Beach sand and the potential for infectious disease transmission: observations and recommendations

Recent studies suggest that sand can serve as a vehicle for exposure of humans to pathogens at beach sites, resulting in increased health risks. Sampling for microorganisms in sand should therefore be considered for inclusion in regulatory programmes aimed at protecting recreational beach users from infectious disease. Here, we review the literature on pathogen levels in beach sand, and their potential for affecting human health. In an effort to provide specific recommendations for sand sampling programmes, we outline published guidelines for beach monitoring programmes, which are currently focused exclusively on measuring microbial levels in water. We also provide background on spatial distribution and temporal characteristics of microbes in sand, as these factors influence sampling programmes. First steps toward establishing a sand sampling programme include identifying appropriate beach sites and use of initial sanitary assessments to refine site selection. A tiered approach is recommended for monitoring. This approach would include the analysis of samples from many sites for faecal indicator organisms and other conventional analytes, while testing for specific pathogens and unconventional indicators is reserved for high-risk sites. Given the diversity of microbes found in sand, studies are urgently needed to identify the most significant aetiological agent of disease and to relate microbial measurements in sand to human health risk.

Massive shift in the pneumococcal nasopharyngeal flora after the 7-valent conjugate vaccine: epidemiological studies and testing pathogenic potential in animal models

Dissertation presented to obtain the PhD degree in Biology/Molecular Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica

Staphylococcus aureus: towards a comprehensive view on epidemiology and clonal spread

Dissertation presented to obtain a PhD degree in Biology/ Molecular Biology by the Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica

Insights into cell wall synthesis and cell division in Staphylococcus aureus

Dissertation presented to obtain the Ph.D degree in Biology

Enterococcus faecalis V583 prophages: Dynamic interactions and contribution to bacterial pathogenic traits

Dissertation presented to obtain the Ph.D degree in Biology.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Portugal:

Methicillin-resistant Staphylococcus aureus (MRSA), a human pathogen confined to hospitals (HAMRSA) for over 30 years have been emerging worldwide in the last two decades as a leading cause of severe infections in healthy individuals in the community (CA-MRSA). Despite its clinical significance, in the beginning of our studies no information existed on the prevalence, and population structure of CA-MRSA in Portugal. Moreover, it remained to be clarified how CA-MRSA emerged in our country. In particular, it was not known if CA-MRSA emerged locally by acquisition of the staphylococcal cassette chromosome mec (SCCmec) by established methicillin-susceptible S. aureus (MSSA) in the community, if they were imported from abroad or have escaped from the hospital.(...)

The role of pneumococcal carbon metabolism in colonisation and invasive disease

Streptococcus pneumoniae is a common asymptomatic commensal of the human nasopharynx. However, it is better known as a threatening pathogen that causes serious diseases such as pneumonia, meningitis and sepsis, as well as other less severe but more prevalent infections (e.g. otitis media). With the increase of antibiotic resistance and the limited efficacy of vaccines, pneumococcal infections remain a major problem. Therefore, the discovery of new therapeutic targets and preventive drugs are in high demand.(...)

A infecção por Helicobacter pylori em populações de Angola

RESUMO: A infecção por H. pylori, enquadra-se nas doenças infecciosas gastroduodenais e estima-se que mais de 50% da população mundial esteja infectada. A história natural da infecção por H. pylori, sofre interferências relacionadas com a genética do hospedeiro, a estirpe e as características da toxicidade da bactéria. Associam-se a estes factores, o tempo de exposição à infecção, assim como as condições sociais e higiéno-sanitárias. Paralelamente, o H. pylori é considerado o principal agente patogénico das doenças gastroduodenais. Este estudo teve como objectivo principal caracterizar a infecção por H. pylori em populações de Angola e sua avaliação como problema de Saúde Pública. Trata-se de um estudo prospectivo dirigido a dois grupos populacionais, um constituído por indivíduos aparentemente saudáveis, sem queixas gástricas específicas, em ambiente de comunidade, Grupo I, e outro, Grupo II, constituído por doentes que acorreram ao serviço de Gastrenterologia do Hospital Militar Principal de Luanda (HMP). No que diz respeito ao estudo na comunidade a pesquisa de H. pylori foi realizada pelo método ELISA de pesquisa de antigénios nas fezes. Por sua vez, a nível hospitalar, os métodos de diagnóstico da infecção por H. pylori foram: a endoscopia digestiva alta para a colheita de biópsias da mucosa gástrica destinadas ao exame anatomopatológico, ao exame citobacteriológico e aos métodos moleculares. Como método não invasivos foi utilizado o teste respiratório com ureia marcada. Grupo I: o diagnóstico da infecção por H. pylori, realizado pela pesquisa de antigénios deste microrganismo nas fezes, revelou uma frequência de 69,6% na população em estudo. Considerando em cada região, verificou-se que a região do Sambizanga possuía o valor mais elevado de frequência, 81,2%, seguida do Dinge com 79,5%, estatisticamente significativas (p 0,001). A avaliação da distribuição da frequência da infecção por grupo etário, revelou que os indivíduos com idade inferior a 15 anos, possuíam uma frequência de infecção de 63,5% e sendo de 76% nos indivíduos com idade superior a 15 anos. Este estudo permitiu concluir que a frequência da infecção por H. pylori nas regiões estudadas, é de 70% à excepção do Capulo, zona litoral em que não obstante as precárias condições de saneamento, a frequência da infecção por H. pylori é baixa. Grupo II: dos 309 doentes avaliados, verificou-se que 22 (7%), apresentavam uma mucosa normal e 287 (93%) uma mucosa alterada. A avaliação histológica das biópsias do antro, em 270 amostras de acordo com o Sistema de Sidney, em 235 (87,0%), revelou a presença de gastrite, 13 (4,8%) a presença de úlcera e em 9 (3,3%), uma lesão tumoral. A avaliação histológica da actividade nas 226 amostras do antro gástrico, verificou-se que 129 (57%) possuíam actividade e 97 (43%) não possuíam. O estudo das 255 biópsias do corpo, revelou em 212 (83,1%), a presença de lesões de gastrite, em 7 (2,7%), observaram-se lesões tumorais e 2 (0.8%) apresentaram úlcera. Dos 263 doentes avaliados histologicamente para pesquisa do H. pylori, 148 (58,2%) revelaram a presença positiva desta bactéria e 106 (41,7%) foram negativas. No que diz respeito à susceptibilidade aos macrólidos, do universo de 158 doentes com H. pylori positivo, 125 (79,1%) doentes apresentaram estirpes sensíveis aos macrólidos e 33 (20,9%) estirpes resistentes. Em relação aos factores de virulência, na avaliação conjunta dos dois factores de virulência estudados (cagA e vacA), em relação ao tipo de lesões encontradas na mucosa gástrica, verificou-se que dos 11 doentes com úlcera, 7 (63,6%), apresentavam uma estirpe cagA negativa, sendo 6 vacA s1 (85,7%), uma s2 e 4 (36,3%) com uma estirpe cagA positiva e vacA s1. Por sua vez dos 2 doentes com tumor, ambas as estirpes eram cagA negativas, sendo uma vacA s1 e outra vacA s2. Em relação aos factores de virulência nos doentes aos quais se diagnosticou úlcera e tumor apresentavam estirpe cagA negativa, vacAs1. Em relação ás lesões gástricas inflamatórias, os doentes com gastrite apresentavam cagA positivo. Do presente trabalho, em atenção aos resultados obtidos no que concerne a prevalência em populações sem queixas gastrenterológicas, recomenda-se que o mesmo se possa vir a replicar numa abrangência maior, realizando-se, por exemplo, estudos comparativos de prevalência entre as populações residentes no litoral (beira-mar) e as do interior. Pelas características genotípicas de H. pylori, em correspondência com as lesões encontradas, após novos estudos mais abrangentes, recomenda-se a avaliação de uma terapêutica mais acessível para o doente e que seja de maior eficácia. Face à escassez de médicos especialistas em gastrenterologia em Angola e de meios de diagnóstico, recomenda-se um estudo mais alargado da eficácia do seguimento do doente dispéptico, conforme protocolo avaliado pelo Colégio da Especialidade de Gastrenterologia da Ordem dos Médicos de Angola e já em prática em algumas instituições de saúde.--------------------------- ABSTRACT: H.pylori infection, is part of the gastroduodenal infectious diseases and it is estimated that over 50% of the world population is infected. The natural history of H.pylori infection, is influenced by host genetic, strain type, of bacterial virulence factors, time of exposure to the infection, as well as social and hygienic-sanitary conditions. In parallel, H.pylori is considered the main pathogen of gastroduodenal diseases. This study's main objective was to characterize H.pylori infection in populations of Angola and its evaluation as a public health problem. This is a prospective study conducted in two population groups, one in community environment composed by healthy individuals without specific gastric complaints - Group I, and Group II consisting of patients who went to the Gastroenterology Service of the Hospital Military of Luanda (HMP). As regards to the study in the community detection of H.pylori was carried out by antigen search in faeces using ELISA method. At hospital level H.pylori infection diagnostic methods were: upper gastrointestinal endoscopy to obtain gastric mucosal biopsies for histology, culture and molecular methods. As a non-invasive breath test with labelled urea was used. Group I: the diagnosis of H.pylori infection, by antigens detection in faeces, revealed a frequency of 69.6% in the study population. Whereas in each region, it was found that the Sambizanga region had the highest frequency of positive cases, 81.2% , followed by Dinge with 79.5%, Funda with 78.7 and Capulo with 39.8% being differences statistically significant (p=0.001). The evaluation of the distribution of the infection frequency by age group, revealed that individuals younger than 15 years had a frequency of 63.5% and in individuals older than 15 years, 76%. This study showed that the frequency of H.pylori infection in the regions studied was 70% exception due to Capulo, a coastal zone where despite the poor sanitation conditions; the frequency of H.pylori infection is lower. Group II: from the 309 patients evaluated, it was found that 22 (7%) had a normal mucosa and 287 (93%) a modified mucosa. Histological evaluation of antrum biopsies in 270 samples according to the Sydney System revealed the presence of gastritis in 235 (87.0%), the presence of ulcers in 13 (4.8%) and a tumour in 9 (3 3%). Histological assessment of activity in the gastric antrum of 226 samples, revealed that 129 (57%) had activity and 97 (43%) did not. The evaluation of the 255 corpus biopsies showed in 212 (83.1%), the presence of lesions of gastritis, in 7 (2.7%) tumour lesions and in 2 (0.8%) an ulcer. Of the 263 patients histological evaluated for H.pylori, 148 (58.2%) revealed the presence of this bacteria and 106 (41.7%) were negative. As regards susceptibility to macrolides from the universe of 158 patients with H.pylori, 125 (79.1%) patients had macrolides susceptible strains and 33 (20.9%) resistant strains. Regarding virulence factors (vacA and cagA), it was found that from the 11 patients with ulcers, 7 (63.6%), had a cagA negative strain, being 6 vacA s1, (85.7%) one vacA s2 and 4 (36.3%) with a cagA positive strain vacA s1. Concerning the 2 patients with tumour, both strains were cagA negative, one vacA s1 and other vacA s2. Patients with ulcer and tumour had cagA negative strains vacAs1. From this work, considering the prevalence of H.pylori obtained in health population, it is recommended that the same study should be performed in larger scale to confirm these results. The results of H.pylori genotyping suggest that more comprehensive studies are needed. Given the reduce number gastroenterology specialist in Angola and the lack of diagnostics methods, we recommend a larger study of the effectiveness of follow-up the patient dyspeptic, according to the protocol assessed by the College of Gastroenterology Specialty of the Order of Doctors and Angola already in place in some health institutions.

Evolutionary dynamics of Chlamydia trachomatis genome and identification of molecular patterns of hypothetical protein coding genes

The obligate intracellular bacterium Chlamydia trachomatis is a human pathogen of major public health significance. Strains can be classified into 15 main serovars (A to L3) that preferentially cause ocular infections (A-C), genital infections (D-K) or lymphogranuloma venereum (LGV) (L1-L3), but the molecular basis behind their distinct tropism, ecological success and pathogenicity is not welldefined. Most chlamydial research demands culture in eukaryotic cell lines, but it is not known if stains become laboratory adapted. By essentially using genomics and transcriptomics, we aimed to investigate the evolutionary patterns underlying the adaptation of C. trachomatis to the different human tissues, given emphasis to the identification of molecular patterns of genes encoding hypothetical proteins, and to understand the adaptive process behind the C. trachomatis in vivo to in vitro transition. Our results highlight a positive selection-driven evolution of C. trachomatis towards nichespecific adaptation, essentially targeting host-interacting proteins, namely effectors and inclusion membrane proteins, where some of them also displayed niche-specific expression patterns. We also identified potential "ocular-specific" pseudogenes, and pointed out the major gene targets of adaptive mutations associated with LGV infections. We further observed that the in vivo-derived genetic makeup of C. trachomatis is not significantly compromised by its long-term laboratory propagation. In opposition, its introduction in vitro has the potential to affect the phenotype, likely yielding virulence attenuation. In fact, we observed a "genital-specific" rampant inactivation of the virulence gene CT135, which may impact the interpretation of data derived from studies requiring culture. Globally, the findings presented in this Ph.D. thesis contribute for the understanding of C.trachomatis adaptive evolution and provides new insights into the biological role of C. trachomatishypothetical proteins. They also launch research questions for future functional studies aiming toclarify the determinants of tissue tropism, virulence or pathogenic dissimilarities among C. trachomatisstrains.

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.

The role of α2,3- and α2,6-sialylation

RESUMO: Os glicoconjugados que decoram a superfície celular e os lípidos e proteínas secretados ocupam o ponto de encontro onde normalmente ocorrem interacções críticas homólogas (hospedeiro-hospedeiro) e heterólogas (hospedeiro-patogénio). Apesar de ser largamente aceite que os glicanos são parte integrante do processo de imunidade, continua a não ser claro qual o papel que os glicanos, em toda a sua diversidade, tomam no quadro geral da imunidade. Os glicanos, que são frequentemente terminados por resíduos de ácido siálico, podem ser alterados por factores externos, tais como patogénios, ou por acontecimentos fisiológicos celulares específicos. Normalmente em posição terminal, as glico-estruturas que contêm ácido siálico assumem um papel fundamental numa quantidade substancial de receptores imunes envolvidos na adesividade e tráfico celular, tal como as Selectinas e as Siglecs, das quais se sabe apresentarem uma relevante função imune. À altura do início desta tese, era sabido que os ácidos siálicos expressos à superfície das células poderiam modular mecanismos importantes nas respostas imunes adaptativas. Considerando a posição de charneira que as células dendríticas (DCs) ocupam na transição da resposta imune inata para a adaptativa, antecipámos que os ácidos siálicos poderiam também modular mecanismos relevantes nas DCs humanas. As DCs têm uma função muito relevante na verificação e captura antigénica, migração para os gânglios linfáticos e apresentação antigénica aos linfócitos, uma sequência de funções que conduz, em ultima instância, à indução da resposta inata adaptativa. Considerando estas premissas, a nossa hipótese principal foi que os ácidos siálicos podem influenciar funções relevantes das DCs, tais como captura de antigénios, maturação, migração para os gânglios linfáticos e apresentação antigénica às células Para testar esta hipótese, dividimos o trabalho em quatro partes: 1) Analisámos os glicanos sialilados de superfície, expressos durante a diferenciação de monócitos humanos em DCs (moDCs). Os nossos dados mostraram que a expressão dos glicanos com ligações em O (O-glicanos) e sialilados em α2,3, assim como glicanos com ligações em N (N-glicanos) sialilados em α2,6 e α2,3 aumentou durante o processo de diferenciação das moDCs. Contribuindo para esta nova configuração glicosídica, três sialiltransferases (STs) poderão estar envolvidas: a ST6Gal-1 correlaciona-se com a expressão aumentada de N-glicanos sialilados em α2,6; a ST3Gal-1 contribui para a sialilação em α2,3 de O-glicanos, em especial de antigénios T; e a ST3Gal-4 poderá ser responsável pelo aumento de N-glicanos sialilados em α2,3. Após estímulo e consequente maturação das moDCs, ambos os níveis de expressão génica de ST6Gal-1 e ST3Gal-4 são negativamente modificados sendo, também, que a expressão de ST3Gal-1 varia consoante o estímulo. 2) Estudámos posteriormente as consequências da modulação dos ácidos siálicos de superfície nas funções das DCs. Observámos que a remoção dos ácidos siálicos de superfície diminui significativamente a capacidade de macropinocitose e endocitose mediada por receptores nas moDCs. Em contrapartida, o tratamento com sialidase aumentou significativamente a capacidade das moDCs para fagocitar Escherichia coli. Determinou-se também que este mecanismo requer a existência de ácido siálico presente nas E. coli indicando um mecanismo de interacção hospedeiro-patogénio dependente de ácido siálico em ambas as partes envolvidas. As moDCs tratadas com sialidase também apresentam um nível superior de expressão de moléculas de MHC e moléculas co-estimulatórias, sugerindo um fenótipo celular mais maduro. Recorrendo ao modelo de ratinho, utilizaram-se DCs derivadas de células da medula (BMDCs) de ratinhos deficientes em ST3Gal-1 e ST6Gal-1. Estes ensaios revelaram que quer a endocitose quer a maturação são influenciadas por modificações 37 nos glicanos sialilados em α2,3 ou α2,6. A detecção e quantificação de proteínas Nglicosiladas e sialiladas em α2,6 apontou para um potencial envolvimento de integrinas β2 nestes mecanismos. 3) O efeito da sialilação em α2,6 na migração das DCs para os gânglios linfáticos foi também analisado. Observámos que BMDCs deficientes para ST6Gal-1 apresentam uma redução de cerca de 50% nos níveis de migração das DCs para os gânglios linfáticos, tal como aferido em ensaios de inflamação in situ e estudos de transferência adoptiva de células. Uma redução dos níveis deste tipo de migração foi também observada quando BMDCs nativas foram transferidas para ratinhos receptores deficientes em ST6Gal-1. São, contudo, necessários mais ensaios de forma a identificar as moléculas envolvidas neste processo. 4) Por último, analisámos o impacto da sialilação na estimulação antigénica das DCs às células T. Assim, concluiu-se que moDCs tratadas com sialidase apresentam um nível de expressão superior de IL-12, TNF-ɑ, IL-6 e IL-10, e activação do factor de transcrição nuclear kappa B (NF-κB). As DCs tratadas com sialidase induziram uma maior proliferação nas células T, com expressão correspondente de interferão-γ. Este dado sugere que a remoção de ácidos siálicos de superfície contribui para o desenvolvimento de uma resposta pro-inflamatória do tipo 1 por células T auxiliares (resposta Th1). Considerando estes dados no seu todo, concluímos que o ácido siálico tem um papel marcante nas funções imunes das DCs. Alterações à concentração de ácido siálico à superfície das células podem alterar a endocitose/fagocitose, maturação, migração para os tecidos e gânglios linfáticos e capacidade estimulatória para com as células T. Complementando estes dados, as ligações glicosídicas de ácidos siálicos criados por ST6Gal-1 e ST3Gal-1 são funcionalmente relevantes. A modulação programada da sialilação do glicocálice, mediada por sialidases individuais ou sialiltransferases é uma possibilidade aceitável para a melhoria da fagocitose por DCs e da sua potência imunológica. Este facto tem um significado particular para imunoterapias baseadas em DCs, podendo provar-se decisivo para a sua eficiência e aplicabilidade num futuro muito próximo.-------------------------------ABSTRACT: Glycans decorating cell surface and secreted proteins and lipids occupy the junction where critical host–host and host-pathogen interactions occur. In spite of the wide acceptance that glycans are centrally implicated in immunity, exactly how glycans and their variety and variability contribute to the overall immune response remains poorly defined. Glycans, frequently terminated by sialic acid residues, may be modified by external factors such as pathogens or upon specific physiological cellular events. The terminal, privileged positions of sialic acid-modified structures makes them key, fundamental determinants for a number of immune receptors with known involvement in cellular adhesiveness and cell trafficking, such as Selectins and Siglecs, with known relevant immune functions. At the time this thesis was initiated, it was established that sialic acids expressed at cell surface could modulate important mechanisms of the adaptive immune responses. Given the key role of dendritic cells (DCs) in the transition from innate to the adaptive immune responses, we anticipated that sialic acids could also modulate important mechanisms of human DCs. DCs have a relevant role in antigen screening and uptake, migration to lymph nodes and antigen presentation to lymphocytes, ultimately triggering the adaptive immune response. Therefore, our primary hypothesis was that sialic acids may modulate DC functions, such as antigen uptake, maturation, homing to lymph nodes and antigen presentation to T cells. To test this hypothesis, we divided our work in four parts. 1) Surface sialylated glycans expressed during differentiation from human monocytes to DCs (moDCs) were analyzed. Our data showed that α2,3-sialylated O-glycans and α2,6- and α2,3-sialylated N-glycans expression increased during moDC differentiation. Three main sialyltransferases (STs) are committed with this new glycan configuration: ST6Gal- 1 correlates with the increased expression of α2,6-sialylated N-glycans; ST3Gal-1 32 contributes for the α2,3-sialylation of O-glycans, especially T antigens; and ST3Gal-4 may contribute for the increased α2,3-sialylated N-glycans. Upon moDC maturation, ST6Gal-1 and ST3Gal-4 are downregulated and ST3Gal-1 is altered in a stimulus dependent manner. 2) We subsequently analyzed the consequences of the modulation of cell surface sialic acids in DC functions. We observed that removing surface sialic acid by sialidase significantly decreased the capacity of moDCs to micropinocytose and receptormediated endocytose. In contrast, treatment with a sialidase significantly improved the capacity of moDCs to phagocytose Escherichia coli. The improved phagocytosis mechanism required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. Sialidase-treated moDCs have increased expression of MHC and co-stimulatory molecules, suggesting a more mature phenotype. Experiments using mouse bone marrow-derived DCs (BMDCs) from ST3Gal-1-/- and ST6Gal-1-/- strains indicated that endocytosis and maturation are influenced by changes in either α2,3 or α2,6-sialylated glycans. The analysis of α2,6-sialylated, N-glycosylated proteins, strongly suggested the potential involvement of β2 integrins, underlying these mechanisms. 3) The effect of α2,6-sialylation in DC homing to lymph nodes was also analyzed. We observed that BMDCs deficient for ST6Gal-1 have an almost 50% reduction in DC homing, as assayed by in situ inflammation and adoptive transfer studies. A reduction in DC homing was also observed when wild type BMDCs were transferred into ST6Gal-1-/- recipient mice. Further investigations are necessary to identify the molecules involved in this process. 4) Finally, we also analyzed the impact of sialylation on DCs ability to prime T cells. Sialidase-treated moDCs show increased gene expression of IL-12, TNF-α, IL-6 and IL- 10 cytokines, and activation of the transcription factor nuclear factor-κB. Sialidase33 treated DCs induced a higher proliferative response of T cells with concomitant higher expression of interferon-γ, suggesting that the clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. Together, our data strongly support sialic acid’s relevance in DC immune functions. Alterations of cell surface sialic acid content can alter the endocytosis/phagocytosis, maturation, migration/homing and the ability for T cell priming in human DCs. Moreover, sialic acid linkages created by ST6Gal-1 and ST3Gal-1 are functionally relevant. The engineering of cell surface sialylation, mediated by individual sialidases or sialyltransferases is a likely possibility to fine tune DC phagocytosis and immunological potency, with particular significance to DC-based therapies.

La résistance bactérienne aux antibiotiques.

50 years ago, the introduction of penicillin, followed by many other antibacterial agents, represented an often underestimated medical revolution. Indeed, until that time, bacterial infections were the prime cause of mortality, especially in children and elderly patients. The discovery of numerous new substances and their development on an industrial scale gave us the illusion that bacterial infections were all but vanquished. However, the widespread and sometimes uncontrolled use of these agents has led to the selection of bacteria resistant to practically all available antibiotics. Bacteria utilize three main resistance strategies: (1) modification of their permeability, (2) modification of target, and (3) modification of the antibiotic. Bacteria modify their permeability either by becoming impermeable to antibiotics, or by actively excreting the drug accumulated in the cell. As an alternative, they can modify the structure of the antibiotic's molecular target--usually an essential metabolic enzyme of the bacterium--and thus escape the drug's toxic effect. Lastly, they can produce enzymes capable of modifying and directly inactivating antibiotics. In addition, bacteria have evolved extremely efficient genetic transfer systems capable of exchanging and accumulating resistance genes. Some pathogens, such as methicillin-resistant Staphylococcus aureus and multiresistant Mycobacterium tuberculosis, have become resistant to almost all available antibiotics and there are only one or two substances still active against such organisms. Antibiotics are very precious drugs which must be administered to patients who need them. On the other hand, the development of resistance must be kept under control by a better comprehension of its mechanisms and modes of transmission and by abiding by the fundamental rules of anti-infectious chemotherapy, i.e.: (1) choose the most efficient antibiotic according to clinical and local epidemiological data, (2) target the bacteria according to the microbiological data at hand, and (3) administer the antibiotic in an adequate dose which will leave the pathogen no chance to develop resistance.

Constitutive activation of Wnt signaling favors generation of memory CD8 T cells.

T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Ralpha and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized beta-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized beta-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.

MC1R-dependent, melanin-based colour polymorphism is associated with cell-mediated response in the Eleonora's falcon.

Colour polymorphism in vertebrates is usually under genetic control and may be associated with variation in physiological traits. The melanocortin 1 receptor (Mc1r) has been involved repeatedly in melanin-based pigmentation but it was thought to have few other physiological effects. However, recent pharmacological studies suggest that MC1R could regulate the aspects of immunity. We investigated whether variation at Mc1r underpins plumage colouration in the Eleonora's falcon. We also examined whether nestlings of the different morphs differed in their inflammatory response induced by phytohemagglutinin (PHA). Variation in colouration was due to a deletion of four amino acids at the Mc1r gene. Cellular immune response was morph specific. In males, but not in females, dark nestling mounted a lower PHA response than pale ones. Although correlative, our results raise the neglected possibility that MC1R has pleiotropic effects, suggesting a potential role of immune capacity and pathogen pressure on the maintenance of colour polymorphism in this species.

Role of secretory immunoglobulin A and secretory component in the protection of mucosal surfaces.

The contribution of secretory immunoglobulin A (SIgA) antibodies in the defense of mucosal epithelia plays an important role in preventing pathogen adhesion to host cells, therefore blocking dissemination and further infection. This mechanism, referred to as immune exclusion, represents the dominant mode of action of the antibody. However, SIgA antibodies combine multiple facets, which together confer properties extending from intracellular and serosal neutralization of antigens, activation of non-inflammatory pathways and homeostatic control of the endogenous microbiota. The sum of these features suggests that future opportunities for translational application from research-based knowledge to clinics include the mucosal delivery of bioactive antibodies capable of preserving immunoreactivity in the lung, gastrointestinal tract, the genito-urinary tract for the treatment of infections. This article covers topics dealing with the structure of SIgA, the dissection of its mode of action in epithelia lining different mucosal surfaces and its potential in immunotherapy against infectious pathogens.