance in the USA and elsewhere in the world. The US evidence of diminishing returns to scale is not a universal truth as the performance of funds located outside the USA and funds that invest overseas is not negatively affected by scale. Our findings suggest that the adverse scale effects in the USA are related to liquidity constraints faced by funds that, by virtue of their style, have to invest in small and domestic stocks. Country characteristics also explain fund performance. Funds located in countries with liquid stock markets and strong legal institutions display better performance.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.

Neospora caninum and Toxoplasma gondii serodiagnosis in human immunodeficiency virus carriers

ABSTRACTINTRODUCTION:Neospora caninum and Toxoplasma gondii belong to the Sarcocystidae family, and both have one definitive and various intermediary hosts. Owing to their weak immune systems, immunocompromised persons might be prone to opportunistic infections. The aim of this study was to investigate the presence of anti- N. caninum and anti- T. gondii antibodies in immunocompromised individuals.METHODS:This cross-sectional study investigated the rates of N. caninum and T. gondii , as assessed using immunofluorescent antibody reaction (IFAT) with 1:50 and 1:16 dilution, respectively, in patients with human immunodeficiency virus (HIV).RESULTS:The seropositivity for N. caninum was 26.1% (81/310) in Mato Grosso do Sul and 31.2% (10/32) in Paraná and for T. gondii was 76.8% (238/310) in Mato Grosso do Sul and 68.7% (22/32) in Paraná.CONCLUSIONS:There is evidence of anti- N caninum and anti- T. gondii antibodies in patients with HIV. Other aspects of T. gondii , which is a zoonosis, and N. caninum , which might affect immunodeficient individuals, need to be evaluated and reported.

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Abstract: INTRODUCTION : Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no efficient diagnostic tests that combine high sensitivity and specificity and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. METHODS: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). RESULTS: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specificity of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). CONCLUSIONS: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

Emergent and re-emergent parasites in HIV-infected children: immunological and socio-environmental conditions that are involved in the transmission of Giardia spp. and Cryptosporidium spp.

ABSTRACT INTRODUCTION: Emergent and re-emergent waterborne protozoans have become a worldwide public health problem, especially among vulnerable groups. METHODS: This cross-sectional study evaluated 17 HIV-infected children and their families. RESULTS: A high (76.5%) percentage of parasite-infected children was observed, even among children with CD4+ T-cell counts of >200 cells/mm3. Giardia spp., Cryptosporidium spp. and Cyclospora spp. were observed in 41.2% of these children Low income, poor hygiene practices, and co-infection in domestic, peridomestic and scholastic environments were significant sources of these intestinal infections. CONCLUSIONS: Early diagnosis, timely treatment, and socio-educational interventions may improve the health conditions of this vulnerable population.

Reflux esophagitis and gastroesophageal reflux disease: a cross-sectional study of gastroesophageal reflux disease patients by age group

The purpose of this study was to explore the relationship between the intensity of acid reflux and severity of esophageal tissue damage in a cross-sectional study of patients with gastroesophageal reflux disease (GERD). Seventy-eight patients with were selected in accordance with the strict 24-hour ambulatory esophageal pHmetry (24h-pHM) criteria and distributed into three age groups: Group A: 14 - 24 years of age. Group B: 25 - 54; and Group C: 55 - 64. The 24h-pHM was carried out in accordance with DeMeester standardization, and the Savary-Miller classification for the diagnosis of reflux esophagitis was used. The groups were similar in 24h-pHM parameters (p > 0.05), having above normal values. For the study group as a whole, there was no correlation between age group and intensity of acid reflux, and there was no correlation between intensity of acid reflux and severity of esophageal tissue damage. However, when the same patients were sub-grouped in accordance with the depth of their epithelial injury and then distributed into age groups, there was a significant difference in esophagitis without epithelial discontinuity. Younger patients had less epithelial damage than older patients. Additionally, although there was a significant progression from the least severe to the moderate stages of epithelial damage among the age groups, there was no apparent difference among the age groups in the distribution between the moderate stages and most severe stages. The findings support the conclusion that the protective response of individuals to acid reflux varies widely. Continued aggression by acid reflux appears to lead to the exhaustion of individual mechanisms of epithelial protection in some patients, but not others, regardless of age or duration of the disease. Therefore, the diagnosis and follow-up of GERD should include both measurements of the quantity of refluxed acid and an assessment of the damage to the esophageal epithelium.

Engineered MRI nanoprobes based on superparamagnetic iron oxide nanoparticles

This project aimed to engineer new T2 MRI contrast agents for cell labeling based on formulations containing monodisperse iron oxide magnetic nanoparticles (MNP) coated with natural and synthetic polymers. Monodisperse MNP capped with hydrophobic ligands were synthesized by a thermal decomposition method, and further stabilized in aqueous media with citric acid or meso-2,3-dimercaptosuccinic acid (DMSA) through a ligand exchange reaction. Hydrophilic MNP-DMSA, with optimal hydrodynamic size distribution, colloidal stability and magnetic properties, were used for further functionalization with different coating materials. A covalent coupling strategy was devised to bind the biopolymer gum Arabic (GA) onto MNPDMSA and produce an efficient contrast agent, which enhanced cellular uptake in human colorectal carcinoma cells (HCT116 cell line) compared to uncoated MNP-DMSA. A similar protocol was employed to coat MNP-DMSA with a novel biopolymer produced by a biotechnological process, the exopolysaccharide (EPS) Fucopol. Similar to MNP-DMSA-GA, MNP-DMSA-EPS improved cellular uptake in HCT116 cells compared to MNP-DMSA. However, MNP-DMSA-EPS were particularly efficient towards the neural stem/progenitor cell line ReNcell VM, for which a better iron dose-dependent MRI contrast enhancement was obtained at low iron concentrations and short incubation times. A combination of synthetic and biological coating materials was also explored in this project, to design a dynamic tumortargeting nanoprobe activated by the acidic pH of tumors. The pH-dependent affinity pair neutravidin/iminobiotin, was combined in a multilayer architecture with the synthetic polymers poy-L-lysine and poly(ethylene glycol) and yielded an efficient MRI nanoprobe with ability to distinguish cells cultured in acidic pH conditions form cells cultured in physiological pH conditions.

Elucidating the role of glycans in leucocyte function

RESUMO: O processo de glicosilação é a modificação pós-traducional de proteínas mais comum e está envolvido em vários processos fisiológicos e patológicos. Especificamente, certos perfis glicosídeos estão correlacionados a estados específicos de diferenciação celular, e podem modular vários eventos celulares, como sinalização celular, migração celular e interações hospedeiro-patogénio. Assim sendo, a glicosilação desempenha um papel crucial na modulação de vários processos imunológicos. No entanto, permanece por esclarecer como as estruturas glicosídicas influenciam a imunidade. Especificamente, algumas estruturas glicosídicas terminais que estão modificadas pela ligação de ácido siálico desempenham um papel importante em várias funções do sistema imune, nomeadamente migração leucocitária em contexto de inflamação e ativação de células imunes. Como tal, este trabalho teve como objectivo investigar como a expressão de certos glicanos influencia componentes importantes da resposta imune inata e adaptativa. Este trabalho está dividido em três componentes principais: 1) A imunidade está amplamente dependente da habilidade das células circulantes migrarem para os tecidos inflamados, sendo que a ligação de leucócitos à Eselectina endotelial é o primeiro passo. Assim, nós analisámos a estrutura e função dos ligandos de E-selectina que são expressos pelas células humanas mononucleares de sangue periférico (PBMCs), fornecendo novos conhecimentos para a compreensão dos intervenientes moleculares que mediam a ligação dos monócitos, células CD4+ e CD8+T e células B ao endotélio vascular. Surpreendentemente, os monócitos apresentaram maior capacidade de ligação à E-selectina comparativamente aos linfócitos. Esta observação pode ser explicada pelo facto de os monócitos humanos expressarem, uniformemente, um vasto reportório de glicoproteínas que exibem afinidade de ligação à E-selectina, nomeadamente: as glicoformas do CD43 (CD43E) e do CD44 (HCELL), em adição à já previamente reportada glicoforma da PSGL-1 (CLA). Consistentemente, a diferente capacidade que as diversas populações linfocitárias apresentam de se ligar à E-selectina, está integralmente relacionada com a sua expressão de glicoproteínas com afinidade de ligação à E-selectina. Enquanto que as células CD4+T apresentam uma elevada reatividade à E-selectina, as células CD8+T e B demonstram pouca ou nenhuma capacidade de ligação à E-selectina. Esta atividade de ligação à E-selectina das células CD4+T é conferida pela expressão de HCELL, em adição às já previamente reportadas CLA e CD43E. As células CD8+ T não expressam HCELL e apenas expressam pequenas quantidades de CLA e CD43E, enquanto que as células B não expressam ligandos de Eselectina. Mais, a exofucosilação da superfície destas células, levou ao dramático aumento da expressão dos ligandos de E-selectina em todos as populações leucocitárias, verificando-se que a criação de certos ligandos de E-selectina está dependente do tipo de célula, após fucosilação. Colectivamente, estes resultados redefinem o nosso conhecimento acerca dos mecanismos moleculares que governam o tráfico das células mononucleares de sangue periférico em contexto de inflamação. 2) A habilidade das células dendríticas (DCs) para extravasarem em locais de inflamação é crucial para o sucesso da terapia com DCs. Assim, analisámos a estrutura e função das moléculas de adesão que mediam a migração transendotelial (TEM) das DCs. Para isso, foram usadas DCs geradas a partir da diferenciação de monócitos (mo-DCS), obtidos quer pelo métodos de separação imuno-magnética de células CD14+ (CD14-S) ou por isolamento por aderência ao plástico (PA-S). Os resultados obtidos indicam que as glicoformas de ligação à Eselectina de PSGL-1, CD43 e CD44 são expressas pelas CD14-S mo-DCs, enquanto que as PA-S mo-DCs expressam apenas CLA. É importante notar que a ligação do CD44 nas mo-DCs, mas não nas PA-S mo-DCs, desencadeia a ativação e consequente adesão da VLA-4 ao endotélio na ausência de um gradiente de quimiocinas. Procedeu-se também à análise dos ligandos E-selectina expressos em mo-DCs geradas a partir de monócitos do sangue do cordão umbilical (UCB) e, inesperadamente, as UCB mo-DCs não expressam qualquer glicoproteína com reatividade à E-selectina. Além disso, a exofucosilação das mo- DCs humanas utilizando uma α(1,3)-fucosiltransferase aumenta significativamente a expressão de HCELL e, portanto, estas células apresentam uma capacidade aumentada para se ligarem à E-selectina em condições de fluxo hemodinâmico. Estes resultados destacam o papel do HCELL no desencadeamento do TEM das CD14-S mo-DCs e sugerem que estratégias para potenciar a expressão de HCELL poderão impulsionar o recrutamento de mo-DCs para locais de inflamação. 3) Outro obstáculo para alcançar o sucesso promissor de vacinas baseadas em DCs é o estabelecimento de abordagens eficientes que poderão melhorar o estado de maturação e apresentação antigénica das DCs. Por conseguinte, foram investigadas abordagens alternativas que podem superar este obstáculo. Através da remoção de ácido siálico de superfície celular das DCs, conseguiu-se induzir a maturação de DC humanas e de ratinhos. Notavelmente, tanto as DCs humanas como as de ratinho, ao serem desialiladas mostraram uma capacidade aumentada para induzir a proliferação de células T, para secretar citocinas Th1 e para induzir a morte específica de células tumorais. Em adição, as DCs desialiladas apresentam uma maior capacidade de apresentação cruzada de antigénios tumorais às células T citotóxicas. Colectivamente, o presente estudo oferece uma visão chave para optimizar a capacidade das DCs em induzir respostas imunitárias anti-tumorais, e indica que o tratamento com sialidase é uma nova tecnologia para melhorar a eficácia e aplicabilidade das vacinas baseadas em DCs. Coletivamente, os nossos resultados demostram como a glicosilação e a sua manipulação podem modular a imunidade. Concretamente, através de uma reação de exofucosilação conseguimos aumentar fortemente a capacidade de os leucócitos extravasarem para os tecidos afectados, enquanto que a remoção dos níveis de ácido siálico da superfície celular das DCs, induz potentes respostas anti-tumorais mediadas por células T citotóxicas. ------------------------------------ ABSTRACT: Glycosylation is the most widely form of protein post-translational modification and is involved in many physiological and pathological processes. Specifically, certain patterns of glycosylation are associated with determined stages of cell differentiation and can modulate processes like cell-signaling and migration and host-pathogen interactions. As such, glycosylation plays a crucial role in the modulation of several immune events. However, how glycans execute this immune-modulation and, therefore, influence immunity is still poorly unknown. Specifically, some terminal sialic acid-modified determinants are known to be involved in several physiological immune processes, including leukocyte trafficking into sites of inflammation and cell immune activation. Therefore, in this work, we sought to investigate more deeply how the expression of these glycosidic structures affects events form both innate and adaptive immune responses. To this end, we divided our work into three main parts: 1) Immunity critically depends on the ability of sentinel circulating cells to infiltrate injured sites, of which leukocyte binding to endothelial E-selectin is the critical first step. Thus, we first analyzed the structure and function of the E-selectin ligands expressed on native human peripheral blood mononuclear cells (PBMCs), providing novel insights into the molecular effectors governing adhesion of circulating monocytes, and of circulating CD4+T, CD8+T and B cells, to vascular endothelium under hemodynamic shear conditions. Strikingly, monocytes show a higher ability to tether and roll on endothelial cells than lymphocyte subsets. This is due to the fact that human circulating monocytes uniformly display a wide repertoire of E-selectin binding glycoproteins, namely the E-selectin-binding glycoforms of CD43 (CD43E) and CD44 (HCELL), in addition to the previously described E-selectin-binding glycoform of PSGL-1 (CLA). In addition, we also observed a differential ability of the different lymphocyte subsets to bind to Eselectin under hemodynamic shear stress conditions, and these differences were highly correlated with their individual expression of E-selectin binding glycoproteins. While CD4+T cells show a robust E-selectin binding ability, CD8+T and B cells show little to no E-selectin reactivity. CD4+T cell potent Eselectin rolling activity is conferred by HCELL expression, in addition to the previously reported E-selectin-binding glycoproteins CD43E and CLA. CD8+T cells display no HCELL and low amounts of CLA and CD43E, whereas B cells lack E-selectin ligand expression. Moreover, enforced exofucosylation of cell surface of these cells noticeably increases expression of functional E-selectin ligands among all leukocytes subsets, with cell type-dependent specificity in the protein scaffolds that are modified. Taken together, these findings redefine our understanding of the molecular mechanisms governing the trafficking patterns of PBMCs that are relevant in the context of acute or chronic inflammatory conditions. 2) The ability of circulating dendritic cells (DCs) to extravasate at inflammatory sites is critical to the success of DC-based therapies. Therefore, we assessed the structure and function of adhesion molecules mediating the transendothelial migration (TEM) of human monocyte derived-DCs (mo-DCs), obtained either by CD14 positive immune-magnetic selection (CD14-S) or by plastic adherence of blood monocytes (PA-S). We report for the first time that the E-selectin binding glycoforms of PSGL-1, CD43 and CD44 are all expressed on CD14-S mo-DCs, in contrast to PA-S mo-DCs that express only CLA. Importantly, CD44 engagement on CD14-S mo-DCs, but not on PA-S mo-DCs, triggers VLA-4-dependent adhesiveness and programs TEM in absence of chemokine gradient. We also analyzed the E-selectin ligands expressed on mo-DCs generated from umbilical cord blood (UCB) monocytes, and unexpectedly, UCB mo-DCs do not express any glycoprotein with E-selectin reactivity. Furthermore, exoglycosylation of human mo-DCs using an α(1,3)-fucosyltransferase significantly increases expression of HCELL, and therefore exofucosylated mo-DCs exhibit an augmented ability to bind to E-selectin under hemodynamic shear stress conditions. These findings highlight a role for HCELL engagement in priming TEM of CD14-S mo-DCs, and suggest that strategies to enforce HCELL expression could boost mo-DC recruitment to inflammatory sites. 3) Another obstacle to achieve the promising success of DC-based vaccines is the establishment of efficient approaches that could successfully enhance maturation and cross-presentation ability of DCs. Therefore, we investigated an alternative approach that can overcome this problem. Through removal of sialic acid content from DC cell surface we are able to elicit maturation of both human and mouse DCs. Notably, desialylated human and murine DCs showed enhanced ability to induce autologous T cell to proliferate, to secrete Th1 cytokines and to kill tumor cells. Moreover, desialylated DCs display enhanced cross-presentation of tumor antigens to cytotoxic CD8+ T cells. Collectively, this study offers key insight to optimize the ability of DCs to boost anti-tumor immune responses, and indicates that the treatment with an exogenous sialidase is a powerful new technology to improve the efficacy and applicability of DC-based vaccines. Overall, our findings show how glycosylation and its manipulation can modulate immunity. Concretely, through an exofucosylation reaction we are able to greatly augment the ability of leukocytes to extravasate into injured tissues, while removal of sialic acid moieties from cell surface of DCs, significantly potentiate their ability to induce anti-tumor cytotoxic T cell-mediate responses.

Elucidating the role of glycans in leucocyte function

RESUMO:O processo de glicosilação é a modificação pós-traducional de proteínas mais comum e está envolvido em vários processos fisiológicos e patológicos. Especificamente, certos perfis glicosídeos estão correlacionados a estados específicos de diferenciação celular, e podem modular vários eventos celulares, como sinalização celular, migração celular e interações hospedeiro-patogénio. Assim sendo, a glicosilação desempenha um papel crucial na modulação de vários processos imunológicos. No entanto, permanece por esclarecer como as estruturas glicosídicas influenciam a imunidade. Especificamente, algumas estruturas glicosídicas terminais que estão modificadas pela ligação de ácido siálico desempenham um papel importante em várias funções do sistema imune, nomeadamente migração leucocitária em contexto de inflamação e ativação de células imunes. Como tal, este trabalho teve como objectivo investigar como a expressão de certos glicanos influencia componentes importantes da resposta imune inata e adaptativa. Este trabalho está dividido em três componentes principais: 1) A imunidade está amplamente dependente da habilidade das células circulantes migrarem para os tecidos inflamados, sendo que a ligação de leucócitos à Eselectina endotelial é o primeiro passo. Assim, nós analisámos a estrutura e função dos ligandos de E-selectina que são expressos pelas células humanas mononucleares de sangue periférico (PBMCs), fornecendo novos conhecimentos para a compreensão dos intervenientes moleculares que mediam a ligação dos monócitos, células CD4+ e CD8+T e células B ao endotélio vascular. Surpreendentemente, os monócitos apresentaram maior capacidade de ligação à E-selectina comparativamente aos linfócitos. Esta observação pode ser explicada pelo facto de os monócitos humanos expressarem, uniformemente, um vasto reportório de glicoproteínas que exibem afinidade de ligação à E-selectina, nomeadamente: as glicoformas do CD43 (CD43E) e do CD44 (HCELL), em adição à já previamente reportada glicoforma da PSGL-1 (CLA). Consistentemente, a diferente capacidade que as diversas populações linfocitárias apresentam de se ligar à E-selectina, está integralmente relacionada com a sua expressão de glicoproteínas com afinidade de ligação à E-selectina. Enquanto que as células CD4+T apresentam uma elevada reatividade à E-selectina, as células CD8+T e B demonstram pouca ou nenhuma capacidade de ligação à E-selectina. Esta atividade de ligação à E-selectina das células CD4+T é conferida pela expressão de HCELL, em adição às já previamente reportadas CLA e CD43E. As células CD8+ T não expressam HCELL e apenas expressam pequenas quantidades de CLA e CD43E, enquanto que as células B não expressam ligandos de Eselectina. Mais, a exofucosilação da superfície destas células, levou ao dramático aumento da expressão dos ligandos de E-selectina em todos as populações leucocitárias, verificando-se que a criação de certos ligandos de E-selectina está dependente do tipo de célula, após fucosilação. Colectivamente, estes resultados redefinem o nosso conhecimento acerca dos mecanismos moleculares que governam o tráfico das células mononucleares de sangue periférico em contexto de inflamação. 2) A habilidade das células dendríticas (DCs) para extravasarem em locais de inflamação é crucial para o sucesso da terapia com DCs. Assim, analisámos a estrutura e função das moléculas de adesão que mediam a migração transendotelial (TEM) das DCs. Para isso, foram usadas DCs geradas a partir da diferenciação de monócitos (mo-DCS), obtidos quer pelo métodos de separação imuno-magnética de células CD14+ (CD14-S) ou por isolamento por aderência ao plástico (PA-S). Os resultados obtidos indicam que as glicoformas de ligação à Eselectina de PSGL-1, CD43 e CD44 são expressas pelas CD14-S mo-DCs, enquanto que as PA-S mo-DCs expressam apenas CLA. É importante notar que a ligação do CD44 nas mo-DCs, mas não nas PA-S mo-DCs, desencadeia a ativação e consequente adesão da VLA-4 ao endotélio na ausência de um gradiente de quimiocinas. Procedeu-se também à análise dos ligandos E-selectina expressos em mo-DCs geradas a partir de monócitos do sangue do cordão umbilical (UCB) e, inesperadamente, as UCB mo-DCs não expressam qualquer glicoproteína com reatividade à E-selectina. Além disso, a exofucosilação das mo- DCs humanas utilizando uma α(1,3)-fucosiltransferase aumenta significativamente a expressão de HCELL e, portanto, estas células apresentam uma capacidade aumentada para se ligarem à E-selectina em condições de fluxo hemodinâmico. Estes resultados destacam o papel do HCELL no desencadeamento do TEM das CD14-S mo-DCs e sugerem que estratégias para potenciar a expressão de HCELL poderão impulsionar o recrutamento de mo-DCs para locais de inflamação. 3) Outro obstáculo para alcançar o sucesso promissor de vacinas baseadas em DCs é o estabelecimento de abordagens eficientes que poderão melhorar o estado de maturação e apresentação antigénica das DCs. Por conseguinte, foram investigadas abordagens alternativas que podem superar este obstáculo. Através da remoção de ácido siálico de superfície celular das DCs, conseguiu-se induzir a maturação de DC humanas e de ratinhos. Notavelmente, tanto as DCs humanas como as de ratinho, ao serem desialiladas mostraram uma capacidade aumentada para induzir a proliferação de células T, para secretar citocinas Th1 e para induzir a morte específica de células tumorais. Em adição, as DCs desialiladas apresentam uma maior capacidade de apresentação cruzada de antigénios tumorais às células T citotóxicas. Colectivamente, o presente estudo oferece uma visão chave para optimizar a capacidade das DCs em induzir respostas imunitárias anti-tumorais, e indica que o tratamento com sialidase é uma nova tecnologia para melhorar a eficácia e aplicabilidade das vacinas baseadas em DCs. Coletivamente, os nossos resultados demostram como a glicosilação e a sua manipulação podem modular a imunidade. Concretamente, através de uma reação de exofucosilação conseguimos aumentar fortemente a capacidade de os leucócitos extravasarem para os tecidos afectados, enquanto que a remoção dos níveis de ácido siálico da superfície celular das DCs, induz potentes respostas anti-tumorais mediadas por células T citotóxicas. ---------------------------- ABSTRACT: Glycosylation is the most widely form of protein post-translational modification and is involved in many physiological and pathological processes. Specifically, certain patterns of glycosylation are associated with determined stages of cell differentiation and can modulate processes like cell-signaling and migration and host-pathogen interactions. As such, glycosylation plays a crucial role in the modulation of several immune events. However, how glycans execute this immune-modulation and, therefore, influence immunity is still poorly unknown. Specifically, some terminal sialic acid-modified determinants are known to be involved in several physiological immune processes, including leukocyte trafficking into sites of inflammation and cell immune activation. Therefore, in this work, we sought to investigate more deeply how the expression of these glycosidic structures affects events form both innate and adaptive immune responses. To this end, we divided our work into three main parts: 1) Immunity critically depends on the ability of sentinel circulating cells to infiltrate injured sites, of which leukocyte binding to endothelial E-selectin is the critical first step. Thus, we first analyzed the structure and function of the E-selectin ligands expressed on native human peripheral blood mononuclear cells (PBMCs), providing novel insights into the molecular effectors governing adhesion of circulating monocytes, and of circulating CD4+T, CD8+T and B cells, to vascular endothelium under hemodynamic shear conditions. Strikingly, monocytes show a higher ability to tether and roll on endothelial cells than lymphocyte subsets. This is due to the fact that human circulating monocytes uniformly display a wide repertoire of E-selectin binding glycoproteins, namely the E-selectin-binding glycoforms of CD43 (CD43E) and CD44 (HCELL), in addition to the previously described E-selectin-binding glycoform of PSGL-1 (CLA). In addition, we also observed a differential ability of the different lymphocyte subsets to bind to Eselectin under hemodynamic shear stress conditions, and these differences were highly correlated with their individual expression of E-selectin binding glycoproteins. While CD4+T cells show a robust E-selectin binding ability, CD8+T and B cells show little to no E-selectin reactivity. CD4+T cell potent Eselectin rolling activity is conferred by HCELL expression, in addition to the previously reported E-selectin-binding glycoproteins CD43E and CLA. CD8+T cells display no HCELL and low amounts of CLA and CD43E, whereas B cells lack E-selectin ligand expression. Moreover, enforced exofucosylation of cell surface of these cells noticeably increases expression of functional E-selectin ligands among all leukocytes subsets, with cell type-dependent specificity in the protein scaffolds that are modified. Taken together, these findings redefine our understanding of the molecular mechanisms governing the trafficking patterns of PBMCs that are relevant in the context of acute or chronic inflammatory conditions. 2) The ability of circulating dendritic cells (DCs) to extravasate at inflammatory sites is critical to the success of DC-based therapies. Therefore, we assessed the structure and function of adhesion molecules mediating the transendothelial migration (TEM) of human monocyte derived-DCs (mo-DCs), obtained either by CD14 positive immune-magnetic selection (CD14-S) or by plastic adherence of blood monocytes (PA-S). We report for the first time that the E-selectin binding glycoforms of PSGL-1, CD43 and CD44 are all expressed on CD14-S mo-DCs, in contrast to PA-S mo-DCs that express only CLA. Importantly, CD44 engagement on CD14-S mo-DCs, but not on PA-S mo-DCs, triggers VLA-4-dependent adhesiveness and programs TEM in absence of chemokine gradient. We also analyzed the E-selectin ligands expressed on mo-DCs generated from umbilical cord blood (UCB) monocytes, and unexpectedly, UCB mo-DCs do not express any glycoprotein with E-selectin reactivity. Furthermore, exoglycosylation of human mo-DCs using an α(1,3)-fucosyltransferase significantly increases expression of HCELL, and therefore exofucosylated mo-DCs exhibit an augmented ability to bind to E-selectin under hemodynamic shear stress conditions. These findings highlight a role for HCELL engagement in priming TEM of CD14-S mo-DCs, and suggest that strategies to enforce HCELL expression could boost mo-DC recruitment to inflammatory sites.3) Another obstacle to achieve the promising success of DC-based vaccines is the establishment of efficient approaches that could successfully enhance maturation and cross-presentation ability of DCs. Therefore, we investigated an alternative approach that can overcome this problem. Through removal of sialic acid content from DC cell surface we are able to elicit maturation of both human and mouse DCs. Notably, desialylated human and murine DCs showed enhanced ability to induce autologous T cell to proliferate, to secrete Th1 cytokines and to kill tumor cells. Moreover, desialylated DCs display enhanced cross-presentation of tumor antigens to cytotoxic CD8+ T cells. Collectively, this study offers key insight to optimize the ability of DCs to boost anti-tumor immune responses, and indicates that the treatment with an exogenous sialidase is a powerful new technology to improve the efficacy and applicability of DC-based vaccines. Overall, our findings show how glycosylation and its manipulation can modulate immunity. Concretely, through an exofucosylation reaction we are able to greatly augment the ability of leukocytes to extravasate into injured tissues, while removal of sialic acid moieties from cell surface of DCs, significantly potentiate their ability to induce anti-tumor cytotoxic T cell-mediate responses.