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Background: Autophagy is a lysosome mediated catabolic process that promotes cell survival during stress conditions. Aberrant regulation of autophagy is involved in the pathogenesis of a widening spectrum of autoimmune disorders: in particular, activation of autophagy pathway is implicated in aberrant survival of activated T cells. We recently demonstrated that autophagy is up-regulated in CD4+ T lymphocytes from patients with Sjogren’s Syndrome (SS) both in peripheral blood and tissue. Moreover, aberrant autophagy correlates with systemic disease activity. Little is known on the role of autophagy in B cells homeostasis in SS. Objectives: To investigate the activation of the autophagy pathway in lymphocytes infiltrating salivary gland in SS and peripheral blood from patients with SS. Methods: Frozen SS minor salivary glands (MSG) from 20 patients were sectioned and evaluated with immunohistochemistry (IHC) by staining for CD3+, CD20+, and CD21+. MSG sections, sequential to IHC, were stained by Cresyl Violet and microdissected (Laser Capture Microdissection) in order to isolate and collect small infiltrates, large CD21- infiltrates, and large CD21+ infiltrates (GC-like structures). Microdissected GC from frozen human tonsils were used as control. The expression of autophagy genes Atg5 and MAP1LC3II (expressed as 2^deltaCT normalized to GADPH) was determined by qPCR on microdissected tissue. For studies on peripheral blood, 19 SS patients and 11 healthy controls were enrolled, and autophagy, expressed as the ratio between Mean Fluorescence Intensity and the isotopic control (rMFI), was evaluated by Cyto-ID® Autophagy detection kit in T lymphocytes (CD4+ and CD8+) and B lymphocytes (CD19+) using a FACSCalibur cytometer. Results: Expression of the autophagy genes Atg5 and MAP1LC3 was significantly higher in the GCs from SS salivary glands compared to control tonsils (p Immunophenotyping studies revealed activation of the autophagy pathway in both circulating CD19+ and CD4+ lymphocytes from SS (p=0.04 and p=0.03 compared to HC); conversely, no pathway activation was detected in CD8+ cells. Of note, in patients with SS activation of autophagy was most marked in circulating CD19+ B cells compared the other subsets [(CD19+ vs CD4+ (p Conclusion: Our findings revealed that the autophagy pathway is aberrantly activated in lymphocytes infiltrating salivary glands of SS patients. Of note, progressively higher expression levels of autophagy genes mirrored the severity and extension of inflammatory infiltrates. Immunophenotyping studies revealed that activation of autophagy is preeminent in B cells. Thus, activation of autophagy emerges as a feature of both infiltrating and circulating SS lymphocytes, and as mechanism possibly implicated in the activation and survival o?f autoreactive cells. Disclosure of Interests: Serena Colafrancesco: None declared, Antonina Minniti: None declared, Roberta Priori: None declared, Marta Vomero: None declared, cristiana barbati: None declared, Francesca Arienzo: None declared, Valentina Iannizzotto: None declared, Elena Pipi: None declared, Joana Campos: None declared, Saba Nayar: None declared, Francesco Ciccia Grant/research support from: CELGENE, PFIZER, Consultant for: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, Paid instructor for: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, JANSSEN, Speakers bureau: UCB, NOVARTIS, CELGENE, PFIZER, LILLY, JANSSEN, MSD, ROCHE, AMGEN, Francesca Barone Grant/research support from: GlaxoSmithKline, Roche, UCB Pharma, Actelion, ONO Pharmaceutical, Consultant for: GlaxoSmithKline, Roche, Actelion, ONO Pharmaceutical, Guido Valesini: None declared, cristiano alessandri: None declared

The identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune-mediated inflammatory diseases (IMIDs)1,2. However, it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue-driven processes observed in IMIDs, such as inflammation and damage3-5. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of fibroblast activation protein-α (FAPα)+ fibroblasts suppressed both inflammation and bone erosions in mouse models of resolving and persistent arthritis. Single-cell transcriptional analysis identified two distinct fibroblast subsets within the FAPα+ population: FAPα+THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+THY1- destructive fibroblasts restricted to the synovial lining layer. When adoptively transferred into the joint, FAPα+THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation, whereas transfer of FAPα+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell-based therapies aimed at modulating inflammation and tissue damage.

Objetivo: Descrever o cenário de ocupação dos leitos de UTI dedicados à COVID-19 no período próximo ao carnaval e as medidas adotadas para a contenção do vírus, no estado de Goiás. Metodologia: Trata-se de um estudo retrospectivo, observacional e descritivo. Foram usadas as taxas de ocupação de leitos de UTI destinados à COVID-19, por região no estado de Goiás retiradas dos mapas de riscos de 17, 27 e 05 de março de 2021; além de decretos e normas desde 11 de fevereiro a junho de 2021, retirados do Portal da SES-GO e Casa Civil de Goiás. Resultados e Discussão: A média das taxas de ocupação de leitos de UTI aumentou cerca de 12% após o carnaval. Março foi considerado pela mídia o pior mês da pandemia até então, devido à variante P1. Entre as medidas de contenção do vírus, decretos e normas foram emitidos, mas dá-se destaque à crescente distribuição e aplicação de vacinas, o que trouxe um cenário mais favorável em junho. Conclusão: Durante o período próximo ao carnaval, cresceram as taxas de ocupação de leitos de UTI, em Goiás. Entre as medidas, nota-se distanciamento físico, realocação de leitos, higienização das mãos, o uso de máscara e a vacinação.

To address the industry's interest in finding novel biobased glues, the adhesive properties of the bacterial polysaccharide FucoPol were evaluated through shear bond strength tests. A FucoPol solution was used to bond different materials, namely, wood, glass, cardboard and cellulose acetate film. The shear strength was compared to that of the same adherends bonded with commercial synthetic glues. Wood-wood joints bonded with FucoPol formulation withstood 742.2±9.8kPa shear strength without detachment. FucoPol adhesive capacity for cardboard was comparable to that of the tested commercial glues (425±8.9kPa), yielding similar shear strength values (416.0±12.9kPa), while improved performance was shown for glass (115.1±26.2kPa) and cellulose acetate film (153.7±11.3kPa) comparing to the commercial glues (67.7-97.5kPa and 79.4-92.7kPa, respectively). This study demonstrates the adhesive properties of FucoPol, opening up the opportunity of using this bacterial polysaccharide for the development of new natural water-based glues, suitable to bond different materials.

Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. However, the kinetics and dynamic changes occurring to the lymphatic vascular network during TLS development have not been studied. Using a viral-induced, resolving model of TLS formation in the salivary glands of adult mice we demonstrate that the expansion of the lymphatic vascular network is tightly regulated. Lymphatic vessel expansion occurs in two distinct phases. The first wave of expansion is dependent on IL-7. The second phase, responsible for leukocyte exit from the glands, is regulated by lymphotoxin (LT)βR signaling. These findings, while highlighting the tight regulation of the lymphatic response to inflammation, suggest that targeting the LTα1β2/LTβR pathway in TLS-associated pathologies might impair a natural proresolving mechanism for lymphocyte exit from the tissues and account for the failure of therapeutic strategies that target these molecules in diseases such as rheumatoid arthritis.

Primary Sjögren syndrome (pSS) can be considered a systemic autoimmune disease with a strong organ bias. The involvement of the exocrine glands is prevalent and drives the pathognomonic manifestations of dryness that define the sicca syndrome. The salivary glands also represent the hub of pSS pathology. Elements belonging to both innate and acquired immune responses have been described at this site that contribute to disease establishment and progression. The interaction between those elements and their relative contributions to the clinical manifestations and lymphoma progression largely remain to be addressed.

Acute Myeloid Leukemia (AML) is a typically-lethal molecularly heterogeneous disease, with few broad-spectrum therapeutic targets. Unusually compared to many other cancers, over 90% of AML patients retain wild type TP53, encoding pro-apoptotic tumor suppressor p53. However, wild-type p53 functions are frequently suppressed by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. MDM2 inhibitors (MDM2i), which activate wild-type p53, show encouraging pre-clinical activity, but limited clinical activity. In an effort to find targets that synergize with p53 activation via MDM2i and minimize toxicity, we performed a cell-based synthetic lethal drug screen and a CRISPR viability screen. These screens identified BRD4 inhibition as a candidate synthetic lethal partner of MDM2i. BRD4 is a member of the Bromodomains and Extraterminal (BET) family of proteins, a transcriptional co-activator and already a candidate AML therapeutic target. Surprisingly, we found inhibition of BRD4 alone induces expression of some of p53 target genes. We unexpectedly reveal that BRD4 binds to p53 target genes and acts as a transcriptional repressor of these genes. Synergistic cell killing by the drug combination (MDM2i + BET inhibitor (BETi)) depends on synergistic activation of p53 target genes, such as PUMA and NOXA, due to simultaneous stabilization of p53 by MDM2i and relief of BRD4-mediated repression by BETi. Our combined therapy of MDM2i and BETi is synergistically lethal to human AML cell lines harboring wild type TP53in vitro, against two mouse models of AML in vivo, and against primary human patient blasts in vitro. Furthermore, we used BET PROTACs to selectively and completely induce degradation of BRD4 in cells. Consistent with results from BETi, BET degraders and MDM2i synergize to suppress cell viability with superior potency. Taken together, our data show BRD4 represses p53-mediated transcription activation and apoptosis in AML. Therefore, co-targeting wild-type TP53 and a transcriptional repressor function of BRD4 represent a novel synthetic lethal vulnerability in AML. Disclosures Latif: AbbVie: Consultancy, Honoraria; Takeda UK: Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Kite: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria. Higgins:Roche: Current Employment, Current equity holder in publicly-traded company, Other: Support of parent study and funding of editorial support. Copland:Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cyclacel Ltd: Research Funding; Roche: Research Funding; Epizyme: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau; Gilead: Speakers Bureau.

Career situation of first and presenting author Post-doctoral fellow. Introduction Sjogren’s syndrome (SS) is an autoimmune condition characterised by systemic B cell activation, autoantibody production and ectopic germinal centers (GC) formation within salivary gland (SG). The extent of SG infiltrate has been proposed as biomarker of disease severity. Plasma levels of CXCL13 correlate with GC activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B cell activation. Objectives To evaluate the potential role of CXCL13 as biomarker of SG pathology in two independent SS cohorts. Methods 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n=60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n=49). Both sera and matched paraffin-embedded minor SG biopsy were available. Sicca (n=57) and healthy subjects (HS) (n=19) sera were used as control. CXCL13 gene expression was also assessed in 25 frozen SGs. Results CXCL13 serum levels were higher in SS patients [90.3 (84.2) pg/ml], compared to both sicca [61.9 (38.6) pg/ml), p=0.0005] and HS [36.5 (40.18) pg/ml, p Conclusions Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies. Both serum and tissue expression of CXCL13 correlate with SS histological severity, suggesting a major role of this chemokine in SG lymphocytes recruitment and organization. Disclosure of Interest None declared.

Career situation of first and presenting author Post-doctoral fellow. Introduction The development of the B cell repertoire is regulated by the process of affinity maturation that occurs within the inner part of the B cell follicles within secondary lymphoid organs (SLOs). In autoimmunity process might occur in ectopic lymphoid structures (ELS), aggregates of lymphocytes that form in target organs of disease (i.e. the salivary glands of patients with Sjogren’s Syndrome (SS)). The phenotypical and functional features supporting ELS pathogenic properties have not been identified. Moreover, the functional proof that ELS independently from SLOs contribute to the autoimmune response has not been provided. Objectives To characterise the transcriptome profile of human ELS isolated from SS salivary glands in comparison with SLO and to dissect, in an animal model of ELS development, the ability of ELS to contribute to the autoreactive response. Methods Frozen salivary gland biopsies were obtained from SS patients and selected for presence of germinal center +ELS. Samples were stained and microdissected, RNA isolated and transcribed and used for RNAseq using ClonTech SMARTseq v4 kit. Salivary gland ELS and autoimmunity was established as previoully described1 in FAP-DTR mice. Animals were locally treated (day 2 and 6) with DTX to induce selective ELS disaggregation; glands and serum were harvested for analysis at day 15. Results Transcript analysis provide evidence that the ectopic ELS are characterised by upregulation of TNF, INFg, BAFF, APRIL, CXCL12 and CXCL13, FAS and FASL as compared to SLOs. Sequencing unveiled an alterated cell-proliferation profile with downregulation of BCL6 and AID, the enzymes responsible for B cell affinity maturation. Selective depletion of FAP +cells induced loss of anatomical segregation of ELS and profoundly compromised their anatomy with significant impact on the local autoimmune response. Conclusions Our study provides the first evidence of a significant molecular difference between ELS and SLOs. We demonstrated that, although characterised by similar anatomical organization, ELS pathogenic cytokine signature is associated with low levels of bcl6 and AID and aberrant apoptosis, unveiling impaired regulation of the B cell cycle, responsible for the survival of autoreactive, poorly selected B cell clones. Accordingly, selective depletion of ELS in vivo profoundly impact local and systemic autoimmunity. Reference Barone F, et al. PNAS 2015. Disclosure of Interest None declared.

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Sjogren’s syndrome (SS) is a chronic autoimmune disease characterised by B-cell hyper-activation and exocrine gland infiltration that results in loss of glandular function, systemic manifestations and autoantibody production. The phosphatidylinositol 3–kinase delta isoform (PI3Kδ) belongs to a family of intracellular lipid kinases that regulate metabolism, survival, proliferation, apoptosis, growth, and cell migration and has been successfully targeted in B-cell malignancies. Given the central role of the B-cells in the pathogenesis of SS we investigated evidence for the engagement of the PI3Kδ pathway in SS and the functional consequences of blocking PI3Kδ in an animal model of SS. PI3K pathway activation was investigated in salivary gland (SG) biopsies from patients with SS or non-specific chronic sialoadenitis (NSCS). Samples were stained for phosphorylated ribosomal protein S6 (pS6), a downstream effector in the PI3Kδ pathway. UCB5857, a small molecule inhibitor of PI3Kδ, was used either prophylactically or therapeutically in vivo in an inducible model of ectopic lymphoneogenesis in murine SGs that mimics SS. Flow cytometry, immunofluorescence and quantitative PCR was used on the murine isolated SG to evaluate the samples at peak of inflammation which is day 15 post-cannulation. Histological staining for pS6 showed significant expression of pS6 in SS samples relative to NSCS control, confirming engagement of the PI3K pathway. pS6 was detected within lymphoid aggregates in both T- and B-cell areas and on the periphery of the lymphoid foci in SG biopsies. pS6 staining was predominantly found on CD138 + plasma cells in SG biopsies. Mice treated either prophylactically or therapeutically with UCB5857 displayed decreased lymphocyte infiltration in cannulated salivary glands relative to vehicle treated mice. Additionally, a gene expression profile associated with ectopic lymphoneogenesis (CXCL13, CCL19, CCL21) was significantly impaired in mice treated with UCB5857. Lymphoid aggregates in these mice were characterised by decrease in focus score, smaller size and reduced T/B-cell follicular organisation. Preliminary data implicates activation of PI3Kδ pathway in several cells within the SGs of SS patients that may contribute towards pathology. Accordingly, prophylactic and therapeutic blocking of PI3Kδ results in disaggregation of the inflammatory foci and resolution of SG inflammation in an animal model of SS.

Studying the diet of crustaceans based on the direct visual inspection of the gut contents can be difficult because they can ingest only a portion of the prey and further crush it into very small pieces, making a very few food items identifiable. Here, the usefulness of molecular methods to identify the species present in the gut contents of two crustaceans, the shore crab Carcinus maenas and the brown shrimp Crangon crangon, was tested. Stomach contents were removed, and their total genomic DNA extracted followed by amplification (PCR) with four specific prey mtDNA primers. Since both crustaceans are opportunistic predators, preying upon around 40 different species, the study was restricted to common potential preys in the sampled area, the Minho Estuary (Portugal): the common goby Pomatoschistus microps, the ragworm Hediste diversicolor, the flounder Platichthys flesus and the peppery furrow shell Scrobicularia plana. After testing the most appropriate methodology for DNA extraction and PCR amplification to detect the presence of each potential prey with positive control samples, the same methodology was applied to the stomach content samples. The molecular methods allowed for detection of the presence of P. microps, P. flesus and H. diversicolor in the stomachs of both crustaceans. Therefore, once potential prey and their availability in the system are known, PCR technique can be an alternative tool to overcome some of the limitations of traditional methods in the study of the crustaceans’ diet, even when stomach contents include a great part of ‘detritus’. This study reflects also the need to develop more tools (primers) to improve the assessment of predator–prey interactions in aquatic ecosytems.

Residential care (RC) in Portugal accounts for almost 90% of out-of-home placements of children. Since the Youth Self-Report (YSR) and the Strengths and Difficulties Questionnaire (SDQ) are the most commonly used psychological (mal)adjustment screening instruments, it is important to analyze their comparative validity specifically in the context of RC. This study aims to analyze the comparative effectiveness of YSR/SDQ as screening tools of psychological (mal)adjustment, looking for differences in the way psychological problems and difficulties are identified by these two measures in adolescents in RC. Sixty-one adolescents, aged 12–20, living in five RC settings participated in this study. Participants’ characteristics at the time of admission were collected using the previous request for infomation (PIP) from the Portuguese Comprehensive Assessment System for Residential Care Quality (ARQUA-P). Participants filled out both the YSR and the self-report form of SDQ. Significant correlations between the two self-report measures of adolescents’ psychological adjustment were observed. Additionally, the utility of each measure differed based on youngsters’ characteristics, potentially supporting the simultaneous use of both instruments. Implications for further research and professional practice in RC are discussed, namely the differential utility of using YSR and/or SDQ as a screening tool with this population.

Research suggests that mental health problems in adolescents in residential care (RC) are more prevalent than for youth in the community, and that psychosocial competencies are protective factors of mental health. This study compared adolescents in RC in Portugal, with a Portuguese community sample on the incidence of mental health problems and psychosocial skills, explored gender differences and the relationships between mental health problems and psychosocial competencies. Four hundred and forty-three adolescents in RC participated. Results showed that the RC adolescents had higher levels of mental health problems lower academic achievement, a poorer social support network, but were more involved in sports and hobbies, household chores than the adolescents in the normative sample. Gender differences were observed. Psychosocial skills were negatively correlated with mental health problems. These findings indicate an urgent need in Portugal for early screening of mental health problems and socio-cognitive interventions for youth in RC.

Em Portugal existem mais de 8000 crianças em Acolhimento Residencial (AR). Neste estudo participaram 77 crianças em AR (63,6% rapazes). Através da Bateria ASEBA foi possível observar que os participantes apresentaram níveis clínicos de problemas de saúde mental (maioritariamente as raparigas). Estes estão relacionados com o motivo de acolhimento e com o tipo de apoio recebido. A dimensão da casa medeia a relação entre a sintomatologia e o número de apoios recebidos. Estes resultados apelam à necessidade de instituir procedimentos de avaliação psicológica no momento de admissão da criança, sensibilizar os profissionais das casas de AR para estes sinais e sintomas e implementar programas de prevenção e promoção da saúde mental., In Portugal there are more than 8,000 children in Residential Care (RC). Seventy-seven children in RC (63.6% boys) participated in this study. Using the ASEBA Battery it was possible to observe that the participants presented clinical levels of mental health problems (mainly the girls), which were related to the reasons of being in care and the kind of support received. The RC Centre’s dimension mediated the relationship between the amount of support and the child’s symptomatology. These results reinforce the need to provide psychological assessment procedures at the moment when the child enters care, to sensitize RC professionals for these signs and symptoms, and to implement programs for the prevention and promotion of mental health., Il existe au Portugal plus de 8000 enfants en Accueil Institutionnel (AI). Ont participé à cette étude 77 enfants en AI (63,6% garçons). Utilisant la Batterie ASEBA il a été possible d'observer que les participants présentaient des niveaux cliniques de problèmes de santé mentale (surtout les filles). Ceux-ci sont liés au motif de la mise en accueil et au type d’appui reçu. La dimension du centre d’accueil est médiateur du rapport entre la symptomatologie et le nombre d’appuis. Ces résultats renforcent le besoin d'instituer des procédures d'évaluation psychologique au moment de l'admission de l'enfant, de sensibiliser les professionnels de l’AI à ces symptômes et mettre en œuvre des program- mes de prévention et de promotion de la santé mentale.

A thorough understanding of ecological networks relies on comprehensive information on trophic relationships among species. Since unpicking the diet of many organisms is unattainable using traditional morphology‐based approaches, the application of high‐throughput sequencing methods represents a rapid and powerful way forward. Here, we assessed the application of DNA metabarcoding with nearly universal primers for the mitochondrial marker cytochrome c oxidase I in defining the trophic ecology of adult brown shrimp, Crangon crangon, in six European estuaries. The exact trophic role of this abundant and widespread coastal benthic species is somewhat controversial, while information on geographical variation remains scant. Results revealed a highly opportunistic behaviour. Shrimp stomach contents contained hundreds of taxa (>1,000 molecular operational taxonomic units), of which 291 were identified as distinct species, belonging to 35 phyla. Only twenty ascertained species had a mean relative abundance of more than 0.5%. Predominant species included other abundant coastal and estuarine taxa, including the shore crab Carcinus maenas and the amphipod Corophium volutator. Jacobs’ selectivity index estimates based on DNA extracted from both shrimp stomachs and sediment samples were used to assess the shrimp's trophic niche indicating a generalist diet, dominated by crustaceans, polychaetes and fish. Spatial variation in diet composition, at regional and local scales, confirmed the highly flexible nature of this trophic opportunist. Furthermore, the detection of a prevalent, possibly endoparasitic fungus (Purpureocillium lilacinum) in the shrimp's stomach demonstrates the wide range of questions that can be addressed using metabarcoding, towards a more robust reconstruction of ecological networks.

Aim: To assess the prevalence, quality of life (QoL), and the damages caused by PH, in the professional and personal scope of Physical Education academics with Primary Hyperhidrosis (PH). Methods: A descriptive, exploratory, qualitative and quantitative cross-sectional study was carried out. Twenty-five students from the physical education course at Tiradentes University, Aracaju-SE, were interviewed from August to November 2017. Validated questionnaires were used on PH’s influence in academic activities and QoL. absolute and relative frequencies in the case of categorical variables and measures of trend and central variability in the case of numerical variables. Results: The prevalence of PH in students of Physical Education was 11.11%, mainly in combined sites, such as palmoplantar, and with no difference between the sexes. The symptoms started mostly during childhood and adolescence. Most of the interviewees (92%) reported difficulties with activities such as sports, use of personal protective equipment, handling of work equipment and instruments, and exacerbation in stress situations. They reported significant harm to QoL in situations of greeting people with handshakes, writing, wearing socks and dancing socially. Conclusion: Although PH is a disease that negatively impacts the QoL, it is still little known in the academic world with little demand for medical help. Therefore, it is necessary greater dissemination of the disease for its early diagnosis, related to the intensity of the sweating for a better therapeutic approach.

Objectives SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres’ formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres’ activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. Methods 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects’ (n = 19) sera were used as control. Results CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. Conclusion Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

Os termolaminados de alta pressão têm a sua origem no início do século XX, e consistem numa sobreposição de folhas de papel impregnado, que ao serem prensadas a pressão e temperatura elevada, aderem entre si formando estruturas resistentes e com uma vasta gama de aplicações. Estas características fazem dos HPL produtos de valor acrescentado, com um grande potencial económico. A identificação de defeitos superficiais nestes materiais por parte do consumidor pode levar a uma desvalorização do seu valor comercial. De forma a combater este impacto comercial a SIR- SONAE Indústria de Revestimentos, decidiu iniciar o estudo e caracterização destes defeitos superficiais, assim como das suas potenciais causas. Neste sentido foram identificados três tipos de defeitos superficiais: em superfícies claras, a presença de manchas amareladas; em superfícies escuras de alto brilho, a presença de manchas irregulares ou a existência de listas verticais de tonalidade diferente. Os defeitos identificados foram sujeitos análise das amostras, expondo-as a diferentes técnicas, como Espectroscopia de reflectância de Ultravioleta - Visível, FTIR-ATR, Raman, AFM, Pirólise GC/MS, e estudo dos ângulos de contacto pelo método da gota séssil. Com os resultados obtidos concluiu-se que o aparecimento de manchas amareladas, é causado pela migração de compostos fenólicos resultantes de uma impregnação ineficiente de resina melamínica no papel decorativo, não tendo sido, no entanto, possível a identificação total da espécie migratória. No caso das superfícies que apresentavam manchas irregulares concluiu-se, que o aparecimento do defeito não reside na existência de uma espécie externa, mas sim na má adequação da temperatura de ciclo. Por fim nas superfícies listadas verificou-se mais uma vez a ausência de espécies extrínsecas à superfície do laminado, sendo a presença destas listas causadas pela heterogeneidade do processo de impregnação do papel decorativo. The invention of high pressure laminates, HPL, remotes to the XX century, and consists in an overlap of paper impregnated by resins, that after being exposed to high pressure and temperature flows and allows the sheets of paper to bond. Creating a new, resistant and highly stable, material of added value, which represents an elevated commercial value. The detection of flaws, like surface defects, buy the costumers can reduce its high commercial value. So, to diminish the commercial impact of the defects in its economy, SIR- SONAE Indústria de Revestimentos, decided to characterize and chemically analyze them, as well as it’s probable origin. With this in mind, the defects were identified and could be divided in three different types: The light color surfaces, that revealed yellowish regions, the dark color surfaces that showed regions with different texture and shade, and lastly the dark color ones in which is visible the presence of lighter vertical stripes. In order to fulfill the main goal samples of the identified defects were subjected to different optical techniques such as UV-vis reflectance spectroscopy, infrared spectroscopy FTIR-ATR, Raman spectroscopy, AFM, pyrolysis GC/MS and contact angle analysis by the sessile drop method. The obtained results allowed to conclude that in the light colored laminates the yellowish stains where caused by phenolic compounds that migrate to the HPL surface due to an inefficient impregnation process. Despite of this information it was not possible to understand the true identity of this phenolic compounds. As for the black HPL that showed regions with different textures and shades, it was perceptible that the non-uniformity of the regions was not caused by influence of external components. Instead it was caused by an insufficient cycle pressing temperature. Finally, the stripped samples surface analysis did not show the presence of unexpected compounds. Its heterogeneity was caused by a poor impregnated system that leaded to a heterogeneous coated surface. Mestrado em Engenharia Química

At sites of chronic inflammation, recruited immune cells form structures that resemble secondary lymphoid organs (SLOs). Those are characterized by segregated areas of prevalent T- or B-cell aggregation, differentiation of high endothelial venules (HEVs) and local activation of resident stromal cells. B-cell proliferation and affinity maturation towards locally displayed autoantigens have been demonstrated at those sites, known as tertiary lymphoid structures (TLSs). TLS formation has been associated with local disease persistence and progression as well as increased systemic manifestations. While bearing a similar histological structure to SLO, the signals that regulate TLS and SLO formation can diverge, and a series of pro-inflammatory cytokines has been ascribed as responsible for TLS formation at different anatomical sites. Here we review the structural elements as well as the signals responsible for TLS aggregation, aiming to provide an overview to this complex immunological phenomenon.

SUMMARYThe identification of lymphocyte subsets with non-overlapping effector functions has been pivotal to the development of targeted therapies in immune mediated inflammatory diseases (IMIDs). However it remains unclear whether fibroblast subclasses with non-overlapping functions also exist and are responsible for the wide variety of tissue driven processes observed in IMIDs such as inflammation and damage. Here we identify and describe the biology of distinct subsets of fibroblasts responsible for mediating either inflammation or tissue damage in arthritis. We show that deletion of FAPα+ synovial cells suppressed both inflammation and bone erosions in murine models of resolving and persistent arthritis. Single cell transcriptional analysis identified two distinct fibroblast subsets: FAPα+ THY1+ immune effector fibroblasts located in the synovial sub-lining, and FAPα+ THY1- destructive fibroblasts restricted to the synovial lining. When adoptively transferred into the joint, FAP α+ THY1- fibroblasts selectively mediate bone and cartilage damage with little effect on inflammation whereas transfer of FAP α+ THY1+ fibroblasts resulted in a more severe and persistent inflammatory arthritis, with minimal effect on bone and cartilage. Our findings describing anatomically discrete, functionally distinct fibroblast subsets with non-overlapping functions have important implications for cell based therapies aimed at modulating inflammation and tissue damage.

Background Fibroblasts are key effector cells in the persistence of synovial inflammation and joint damage. It is not yet known whether specific subsets of synovial fibroblasts exist, and if so, if they are responsible for the distinct fibroblast mediated features observed in inflammatory arthritis, such as invasion of cartilage, bone damage, and persistence of inflammation. Objectives Here we identify and describe the biology of a functionally distinct pathogenic fibroblast cell type marked by the co-expression of Thy1.2 and Podoplanin (Pdpn), that is responsible for persistence of synovial inflammation. Methods We used the serum transfer arthritis (STA) model to induce joint inflammation. To identify putative subsets of fibroblasts we used flow cytometry using established markers of stromal cells, on enzymatically digested synovial tissue. These cells were localised in tissue sections using immunohistochemistry or immunofluorescence staining. Proliferation of subsets in vivo during the time course of STA was determined by BrdU incorporation studies. Transcriptomic analysis was performed using ultra-low input RNA sequencing on flow sorted populations of cells and single cell analysis performed on CD45 negative gated cells. Results Using FAP as a biomarker of activated synovial fibroblasts we have been able to identify and define distinct subsets of synovial fibroblasts based on their co-expression of Thy1.2 and Pdpn. These subsets reside in distinct compartments of the synovial microanatomy including the lining layer (LL), sub-lining layer (SL) and a subset of pericytes. We found that FAP +cells within the SL are highly proliferative and their expansion in cell number positively correlates with inflammation. Global deletion of FAP +mesenchymal cells in the synovium using a FAP-DTR mouse attenuated synovial inflammation; protected against erosive bone damage and lead to reduced leucocyte accumulation as a result of reduced chemokine and cytokine production by synovial fibroblasts within the membrane. Collectively these data suggest a pathogenic pro-inflammatory role for these cells. Transcriptomic analysis by both ultra-low input bulk RNA sequencing and single cell analysis revealed these subsets to be transcriptionally distinct with the greatest transcriptional differences observed between LL and SL cells, demonstrating a site specific transcriptional program for cells within these compartments. Gene signature analysis of SL FAP +cells was consistent with an immune effector cell phenotype, in contrast to lining layer cells that express genes associated with matrix remodelling. Finally, to test this hypothesis we injected FAP +LL cells and FAP +SL cells into inflamed ankle joints of mice. The injection of FAP +SL cells lead to more severe and prolonged joint inflammation, whereas injection of LL FAP +cells had no effect. Finally, we identified these cell subsets within human synovial tissue and have demonstrated the expansion of SL cells with inflammation. Conclusions Synovial inflammation is associated with the expansion, activation and differentiation of fibroblasts into distinct functional subsets of cells that regulate those specific aspects of inflammatory joint pathology. Direct targeting of specific pathogenic subsets of synovial fibroblasts may provide a novel, non-immunosuppressive approach to the treatment of inflammatory arthritis. Disclosure of Interest None declared

Background Ectopic production of the lymphoid chemokines CXCL13 and CXCL12 has been described in tertiary lymphoid structures (TLS) that harbour in the salivary glands of patients with Sjogren’s Syndrome (pSS). Whilst CXCL13 expression correlates with clinical features, its potential role as a biomarker to monitor the organisation and severity of the salivary gland infiltrate has been hampered by the lack of sensitive tools to describe TLS extent and features. Objectives To investigate CXCL13 and CXCL12 serum and tissue expression and to find any possible association with clinical, histological and laboratory features. Methods We studied histological features of the minor salivary glands (MSG) and sera of respectively fifty and seventy (table 1) unselected consecutive patients with pSS (AECG criteria). Concentration of CXCL13 and CXCL12 were evaluated by ELISA in patient sera and eleven healthy controls (HC). Paraffin embedded MSG were studied by haematoxylin/eosin and anti-CD3, anti-CD20, anti-CD21 staining. Images analysis was used to calculate focus score (FS), mean foci area, percentage of infiltration (%i), segregated foci (%SF),%GCs and lymphoepitelial lesions (%LEL). GCs from MSG and tonsils were microdissected and quantitative PCR was used to test CXCL12 and CXCL13 transcripts. Results Histological analysis unveiled strong correlations between the mean foci area with the%i and the presence of SF; positive correlations were also observed between the%i and both the FS and%SF. This was significantly higher in patients exhibiting SF. The% of SF positively correlated with FS, presence of%GC and%LEL that also correlated with the%i and the%SF (image). Mean CXCL13 and CXCL12 serum levels were significantly higher in pSS compared to HC [(124.12±119.73 pg/ml vs 8.9±15.4 pg/ml (p=0.001) and 34.6±54.2 pg/ml vs 2.5±8.3 pg/ml (p=0.05), respectively]. CXCL13 was significantly higher in patients with SF, with GCs and LEL and correlated with the mean foci area, the%i, the FS and the percentage of LEL. Higher CXCL13 levels were associated with the presence of antibodies and other biological findings including hyperglobulinemia. Higher CXCL13 levels were also able to discriminate patients with lymphoma (p=0.009). CXCL12 levels correlated with the FS,%i and% of LEL. Transcript analysis showed no difference in the expression of CXCL13 between MSG and tonsil GC, whilst CXCL12 was found significantly higher in MSG (p Conclusions Our results suggest the utility to expand the parameters of histologic evaluation of MSG, whilst reinforcing the role of the FS as reliable instrument to reflect the severity of inflammation. Analysis of MSG infiltration and foci segregation was able to identify subjects with increased proliferative risk. We demonstrated that serum CXCL13, is a biomarker of histological severity and is able to stratify patients with lymphoma. The high levels of CXCL12 in MSG GC suggest a differential biology of TLS in the SG, probably implicated in aberrant B cell clone survival. Disclosure of Interest None declared

Summary Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion., Graphical Abstract, Highlights • YTHDF2 is highly expressed across human AML and is essential for leukemia initiation • YTHDF2 shortens the half-life of m6A-modified transcripts in AML • Loss of YTHDF2 expands HSCs but does not derail hematopoiesis • YTHDF2 protects AML cells from apoptosis by downregulating TNFR2, Elimination of cancer stem cells in acute myeloid leukemia (AML) while preserving hematopoiesis is a challenge in leukemia treatment. Paris et al. demonstrate that inactivation of the RNA m6A reader YTHDF2 increases the half-life of m6A-modified transcripts and specifically compromises cancer stem cells, highlighting YTHDF2 as an essential regulator of AML.

Salmonellosis remains one of the most frequent food-borne zoonoses, constituting a worldwide major public health concern. Currently, at a global level, the main sources of infection for humans include meat products, including the consumption of contaminated poultry meat, in spite of the success of Salmonella control measures implemented in food-animal production of industrialized countries. In recent years, a shift in Salmonella serotypes related to poultry and poultry production has been reported in diverse geographical regions, being particularly associated with the spread of certain well -adapted clones. Moreover, antimicrobial resistance in non-typhoidal Salmonella is considered one of the major public health threats related with food-animal production, including the poultry production chain and poultry meat, which is an additional concern in the management of salmonellosis. The circulation of the same multidrug-resistant Salmonella clones and/or identical mobile genetic elements encoding antibiotic resistance genes from poultry to humans highlights this scenario. The purpose of this review was to provide an overview of the role of poultry meat on salmonellosis at a global scale and the main problems that could hinder the success of Salmonella control measures at animal production level. With the increasing globalization of foodstuffs like poultry meat, new problems and challenges might arise regarding salmonellosis control, making new integrated intervention strategies necessary along the food chain. Clinical Microbiology and Infection

Introduction Tertiary lymphoid structures (TLS) often develop in target tissues of autoimmune diseases (AID) such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis, and Sjogren’s syndrome (SS). These structures consist of aggregates of B and T cells with varying degree of organisation and are proposed to promote the generation of autoreactive effector cells and autoantibody production. Modulation of the S1P1 receptor inhibits egress of pathogenic lymphocytes from lymphoid organs and reduces their availability in circulation. This has proven to be an effective target for the treatment of AID including MS and is currently being considered for early phase clinical trials in SLE and SS. Objectives To investigate the functional targeting of the S1P1 receptor in a murine model of SS. Methods Cenerimod, an orally active, selective S1P1 receptor modulator, was administered either preventively (early in inflammation) or therapeutically (established inflammation) in an inducible model of SS to evaluate the efficacy of cenerimod in vivo. Histology, flow cytometry and qPCR were used to analyse the tissue samples. Results Cenerimod induced disaggregation of the lymphocytic structures and resolution of salivary gland (SG) inflammation with a concomitant decrease in focus score, lymphoid structure size and T/B-cell follicular organisation. Mice treated with cenerimod displayed significantly decreased T (naive, central memory and effector) and B (including CD138 +plasma cells) lymphocyte infiltration in cannulated SG, relative to vehicle treated mice. Interestingly, the lymphocytes from cenerimod treated mice exhibited significantly reduced proliferation as well as reduction in pro-inflammatory cytokine RNAs such as IL-17, IL-21, and IL-6. Furthermore, the gene expression profile associated with TLS formation (LTα, LTβ, TNFα, CXCL13, CCL19) was less pronounced in cenerimod treated samples. The cervical lymph nodes draining the salivary glands showed a slight reduction in T lymphocytes, but no significant defects were observed in structure, organisation and production of lymphoid chemokine/cytokines, suggesting that homeostatic regulation of tertiary and physiological lymphoid organs differentially relies on lymphocyte/stromal cell cross-talk during inflammation. Conclusions Together, these data demonstrate that the S1P1 receptor modulator cenerimod regulates TLS in mice and might therefore be a potential treatment option in AID with TLS formation such as SS and SLE. The data also unveil differential requirements for the establishment and maintenance of secondary versus tertiary lymphoid structures. Acknowledgements This research is funded by Idorsia Pharmaceuticals Ltd. Disclosure of interest S. Nayar: None declared, J. Campos: None declared, C. Smith: None declared, C. Buckley: None declared, S. Froidevaux Employee of: Idorsia, K. Wartha Employee of: Idorsia, C. Seemayer Employee of: Idorsia, F. Barone: None declared

Background Salivary glands of primary Sjogren’s syndrome (pSS) are characterised by complex leukocyte infiltration organised into tertiary lymphoid structures (TLS). The mechanisms regulating leukocyte trafficking into inflamed salivary glands are poorly described, but dysregulated T-cell recruitment during inflammation is believed to contribute to disease onset and chronicity. We recently described a homeostatic pathway in which a B cell-derived peptide (PEPITEM), secreted in response to adiponectin, regulates T-cell trafficking during inflammation via sphingosine-1-phosphate activity on endothelial cells1. Disruption of this pathway by downregulation of adiponectin receptors on circulating B cells has been demonstrated in type-1 diabetes and rheumatoid arthritis, suggesting a potential role for PEPITEM in the pathogenesis of autoimmune diseases, and indicating a role for adiponectin receptors as biomarkers in these conditions1. Objectives We aimed to investigate the efficacy of PEPITEM as an inhibitor of T-cell trafficking in an inducible animal model of salivary gland inflammation that mimics the histological features of pSS, and to investigate the potential translatability of this pathway in patients with pSS. Methods Submandibular salivary glands of C57BL/6 mice were intra-ductally cannulated with luciferase-encoding replication-deficient adenovirus to induce TLS formation as previously described2. Mice were administered daily either PBS or PEPITEM by intraperitoneal injection from day 0, and their salivary glands dissected at day 5 post cannulation. T-cell infiltration into salivary glands was assessed using a combination of flow cytometry, immunofluorescence, and qRT-PCR for inflammatory chemokines. Results B cells in sera from cannulated animals express lower levels of both adiponectin receptors 1 and 2 in comparison with non-inflamed control mice. In cannulated animals treated with PEPITEM, histological analysis of salivary glands revealed fewer, as well as less aggregated, infiltrating T cells. Both CD4 +and CD8+numbers were significantly lower in the salivary glands of PEPITEM-treated animals. Furthermore, administration of PEPITEM also decreased mRNA transcripts for lymphotoxin beta, IL-7, lymphoid chemokines (CCL19 and CXCL13) and T-cell chemokine receptor CCR7, cytokines and chemokines known to regulate ectopic lymphoneogenesis in pSS. Samples from pSS patients are currently being assessed to validate the relevance of this pathway in pSS. Conclusions These results demonstrate that administration of exogenous PEPITEM can reduce T-cell influx into salivary glands. This may represent a rescue of the homeostatic regulation of leukocyte trafficking which is disrupted in inflammation. Our work suggests that PEPITEM should be considered to address therapeutic needs in chronic inflammatory conditions, and that the detection of decreased levels of adiponectin receptors could serve as a biomarker in pSS. References [1] Chimen, McGettrick, et al. Nat Med. 2015;21(5):467–475. [2] Bombardieri, Barone, et al. J. Immunol. 2012;189:3767–3776. Disclosure of Interest None declared

Introduction The RANKL (ligand)-RANK-OPG triad, members of the TNF(R) superfamily, is implicated in lymphoid organ development and bone homeostasis. It has recently been demonstrated that RANK-activated astrocytes release CCL20 and attract T cells to the central nervous system in a model of Multiple Sclerosis and that transgenic RANK expression in the skin promotes aberrant epithelial cell proliferation and is sufficient to induce ectopic formation of tertiary lymphoid structures (TLS). Ductal epithelial cells (SGEs) have been implicated in Sjogren’s Syndrome (SS) pathogenesis where they mediate immune recruitment by expression of pro-inflammatory chemokines and support the formation of pre-malignant myoepithelial lesions. Objectives To address the role of RANK-RANKL interaction in primary (p) SS. Methods A combination of human and mouse studies were used to explore the RANK-RANKL interaction in pSS. Salivary glands (SGs) and saliva samples from patients recruited in the OASIS cohort (University of Birmingham) were studied to evaluate this pathway in human disease. Consecutive stimulated saliva samples (n=69) were analysed using Proseek Multiplex INF96 × 96, covering 92 unique inflammation-related protein biomarkers. Taking advantage of a viral induced model of pSS we studied the effect of this pathway with a RANKL blocking antibody and by inducing gain of function with direct cannulation in the salivary glands of recombinant RANKL. Murine SGs were studied by immunofluorescence, flow cytometry and qPCR on total tissue and sorted cells. Results Fourteen proteins in saliva were significantly separated between pSS and sicca controls, and elevated levels of just two proteins, RANKL and TNFβ, could classify pSS or sicca with 75% accuracy. Levels of salivary RANKL and CCL20 were strongly correlated (r=0.6; p Conclusions In vivo RANK/RANKL interaction mediates recruitment of activated T cells that are skewed toward a Th2 phenotype. These, in turn, will favour the establishment of TLS in the SG. Those data were confirmed in human pSS, where expression of RANK is found in inflamed epithelium and RANKL detection in saliva is able to differentiate patients with pSS from sicca controls, thus candidates this pathway both for drug targeting and patient stratification. Disclosure of interest None declared

The role of European fish farms in the spread of antimicrobial-resistance in the environment and food chain, as well as possible sources of their contamination by clinically relevant antimicrobial-resistance bacteria is scarcely known. This study aimed to assess the contribution of Portuguese rural trout farms on dispersion of Enterococcus with antimicrobial-resistance and putative virulence genes in the environment and food chain, as well as to identify farms contamination sources. We also assessed the presence of Enterococcus with low-levels of antimicrobial-resistance using epidemiological cut-offs (ECOFFs). Enterococcus spp. (n = 391) from water/sediment recovered upstream, within and downstream trout tanks, feed, trout (2 aquacultures; no antibiotic use) and marketed trout (8 supermarkets) showed variable resistance to tetracycline, erythromycin, ciprofloxacin, chloramphenicol, quinupristin-dalfopristin, nitrofurantoin or aminoglycosides. Antimicrobial-resistance rates were similar among upstream, within and downstream trout tank samples (P > 0.05), positioning water-supplying aquacultures as a source of multidrug-resistant (MDR) strains. Nevertheless, predominance of MDR E. faecium in feed, trout tanks and trout comparing to upstream samples, suggests feed as an additional aquaculture contamination source. The observation of E. faecium and E. faecalis susceptible to ampicillin and gentamicin by clinical breakpoints but with low-levels of resistance to those antimicrobials by ECOFFs breakpoints is of concern, as they might evolve throughout secondary genetic events to resistance levels with human clinical impact. Multiple MDR clones carrying copper tolerance (tcrB/cueO), putative virulence or other genes often associated with clinical strains (e.g. E. faecium with IS16/ptsD/sgrA) were observed, some in distinct samples (e.g. upstream and within trout tanks). They included major human and animal Enterococcus lineages, suggesting human and non-aquatic animal origins. The results highlight the need to define the maximum acceptance level of antimicrobial-resistance genes/bacteria to assess water quality and to monitor antimicrobial-resistance strains on feed, essential requirements to maintain a sustainable aquaculture production. (c) 2018 Elsevier B.V.

Simpler, quick and low-cost methods for routine Salmonella enterica typing are required for epidemiologic surveillance of this important zoonotic pathogen. In this study, using a comprehensive isolate collection, we investigated the potential of Fourier transform infrared spectroscopy (FTIRS) to discriminate the most clinically-relevant serogroups and serotypes of non-typhoid Salmonella. Moreover, the role of O-units composition on the FTIRS Salmonella discrimination was also explored. S. enterica isolates (n = 325; 2002–2015; different sources and countries), of 57 serotypes and 15 serogroups [including the most frequent ones, B-n = 122; C-n = 108; D-n = 43 and E-n = 33)] were analysed by FTIRS. Infrared spectra were analysed by Partial Least Square Discriminant Analysis (PLSDA) and/or Principal Component Analysis (PCA). The polysaccharides region provided the spectral sharpest differences being used in the subsequent Salmonella typing. Serogroups (B, C, D and E) discrimination was achieved with high accuracy (99.6% of correct assignments; PLSDA model). Differences in the O-unit structures composition of those serogroups are likely justifying the discrimination achieved. Other serogroups (G, H, K, L, M, N, O, T, U, Y, Z) were correctly predicted as not belonging to serogroups B, C, D nor E, except for 3 isolates of serogroups H (S. Sundsvall, n = 1) and K (S. Cerro, n = 2). In fact, O-unit structure of serogroup H and K shows some similarity with sub-serogroup C1 with the remaining serogroups presenting marked differences in this cellular component. The sub-serogroups discrimination was successfully achieved for C1, C2 and C3 (using PCA), and for E1-E2-E3 and E4 (by PLSDA). Appropriate serotype discrimination was obtained for most of S. Rissen from the remaining C1 serotypes (91.5%-PLSDA), and S. Enteritidis (D1) from the remaining D1/D2 serotypes (93.4%-PLSDA). The lack of available O-unit composition for particular serotypes prevents the elucidation of the role of this cellular component on the discrimination at serotype level obtained. FTIRS was able to discriminate relevant serogroups (B, C, D and E), sub-serogroups (C1, C2 and C3; E1-E2-E3 and E4) and particular important serotypes (S. Enteritidis, S. Rissen and S. Senftenberg). Further studies on O-antigen composition would clarify the fundaments of discrimination obtained by FTIRS.

The impact of European aquaculture, namely trout farms, in the spread of antibiotic resistance and/or zoonotic pathogens has been scarcely addressed. Moreover, aquaculture contamination sources and bacterial dissemination routes have been barely explored. In this study, we assessed the contribution of Portuguese land-based intensive rainbow trout farms and retailed market trout to the spread of Salmonella and bacteria carrying clinically-relevant antibiotic resistance genes (ARGs) as well as inflow water and feed as possible sources of those contaminants. Cultural and molecular methods were used to analyse 53 fish farm samples (upstream/downstream water and sediments, tanks and trout) and 25 marketed trout. Plasmid-mediated quinolone resistance (PMQR) genes were found in 21% (n = 11/53) of samples (water/sediment/feed/trout), from all collection points (upstream/within/downstream tanks) and seasons, as well as in 12% (n = 3/25) of marketed trout (3 supermarkets). PMQR genes (qnrS1-S2-S3, qnrB7-B19, qnrD1, oqxAB) were detected in Enterobacteriaceae or Aeromonas hydrophila. An E. coli strain producing extended-spectrum-beta-lactamase SHV-12 was detected in all sampled points of a fish farm. Salmonella (4 serotypes, including S. Newport-ST118) was detected in 26% (n = 14/53) of the samples from both farms (water/sediment upstream/within tanks). The clinically-relevant plasmid-mediated colistin resistance mcr genes were not detected. However, colistin resistant S. Abony with new mutations in the chromosomal pmrA and pmrB genes was observed. Identical Salmonella and SHV-12-producing E. coli strains (by PFGE/MLST) in water upstream and within trout tanks points to inflow-water of trout farms as an important source of pathogenic bacteria and ARG contamination. These results highlight the need to define microbiological standards for water supplying fish farms in the EU and to establish surveillance and control strategies to limit bacterial transmission associated with this fastest growing food sector worldwide. © 2018 Elsevier B.V.

Extended-spectrum cephalosporin (ESC)-resistant Salmonella have been described at a low level in the EU, nevertheless the increasing importation of poultry meat could be an important source of epidemic strains carrying ESC resistance genes. This study evaluated ESC resistance and its genetic platform among Salmonella isolates from poultry meat products imported into Portugal as well as clonal relatedness of the isolates. All Salmonella isolates recovered from samples of fresh meat destined for import into the EU in the scope of Portuguese official border control (2014-2015) were studied. Antibiotic susceptibility and β-lactamase production was determined by disk diffusion/microdilution. Molecular studies included detection of genes encoding acquired AmpC and extended-spectrum β-lactamases, plasmid-mediated quinolone resistance and other antibiotic resistance genes by PCR/sequencing, and clonality by MLST and XbaI-PFGE. Plasmid characterisation was assessed by conjugation assays, replicon typing (PCR-PBRT/pMLST) and hybridisation experiments (I-CeuI/S1-PFGE nuclease). Isolates belonged to Salmonella Heidelberg (n = 6; ST15/eBG26) and Salmonella Minnesota (n = 1; ST548/eBG77) and presented multidrug-resistant profiles, including to ESCs and/or fluoroquinolones. All but one carried blaCMY-2, located on two epidemic plasmids, IncA/C (ST2, n = 5) or transferable IncI1 (ST12, n = 1). Salmonella Heidelberg was associated with five PFGE types, including one similar to an American epidemic clone. This study reveals imported poultry products as a source of uncommon and/or invasive ESC-resistant Salmonella strains in the EU. The increase of clinically relevant poultry-related serotypes in Europe must be taken into account in the current monitoring of antibiotic resistance trends and in re-evaluation of food regulations. This work was supported by FCT/MEC (Fundação para a Ciência e a Tecnologia/Ministério da Educação e Ciência) through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 [grant no. UID/MULTI/04378/2013 – POCI/01/ 0145/FERDER/007728]; the project NORTE-01-0145-FEDER -000011 – Qualidade e Segurança Alimentar—uma abordagem (nano)tecnológica. JC is supported by a PhD fellowship from FCT [grant no. SFRH/BD/93091/2013] and JM is supported by a postdoctoral fellowship from project NORTE-01-0145-FEDER-000024. The authors are greatly indebted to all of the funding sources. info:eu-repo/semantics/publishedVersion

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A expansão romana pelo Mediterrâneo Oriental foi um processo complexo, envolvendo diplomacia, alianças e conflitos com antigos reinos estabelecidos em uma região de enorme diversidade cultural e resultou na gradual absorção desses territórios sob o controle direto de Roma. O Egito ptolomaico foi o último grande reino helenístico conquistado e seu processo de anexação foi adiado em virtude de seu valor diferenciado para Roma, em vários aspectos. A literatura greco-romana pontua alguns momentos da vivência da dinastia ptolomaica, principalmente as ocasiões de contatos e tensões com Roma. A intenção desse artigo é avaliar como tais fontes representam a sucessão dos Ptolomeus, de forma a ressaltar o “caos” interno da dinastia, ao mesmo tempo em que justificariam a necessidade da instalação da “ordem” romana na região. The Roman expansion through the Eastern Mediterranean was a complex process involving diplomacy, alliances and conflicts with ancient kingdoms established in a region of enormous cultural diversity and resulted in the gradual absorption of these territories to direct control by Rome. Ptolemaic Egypt was the last huge Hellenistic kingdom to be conquered and its process of annexation was postponed for its differentiated value to Rome in several aspects. The Greco-Roman literature point to some moments of the dynasty’s life, especially the occasions of contacts and tensions with Rome. The intention of this article is to evaluate how these sources represent the Ptolemaic succession in order to highlight the internal “chaos” of the dynasty while justifying the need for the Roman “order” in the territory.

The objective of this paper is to evaluate the lightning overvoltages that appear on a conventional 69 kV line, a compact overhead distribution line (spacer cable system) rated 11.4 kV and a low voltage line rated 220 V sharing the same tower. The simulations were performed using the software ATP (Alternative Transient Program) considering lightning strikes at the top of the tower, the mid span of the ground wire and one of the phases of the high voltage line.

Pulmonary thromboembolism and deep venous thrombosis occur in pediatric age, with unknown incidence, morbidity and mortality. Our aim is to review the epidemiology, clinical presentation, complementary diagnostic tests and prognosis of patients with pulmonary thromboembolism and deep venous thrombosis.Retrospective, descriptive and analytical study of pediatric patients admitted to a Level II hospital for pulmonary thromboembolism and deep venous thrombosis, between 2000 and 2014. Demographic characteristics, clinical history, comorbidities and risk factors were studied.Eleven patients (n = 7 pulmonary thromboembolism, n = 5 deep venous thrombosis, n = 1 both), 64% females and with 16 years old average, were admitted. All patients with pulmonary thromboembolism presented symptoms of chest pain and/or dyspnea, 25% syncope/palpitations and 25% fever. All patients with deep venous thrombosis reported localized pain at the site of obstruction, 83% edema/cyanosis of the affected limb and 17% fever. The study of positive thrombophilia was the most frequent risk factor in both entities. The mean value of D-dimers was 3252 ug/dL and 2660 ug/dL in pulmonary thromboembolism and deep venous thrombosis, respectively. All patients started anticoagulation, three required intensive care, two had sequelae and one died.All patients had at least one risk factor, and hereditary hypercoagulability was most commonly established.The increased incidence in the pediatric population described in some studies can be attributed to an increased awareness of this pathology, medical advances and increasing survival of chronic diseases. There is a lack of evidence-based recommendations identifying patients at risk of thrombosis so that decisions can be made carefully, balancing the risk and benefit in each case.Introdução: O tromboembolismo pulmonar e trombose venosa profunda ocorrem em idade pediátrica com incidência, morbilidade e mortalidade desconhecidas. O objetivo foi rever epidemiologia, apresentação clínica, exames complementares de diagnóstico e prognóstico de doentes com tromboembolismo pulmonar e trombose venosa profunda.Material e Métodos: Estudo retrospetivo, descritivo e analítico de doentes pediátricos internados num hospital de nível II por tromboembolismo pulmonar e trombose venosa profunda, entre 2000 e 2014. Estudaram-se características demográficas, história clínica, comorbilidades e fatores de risco.Resultados: Foram internados 11 doentes (sete com tromboembolismo pulmonar, cinco com trombose venosa profunda e um com ambos), 64% do género feminino e idade média de 16 anos. Todos os doentes com tromboembolismo pulmonar referiam toracalgia/dispneia, 25% síncope/palpitações e 25% febre. Todos os doentes com trombose venosa profunda referiam dor no local da obstrução, 83% edema/cianose do membro afetado e 17% febre. O estudo da trombofilia positivo foi o fator de risco mais frequente nas duas entidades. O valor médio dos D-dímeros foi 3252 ug/L e 2660 ug/L no tromboembolismo pulmonar e trombose venosa profunda, respetivamente. Todos os doentes iniciaram anticoagulação, três necessitaram de cuidados intensivos, três apresentaram sequelas e houve um óbito.Discussão: Todos os doentes tinham pelo menos um fator de risco associado e as condições de hipercoagulabilidade herdadas foram o fator de risco mais frequentemente encontrado nos nossos adolescentes.Conclusão: O aumento da incidência na população pediátrica descrito na literatura pode ser atribuído à crescente sensibilização para esta patologia, aos avanços médicos e aumento da sobrevida de doenças crónicas. Escasseiam recomendações baseadas na evidência que identifiquem os doentes com risco de trombose, para que as decisões possam ser tomadas de forma cuidadosa, equilibrando o risco e benefício em cada caso.

Tese de Doutoramento em Estudos da Criança (Especialidade em Sociologia da Infância), The main goals of this thesis were to study children participation in schools, through their bodily expression and free movement, subverting the discipline of the traditional school inducing passivity. From references mainly sustained by Childhood Studies, with incursions through the sciences that deal with the children’s bodies and its relations with school and learning, this research was a case study using mainly qualitative data, such as participant observation, visual methods and interviews. It was done over three years following a project of learning through movement called THE BODY AT SCHOOL, applied by an artistic association (c.e.m) at a primary school in Mouraria, Lisbon. The results showed that the children learnt curricular, pedagogic, artistic and social contents through the project. The positive feelings the children associated with the project’s sessions helped them remember what they had learnt, enhanced episodic learning and memory in what, we argue, was the creation of positive “somatic markers”. The network of care and affection created through the project between the children and the adults, inside and outside the school and the fact that the sessions were based on open-ended exercises increased the children’s confidence and pleasure in learning, which was particularly important for the children with learning or language difficulties. Children participation at school through the project was especially developed when they taught a class to a group of adults, when they made a book and in their participation in the project’s sessions outside the school, in the ruins of an abandoned building in Mouraria. These “collaboratively emergent” sessions developed in a shared decision-making process that also included the children’s families, former students from the school, the teachers and some inhabitants of Mouraria. The work with the neighbourhood managed to combine the children’s “emplaced knowledge” with “spatial knowledge” and the children’s collective actions with the adults transformed ruins that were not originally designed for children, into a “children’s space”. The use of the arts and the fact that the sessions developed in a sequence of “expansive cycles” (defined by activity theory) permitted the children to manage their own bodies and, in particular, we described how one child achieved a “moment of epiphany” (or “priming event”) that transformed her relationship with the others. A moment that, we argue, would not have happened in a regular class and, in that sense, the project created an approximation to an “ideal speech situation”. We exemplified a moment where the project allowed for the translation of children’s notions of their “incarnated bodies” into the adults’ idea of a “somatic body”. Through these sessions, the children’s “body as project” was potentiated and the children’s formal, informal and non-formal knowledge was included and valued, and we argue that helped the children learn. The “ethical symmetry” between adults and children, developed by the children working together with the adults, created a flexible “network of relationships” in which most children recognised the importance of their participation in the shaping of the sessions, but they also saw this symmetry as being short-lived and limited to the project. The sustainability of the project and its promoting association were threatened by a lack of institutional support. Hence, the research could not help but to focus - despite its original intentions did not predict it - on the difficulty of transformation at school, an important question in the contemporary educational agenda, but that is limited or severely restricted by the lack of political structures capable of sustaining change in education. That is one of this thesis’ dimensions: its undeniable political character, regarding a defence of the public school and its change for the promotion of learning for the children, especially of the poorer children and/or those who do not correspond to the dominant culture., A tese “'Aprendemos de forma diferente. Em vez de estarmos a ouvir, fazíamos.’ Corpo e movimento na educação básica” tem por principais objectivos estudar a participação das crianças em escolas, através da expressão corporal e do seu movimento livre, invertendo a disciplina da escola tradicional indutora da passividade. A partir de um referencial principalmente sustentado nos Estudos da Criança, com incursões pelas ciências que se ocupam do corpo da criança e das suas relações com a escolaridade e a aprendizagem, a investigação da tese assenta num estudo de caso, recorrendo essencialmente a métodos e técnicas qualitativos, como observação participativa, materiais visuais e entrevistas. Feito ao longo de três anos, acompanhou um projecto de aprendizagem pelo movimento chamado O CORPO NA ESCOLA, de uma associação artística (c.e.m) numa escola primária pública da Mouraria, Lisboa. Os resultados mostraram que as crianças aprenderam conteúdos curriculares, pedagógicos, artísticos e sociais através do projecto. Os sentimentos positivos que as crianças associaram às sessões ajudaram-nas a lembrar-se daquilo que tinham aprendido, melhoraram a sua memória episódica e estabeleceram a criação de “marcadores somáticos” positivos. A rede de cuidado e afeto criada através do projecto entre as crianças e os adultos dentro e fora da escola aumentou a confiança das crianças e o seu prazer na aprendizagem, algo particularmente importante para as crianças com dificuldades de aprendizagem ou no domínio da língua, designadamente crianças migrantes. A participação das crianças na escola foi especialmente trabalhada quando ensinaram uma aula a um grupo de adultos, fizeram um livro e participaram nas sessões do projecto fora da escola, nas ruínas de um edifício abandonado. Estas sessões “colaborativamente emergentes”, desenvolveram-se num processo de tomada de decisões partilhado que também incluiu as famílias das crianças, antigos estudantes da escola, as professoras e alguns habitantes do bairro. Foi possível combinar o “conhecimento territorializado” com o “conhecimento espacial” e as acções coletivas das crianças com os adultos transformaram as ruínas, que não lhes eram destinadas, num “espaço de crianças”. O uso das artes e o facto de as sessões se terem desenvolvido numa sequência de “ciclos expansivos” permitiram que as crianças gerissem os seus corpos, gerando, em particular, pelo menos um “momento de epifania” (ou “acontecimento primordial”) capaz de transformar a relação das criança com os outros. Um momento que, argumentamos, não teria acontecido numa aula normal e nesse sentido o projecto criou uma aproximação à “situação de discurso ideal”. Exemplificámos esta situação com um momento em que o projecto permitiu uma tradução das noções das crianças de “encarnated body” na ideia adulta de “corpo somático”. Através das sessões, o “corpo como projecto” das crianças foi potenciado e o conhecimento formal, informal e não formal das crianças foi incluído e valorizado o que argumentamos que ajudou na sua aprendizagem. A “simetria ética” entre os adultos e as crianças, desenvolvida pelo trabalho conjunto, bem como pela investigação colaborativa realizada, criou uma “rede de relações” flexível, na qual a maioria das crianças reconheceu a importância da sua participação na criação das sessões, mas também viu esta simetria como breve no tempo e limitada ao projecto. A sustentabilidade do projeto de intervenção e da sua organização promotora viu-se ameaçada pela ausência de suporte institucional. A investigação, por isso mesmo, não pode deixar de se debruçar - malgrado as suas intenções iniciais não o preverem - sobre a dificuldade da transformação institucional da escola, questão maior na agenda educativa contemporânea, mas que se encontra limitada ou severamente restringida pela ausência de políticas estruturantes capazes de suportar duravelmente a mudança educativa. Encontra-se aqui uma das dimensões da tese: o seu caráter inquestionavelmente político, no sentido da defesa da escola publica e da sua mudança para a promoção das aprendizagens significativa das crianças, especialmente das crianças mais pobres e/ou afastadas da cultura dominante.

Companies like Airbnb, Amazon or Craigslist have challenged the traditional business models and are altering the way people have access to goods and services. This article explores why the concept of online platform is adequate to analyse this new reality from a contractual point of view. It then challenges the idea that all companies where the product or service is supplied by what appears to be a third-party are online platforms, using the example of Uber. Finally, it provides a brief overview at the current EU framework to provide a reflection on how a regime for online platforms could look like.Keywords: sharing economy, online platforms, Uber.Resumen: Empresas como Airbnb, Amazon o Craigslist han desafiado los modelos de negocio tradicionales y están cambiando la forma en que las personas tienen acceso a los bienes y servicios. Este artículo explora por qué el concepto de plataforma en línea es adecuado para analizar esta nueva realidad desde un punto de vista contractual. A continuación, se cuestiona la idea de que todas las empresas en las que el producto o servicio es proporcionado por lo que parece ser un tercero son plataformas en línea, utilizando el ejemplo de Uber. Por último, se aporta una breve visión general del marco actual de la UE para reflexionar sobre cómo podría ser un régimen para las plataformas en línea.Palabras clave: economía colaborativa, plataformas en línea, Uber.

The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein α (C/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop for E2F1, C/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C/EBPα-p42, and in normal granulocyte/macrophage progenitor cells, we detect C/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle or Trib2 knockdown resulted in a block in AML cell proliferation. Our work proposes a novel paradigm whereby E2F1 plays a key role in the regulation of Trib2 expression important for AML cell proliferation control. Importantly, we identify the contribution of dysregulated C/EBPα and E2F1 to elevated Trib2 expression and leukemic cell survival, which likely contributes to the initiation and maintenance of AML and may have significant implications for normal and malignant hematopoiesis.