Search

SUMMARYSeroprevalence of Toxoplasma gondii infection and associated risk factors were investigated in 401 women of childbearing age from the North of Portugal. Of the 98 (24·4%) seropositive women, 92 (93·9%) only had immunoglobulin (Ig)G, two (2·0%) only had IgM, and four (4·1%) others had both IgG and IgM. Risk factors for T. gondii infection in women were: engaging in soil-related activities without gloves [odds ratio (OR) 8·4], consumption of unwashed raw vegetables or fruit (OR 7·6), and consumption of smoked or cured (non-cooked) processed pork products (OR 2·5). Most women of childbearing age from the North Portugal are susceptible to primary infection with T. gondii and, therefore, the risk of congenital toxoplasmosis remains high. [ABSTRACT FROM AUTHOR]

Thelazia callipaeda , also known as the "oriental eye worm", is a zoonotic parasitic nematode with a wide range of hosts, particularly wild and domestic carnivores, but also lagomorphs and humans. Currently, ocular thelaziosis presents an expanding distribution range throughout Europe, including Portugal. This study provides an update on T . callipaeda infection reports (30 studies) in European wildlife comprising 54 host-locality records in 10 host species from nine European countries. The prevalence of T . callipaeda varied widely, with ranges from around 1% in red foxes and European hares to almost 50% in red foxes. The lowest mean intensity was 2.7 nematodes/host in European wildcats and the highest was 38.0 nematodes/host in wolves. In addition, a massive infection with T. callipaeda in a juvenile male red fox from eastern-central Portugal is also described, representing the southernmost report in a wild animal in this country. A total of 188 nematodes (139 females and 49 males) were collected from both eyes and were submitted to morphological and molecular characterization. Collected nematodes were morphologically identified as T . callipaeda . Given the endemicity of T . callipaeda in eastern-central Portugal, surveillance system should be implemented to monitor its presence among wild and domestic animals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Given their role as Guest Editors, Ana Cláudia Coelho, Ana Patrícia Lopes and Luís Cardoso had no involvement in the peer review of this article and have no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Professor Aneta Kostadinova (Editor-in-Chief)., (© 2024 Published by Elsevier B.V.)

Background: Since the seminal publication of the TCGA consortium in 2013, the molecular classification of endometrial cancer has been widely accepted as a new and powerful tool to better understand the natural history of this malignancy. Adoption of routine molecular classification around the world has been limited. We sought to demonstrate our initial experience in incorporating the four molecular subtypes for endometrioid carcinomas., Methods: This was a retrospective analysis at a single center in Portugal. Molecular classification was determined using immunohistochemical staining for MMR and p53 and Sanger Sequencing to determine POLE mutation status as per published PROMISE method. Descriptive statistics were reported., Results: 20 patients with endometrioid histology were included. Median age of the cohort was 64 years (range 45-76). Median Body Mass Index (kg/m 2 ) was 29.81 (range 21.3-43.1). In terms of tumor grading, 16 (80%) of the endometrial carcinomas of the cohort were low-grade (either grade 1 or grade 2). 16 (80%) of the cases were FIGO stage I. Regarding the molecular classification the tumors were classified as: MMRd [n = 6 (30%)]; p53 abn [n = 2 (10%)]; NSMP (n = 10 (50%)), POLE ultramut [n = 2 (10%)]., Conclusion: Despite the small sample size, we were able to show that molecular classification is feasible. To our knowledge this is the first cohort of endometroid endometrial carcinomas fully characterized according to the TCGA classification in Portugal, from one single center., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Casanova, da Costa, Lopes, Catarino, Nave, Sousa and Lima.)

Human leukocyte antigen (HLA) restriction of conventional T-cell targeting introduces complexity in generating T-cell therapy strategies for patients with cancer with diverse HLA-backgrounds. A subpopulation of atypical, major histocompatibility complex-I related protein 1 (MR1)-restricted T-cells, distinctive from mucosal-associated invariant T-cells (MAITs), was recently identified recognizing currently unidentified MR1-presented cancer-specific metabolites. It is hypothesized that the MC.7.G5 MR1T-clone has potential as a pan-cancer, pan-population T-cell immunotherapy approach. These cells are irresponsive to healthy tissue while conferring T-cell receptor(TCR) dependent, HLA-independent cytotoxicity to a wide range of adult cancers. Studies so far are limited to adult malignancies. Here, we investigated the potential of MR1-targeting cellular therapy strategies in pediatric cancer. Bulk RNA sequencing data of primary pediatric tumors were analyzed to assess MR1 expression. In vitro pediatric tumor models were subsequently screened to evaluate their susceptibility to engineered MC.7.G5 TCR-expressing T-cells. Targeting capacity was correlated with qPCR-based MR1 mRNA and protein overexpression. RNA expression of MR1 in primary pediatric tumors varied widely within and between tumor entities. Notably, embryonal tumors exhibited significantly lower MR1 expression than other pediatric tumors. In line with this, most screened embryonal tumors displayed resistance to MR1T-targeting in vitro MR1T susceptibility was observed particularly in pediatric leukemia and diffuse midline glioma models. This study demonstrates potential of MC.7.G5 MR1T-cell immunotherapy in pediatric leukemias and diffuse midline glioma, while activity against embryonal tumors was limited. The dismal prognosis associated with relapsed/refractory leukemias and high-grade brain tumors highlights the promise to improve survival rates of children with these cancers., Competing Interests: Competing interests: ZS and JK are inventors on different patents for γδ TCR sequences, recognition mechanisms and isolation strategies. JK is scientific cofounder and shareholder of Gadeta (www.gadeta.nl). The remaining authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Between 2016 and 2023, a cross-sectional study was conducted in the central region of Portugal in order to better understand the epidemiology and public health risks resulting from the handling and consumption of game animals infected with Brucella spp. The seroprevalence and risk factors for Brucella spp. seropositivity were evaluated. Antibodies against Brucella spp. were determined using a commercial enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. Results showed that in the 650 serum samples collected from red deer ( n = 298) and wild boars ( n = 352) in Portugal, 21.7% ( n = 141; 95% CI: 18.6-25.1%) tested positive. Wild boar had a significantly higher prevalence (35.5%; 95% CI: 30.5-40.8%) than red deer (5.4%, 95% CI: 3.1-8.6%; p ≤ 0.001). Risk factors for seropositivity were investigated using multivariable logistic regression models. The odds of being seropositive was 8.39 (95% CI: 4.75-14.84; p ≤ 0.001) times higher in wild boar than in red deer. Correlations between sex, age, body condition, and seropositivity could not be observed. The higher seroprevalence in wild boar suggests that this species may primarily contribute to the Brucella spp. ecology in central Portugal.

A cross-sectional study was conducted to gain insight into the epidemiology of canine ehrlichiosis and rickettsiosis in northern Portugal. Specific IgG antibodies to Ehrlichia canis were analysed using a commercial enzyme-linked immunosorbent assay (ELISA), and antibodies to Rickettsia conorii were analysed using a commercial indirect immunofluorescence antibody test (IFAT). A total of 113 dogs from two different shelters were sampled, and seroprevalence values of 0.9% (95% confidence (CI): 0.2-4.8%) for E. canis and 9.7 (95% CI: 5.5-16.6%) for R. conorii were found. Multiple logistic regression investigated risk factors for seropositivity. The odds ratios (ORs) of R. conorii seropositivity were higher for female dogs (OR = 6.429; 95% CI: 1.201-34.407). Dogs seropositive for co-infection ( E. canis + R. conorii ) were more frequently observed among females (OR = 7.606; CI 95%: 1.478-39.132) and in Shelter 2 (OR = 18.229; 95% CI: 2.190-151.756). These findings show that shelter dogs in northern Portugal are exposed to E. canis and R. conorii , which can affect both canines and humans. It is imperative to adopt a One Health approach to educate the public about the hazards of canine zoonoses and develop legislation and procedures to control their spread and preserve public health.

Metal contamination poses a significant threat to elasmobranchs, underscoring the need for targeted conservation approaches. The critically endangered Brazilian guitarfish, Pseudobatos horkelii, confronts an array of challenges, notably overexploitation, putting its survival at risk. Our study investigated the potential toxicity arising from arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb) contamination across various adult guitarfish tissues from southeastern Brazil. Serological stress indicators, nutritional metabolites, and creatinine, an organ function marker, were also assessed, and Selenium (Se) levels were also investigated for possible protective effects. Our investigation unveiled significant correlations between metal concentrations and the determined physiological markers, shedding light on potential adverse effects. Remarkably, six correlations were indicative of how Hg and Pb negatively impact hepatic metabolite assimilation, while As was shown to influence renal phosphorus dynamics, Cd to affect rectal gland phosphorus regulation, and Pb to influence creatinine production in muscle tissue. Furthermore, Se demonstrated protective properties against Cd, Hg, and Pb, suggesting a role in alleviating the toxicity of these elements. Despite probable protective Se influences, the detected elemental interactions still suggest potential for organ impairment. These findings gain heightened significance within the context of the cumulative stressors faced by the Brazilian guitarfish, with metal contamination exhibiting the capacity to erode this species resilience against both anthropogenic and environmental pressures, thereby disrupting systemic equilibrium and jeopardizing wild populations. By investigating the intricate balance between metal accumulation and physiological consequences, our study contributes with crucial insights into potential conservation strategy formulations towards pollution for this critically endangered elasmobranch species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Hepatitis E virus (HEV) is a zoonotic foodborne virus with an annual infection prevalence of 20 million human cases, which seriously affects public health and economic development in both developed and developing countries. To better understand the epidemiology of HEV in Central Portugal, a cross-sectional study was conducted from 2016 to 2023 with sera samples from wild ungulates. The seroprevalence and risk factors for HEV seropositivity were evaluated in the present study. Specifically, antibodies against HEV were determined by a commercial enzyme-linked immune-sorbent assay (ELISA). Our results show that in the 650 sera samples collected from 298 wild red deer and 352 wild boars in Portugal, 9.1% red deer and 1.7% wild boar were positive for antibodies to HEV. Regarding age, the seropositivity in juvenile wild ungulates was 1.3%, whereas it was 7.2% in adults. Logistic regression models investigated risk factors for seropositivity. The odds of being seropositive was 3.6 times higher in adults than in juveniles, and the risk was 4.2 times higher in red deer than in wild boar. Both wild ungulate species were exposed to HEV. The higher seroprevalence in red deer suggests that this species may make a major contribution to the ecology of HEV in Central Portugal. Further research is needed to understand how wildlife affects the epidemiology of HEV infections in Portugal.

Objectives: To assess to what extent leflunomide (LEF) and hydroxychloroquine (HCQ) therapy in patients with primary Sjögren's syndrome (RepurpSS-I) targets type I IFN-associated responses and to study the potential of several interferon associated RNA-based and protein-based biomarkers to predict and monitor treatment., Methods: In 21 patients treated with LEF/HCQ and 8 patients treated with placebo, blood was drawn at baseline, 8, 16 and 24 weeks. IFN-signatures based on RNA expression of five IFN-associated genes were quantified in circulating mononuclear cells and in whole blood. MxA protein levels were measured in whole blood, and protein levels of CXCL10 and Galectin-9 were quantified in serum. Differences between responders and non-responders were assessed and receiver operating characteristic analysis was used to determine the capacity of baseline expression and early changes (after 8 weeks of treatment) in biomarkers to predict treatment response at the clinical endpoint., Results: IFN-signatures in peripheral blood mononuclear cell and whole blood decreased after 24 weeks of LEF/HCQ treatment, however, changes in IFN signatures only poorly correlated with changes in disease activity. In contrast to baseline IFN signatures, baseline protein concentrations of galectin-9 and decreases in circulating MxA and Galectin-9 were robustly associated with clinical response. Early changes in serum Galectin-9 best predicted clinical response at 24 weeks (area under the curve 0.90)., Conclusions: LEF/HCQ combination therapy targets type-I IFN-associated proteins that are associated with strongly decreased B cell hyperactivity and disease activity. IFN-associated Galectin-9 is a promising biomarker for treatment prediction and monitoring in pSS patients treated with LEF/HCQ., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The current climate change scenario caused by anthropogenic activities has resulted in novel environmental pressures, increasing the occurrence and severity of fungal infections in the marine environment. Research on fungi in several taxonomic groups is widespread although not the case for elasmobranchs (sharks and rays). In this context, the aim of the present study was to screen the oral fungal microbiota present in artisanally captured Rioraja agassizii , a batoid that, although endangered, is highly fished and consumed worldwide. Oropharyngeal samples were obtained by swabbing and the samples were investigated using morphological and phenotypic methods by streaking on Sabouraud Dextrose Agar (SDA) and subculturing onto CHROMagar Candida (BD Difco) and CHROMagar Candida Plus (CHROMagar TM ), as well as molecular techniques by amplification of the ITS1-5.8S-ITS2 ribosomal DNA region and a MALDI-TOF MS assessment. The findings indicated the presence of Candida parapsilosis (seven isolates), Candida duobushaemulonii (one isolate) and Rhodotorula mucilaginosa (three isolates), several of these reported for the first time in Rioraja agassizii . In addition, a 100% agreement between the MALDI-TOF results and partial ITS region sequencing was noted, demonstrating that the MALDI-TOF MS is a rapid and effective alternative for yeast identification in Rioraja agassizii isolates and potentially in other elasmobranch species. These findings highlight the need for further research to determine the potential impact on elasmobranch health, ecology, and commercial fisheries. Furthermore, this research is paramount in a One Health framework and may be employed to predict elasmobranch responses to an evolving ocean, keep healthy populations in check, monitor species, and assess the public health consequences of consuming these species.

The long-eared owl ( Asio otus ) is a medium-sized owl species that is well-distributed in almost all of the territories in Portugal. Nematodes were found in the oral cavity of a long-eared owl ( A. otus ) admitted to CRASSA (Wildlife Rehabilitation Centre of Santo André). During a physical exam and stabilization of the bird, five nematodes were collected. The worms were examined and measured under light microscopy, and photos were taken. After a morphological analysis was conducted, all the nematodes (five females) were identified as Synhimantus ( Synhimantus ) laticeps . Two specimens were subjected to molecular analysis, which confirmed the result. This study provides a combined morphological and genetic approach to S. laticeps . To the authors' best knowledge, this is the first report including genetic sequencing of S. laticeps in a long-eared owl ( A. otus ) from Portugal.

Most cancer-related deaths are due to metastases. Systemic factors, such as lipid-enriched environments [as low-density lipoprotein (LDL)-cholesterol], favor breast cancer, including triple-negative breast cancer (TNBC) metastasis formation. Mitochondria metabolism impacts TNBC invasive behavior but its involvement in a lipid-enriched setting is undisclosed. Here we show that LDL increases lipid droplets, induces CD36 and augments TNBC cells migration and invasion in vivo and in vitro . LDL induces higher mitochondrial mass and network spread in migrating cells, in an actin remodeling-dependent manner, and transcriptomic and energetic analyses revealed that LDL renders TNBC cells dependent on fatty acids (FA) usage for mitochondrial respiration. Indeed, engagement on FA transport into the mitochondria is required for LDL-induced migration and mitochondrial remodeling. Mechanistically, LDL treatment leads to mitochondrial long-chain fatty acid accumulation and increased reactive oxygen species (ROS) production. Importantly, CD36 or ROS blockade abolished LDL-induced cell migration and mitochondria metabolic adaptations. Our data suggest that LDL induces TNBC cells migration by reprogramming mitochondrial metabolism, revealing a new vulnerability in metastatic breast cancer., Significance: LDL induces breast cancer cell migration that relies on CD36 for mitochondrial metabolism and network remodeling, providing an antimetastatic metabolic strategy., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Q fever is caused by the pathogen Coxiella burnetii and is a zoonosis that naturally infects goats, sheep, and cats, but can also infect humans, birds, reptiles, or arthropods. A survey was conducted for the detection of antibodies against C. burnetii in a sample of 617 free-ranging wild ruminants, 358 wild boar ( Sus scrofa ) and 259 red deer ( Cervus elaphus ), in east-central Portugal during the 2016-2022 hunting seasons. Only adult animals were sampled in this study. Antibodies specific to C. burnetii were detected using a commercial enzyme-linked immunosorbent assay (ELISA; IDVet ® , Montpellier, France) according to the manufacturer's instructions. The seroprevalence of C. burnetii infection was 1.5% ( n = 9; 95% confidence interval [CI]: 0.7-2.8%). Antibodies against C. burnetii were detected in 4/358 wild boar (1.1%; 95% CI: CI: 0.3-2.8%) and 5/259 red deer (1.9%; 0.6-4.5%). Results of the present study indicate that antibodies against C. burnetii were present in wild boar and red deer in Portugal. These findings can help local health authorities to focus on the problem of C. burnetii in wildlife and facilitate the application of a One Health approach to its prevention and control.

Alpha 1-antitrypsin deficiency is an inherited autosomal codominant disorder, which predisposes patients to lung and/or liver disease. Even though it is considered rare, it is one of the most frequent genetic disorders worldwide, albeit remaining underdiagnosed. Several organizations and societies, including the Portuguese Society of Pulmonology have been elaborating guidelines and recommendations for the diagnosis and management of alpha 1-antitrypsin deficiency. Nevertheless, some important matters are yet to be included in those, mainly due to lack of robust scientific evidence, and continue to represent a point of discussion. This article reviews some important scientific publications and expresses the perspectives of a group of Portuguese experts regarding the management of alpha 1-antitrypsin deficiency, namely in terms of the pre and neonatal diagnosis, the impact of the COVID-19 pandemic, the validity of replacement therapy in lung transplant-receiving, and finally, alternative strategies of alpha 1-antitrypsin deficiency treatment to improve the patients' quality of life.

Objectives: Cytosolic DNA-sensing pathway stimulation prompts type I IFN (IFN-I) production, but its role in systemic IFN-I pathway activation in primary SS (pSS) is poorly studied. Here we investigate the responsiveness of pSS monocytes and plasmacytoid dendritic cells (pDCs) to stimulator of interferon genes (STING) activation in relation to systemic IFN-I pathway activation and compare this with SLE., Methods: Expression of DNA-sensing receptors cGAS, IFI16, ZBP-1 and DDX41, signalling molecules STING, TBK1 and IRF3, positive and negative STING regulators, and IFN-I-stimulated genes MxA, IFI44, IFI44L, IFIT1 and IFIT3 was analysed in whole blood, CD14+ monocytes, pDCs, and salivary glands by RT-PCR, monocyte RNA sequencing data, flow cytometry and immunohistochemical staining. Peripheral blood mononuclear cells (PBMCs) from pSS, SLE and healthy controls (HCs) were stimulated with STING agonist 2'3'-cGAMP. STING phosphorylation (pSTING) and intracellular IFNα were evaluated using flow cytometry., Results: STING activation induced a significantly higher proportion of IFNα-producing monocytes, but not pDCs, in both IFN-low and IFN-high pSS compared with HC PBMCs. Additionally, a trend towards more pSTING+ monocytes was observed in pSS and SLE, most pronounced in IFN-high patients. Positive STING regulators TRIM38, TRIM56, USP18 and SENP7 were significantly higher expression in pSS than HC monocytes, while the dual-function STING regulator RNF26 was downregulated in pSS monocytes. STING was expressed in mononuclear infiltrates and ductal epithelium in pSS salivary glands. STING stimulation induced pSTING and IFNα in pSS and SLE pDCs., Conclusion: pSS monocytes and pDCs are hyperresponsive to stimulation of the STING pathway, which was not restricted to patients with IFN-I pathway activation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Background: Trained immunity - or innate immune memory - can be described as the long-term reprogramming of innate immune cells towards a hyperresponsive state which involves intracellular metabolic changes. Trained immunity has been linked to atherosclerosis. A subgroup of patients with primary Sjögren's syndrome (pSS) exhibits systemic type I interferon (IFN) pathway activation, indicating innate immune hyperactivation. Here, we studied the link between type I IFNs and trained immunity in an in vitro monocytic cell model and peripheral blood mononuclear cells (PBMCs) from pSS patients., Methods: The training stimuli heat killed Candida albicans , muramyl dipeptide, IFNβ, and patient serum were added to THP-1 cells for 24 hours, after which the cells were washed, rested for 48 hours and subsequently re-stimulated with LPS, Pam3Cys, poly I:C, IFNβ or oxLDL for 4-24 hours. PBMCs from pSS patients and healthy controls were stimulated with LPS, Pam3Cys, poly I:C or IFNβ for 0.5-24 hours., Results: Training with IFNβ induced elevated production of pro-atherogenic cytokines IL-6, TNFα and CCL2 , differential cholesterol- and glycolysis-related gene expression, and increased glucose consumption and oxLDL uptake upon re-stimulation. Type I IFN production was increased in Candida albicans - and IFNβ-trained cells after LPS re-stimulation, but was reduced after poly I:C re-stimulation. Training with muramyl dipeptide and IFNβ, but not Candida albicans , affected the IFN-stimulated gene expression response to IFNβ re-stimulation. PBMCs from pSS patients consumed more glucose compared with healthy control PBMCs and tended to produce more TNFα and type I IFNs upon LPS stimulation, but less type I IFNs upon poly I:C stimulation., Conclusions: Type I IFN is a trainer inducing a trained immunity phenotype with pro-atherogenic properties in monocytes. Conversely, trained immunity also affects the production of type I IFNs and transcriptional response to type I IFN receptor re-stimulation. The phenotype of pSS PBMCs is consistent with trained immunity. This connection between type I IFN, trained immunity and cholesterol metabolism may have important implications for pSS and the pathogenesis of (subclinical) atherosclerosis in these patients., Competing Interests: LJ is scientific founder of Trained Therapeutix Discovery (TTxD). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Huijser, van Helden-Meeuwsen, Grashof, Tarn, Brkic, Huisman, Wahadat, van de Werken, Lopes, van Roon, van Daele, Kamphuis, Ng, Bekkering, Joosten, Dik and Versnel.)

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pet ownership is common in modern society. In Portugal, 38% and 31% of all households own at least one dog or cat, respectively. Few studies have ascertained the knowledge of pet owners on pet ownership and zoonoses, and none have been carried out in Portugal. The aim of the present study was to assess household knowledge and practices related to pet ownership and zoonoses in northern Portugal. A face-to-face questionnaire was completed by 424 pet owners, from November 2019 to February 2020. Most respondents (97.2%) considered pets as an important part of the family, especially women ( p = 0.036); 73.1% allowed their pets to live an indoor/outdoor life; 41.3% denied sharing the bed with their pets while 29% assumed they did it daily; 20.3% reported never kissing their pets/pets licking their faces. Furthermore, 73.6% considered animals as potential sources of human diseases, but only 25.9% reported knowing the definition of zoonoses; 96.9% considered the role of veterinarians important in protecting public health. The low level of knowledge of pet owners and the occurrence of high-risk behaviors indicates a need to strengthen communication between veterinarians, physicians, pet owners, and the general public towards reduce the risk of acquisition and transmission of zoonoses.

Toxoplasma gondii infection has been reported in numerous species of marine mammals, some of them with fatal consequences. A serosurvey for T. gondii infection was conducted in pinnipeds from an oceanographic park in Portugal ( n = 60); stranded pinnipeds on the Portuguese coast ( n = 10); and pinnipeds captured in Lorenzensplate, Germany ( n = 99). Sera from 169 pinnipeds were tested for the presence of antibodies to T. gondii by the modified agglutination test with a cut-off titre of 25. An overall seroprevalence of 8.9% (95% confidence interval: 5.1-14.2) was observed. Antibody titres of 25, 50, 100, 1600 and ≥3200 were found in five (33.3%), two (13.3%), five (33.3%), one (6.7%) and two (13.3%) animals, respectively. Pinnipeds under human care had a seroprevalence of 20.0% (12/60), in contrast to 2.8% (3/109) in wild pinnipeds ( p < 0.001). General results suggest a low exposure of wild pinnipeds to T. gondii , while the seroprevalence found in pinnipeds under human care highlights the importance of carrying out further studies. This is the first serological survey of T. gondii in pinnipeds in Portugal and the first infection report in South African fur seal ( Arctocephalus pusillus pusillus ).

Toxoplasma gondii is a worldwide zoonotic parasite. According to the "One Health" approach, studies on toxoplasmosis are essential since it affects humans and domestic and wild animals. In the present study, antibodies to T. gondii were determined in serum samples from 263 wild birds located in five wildlife rehabilitation centres in mainland Portugal by using the modified agglutination test (MAT) with a cut-off titre of 20. An overall seroprevalence of 36.5% (95% confidence interval [CI]: 30.7-42.6) was observed. For the first time, antibodies to T. gondii were detected in some avian species, including pallid swift ( Apus pallidus ) (33.3%), black-backed gull ( Larus fuscus ) (39.3%), European turtle-dove ( Streptopelia turtur ) (100%), bee-eater ( Merops apiaster ) (50.0%), carrion crow ( Corvus corone ) (33.3%), and Egyptian vulture ( Neophron percnopterus ) (100%), which expands the list of intermediate hosts of T. gondii . A lower seroprevalence was found in juvenile birds (31.9%) compared to adults (48.7%) ( p = 0.016). The central region of Portugal was considered a risk factor for T. gondii infection in wild birds (odds ratio: 3.61; 95% CI: 1.09-11.91). This pioneer study calls attention to the need for further studies, to provide a clearer understanding of T. gondii epidemiology in Portugal, because it reflects wide dispersion of T. gondii oocysts in the environment.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of the exocrine glands and prominent B cell hyperactivity. Considering the key role of monocytes in promoting B cell hyperactivity, we performed RNA-sequencing analysis of CD14 + monocytes from patients with pSS, non-Sjögren's sicca (nSS), and healthy controls (HC). We demonstrated that the transcriptomic profile of pSS patients is enriched in intermediate and non-classical monocyte profiles, and confirmed the increased frequency of non-classical monocytes in pSS patients by flow-cytometry analysis. Weighted gene co-expression network analysis identified four molecular signatures in monocytes from pSS patients, functionally annotated for processes related with translation, IFN-signaling, and toll-like receptor signaling. Systemic and local inflammatory features significantly correlated with the expression of these signatures. Furthermore, genes highly associated with clinical features in pSS were identified as hub-genes for each signature. Unsupervised hierarchical cluster analysis of the hub-genes identified four clusters of nSS and pSS patients, each with distinct inflammatory and transcriptomic profiles. One cluster showed a significantly higher percentage of pSS patients with higher prevalence of anti-SSA autoantibodies, interferon-score, and erythrocyte sedimentation rate compared to the other clusters. Finally, we showed that the identified transcriptomic differences in pSS monocytes were induced in monocytes of healthy controls by exposure to serum of pSS patients. Representative hub-genes of all four signatures were partially inhibited by interferon-α/β receptor blockade, indicating that the circulating inflammatory mediators, including type I interferons have a significant contribution to the altered transcriptional profile of pSS-monocytes. Our study suggests that targeting key circulating inflammatory mediators, such as type I interferons, could offer new insights into the important pathways and mechanisms driving pSS, and holds promise for halting immunopathology in Sjögren's Syndrome., Competing Interests: TR was a principal investigator in the immune catalyst program of GlaxoSmithKline, which was an independent research program. He did not receive any financial support other than the research funding for the current project. Currently, TR is an employee of Abbvie where he holds stock. TR had no part in the design and interpretation of the study results after he started at Abbvie. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lopes, Bekker, Hillen, Blokland, Hinrichs, Pandit, Kruize, Radstake and van Roon.)

Introduction: CCR9+ Tfh-like pathogenic T helper (Th) cells are elevated in patients with primary Sjögren's syndrome (pSS) and indicated to play a role in pSS immunopathology. Here we delineate the CCR9+ Th cell-specific transcriptome to study the molecular dysregulation of these cells in pSS patients., Methods: CCR9+, CXCR5+ and CCR9-CXCR5- Th cells from blood of 7 healthy controls (HC) and 7 pSS patients were FACS sorted and RNA sequencing was performed. Computational analysis was used to identify differentially expressed genes (DEGs), coherent gene expression networks and differentially regulated pathways. Target genes were replicated in additional cohorts., Results: 5131 genes were differentially expressed between CCR9+ and CXCR5+ Th cells; 6493 and 4783 between CCR9+ and CCR9-CXCR5- and between CXCR5+ and CCR9-CXCR5-, respectively. In the CCR9+ Th cell subset 2777 DEGs were identified between HC and pSS patients, 1416 and 1077 in the CXCR5+ and CCR9-CXCR5- subsets, respectively. One gene network was selected based on eigengene expression differences between the Th cell subsets and pathways enriched for genes involved in migration and adhesion, cytokine and chemokine production. Selected DEGs of interest (HOPX, SOX4, ITGAE, ITGA1, NCR3, ABCB1, C3AR1, NT5E, CCR5 and CCL5) from this module were validated and found upregulated in blood CCR9+ Th cells, but were similarly expressed in HC and pSS patients. Increased frequencies of CCR9+ Th cells were shown to express higher levels of CCL5 than CXCR5+ and CCR9-CXCR5- Th cells, with the highest expression confined to effector CCR9+ Th cells. Antigenic triggering and stimulation with IL-7 of the Th cell subsets co-cultured with monocytes strongly induced CCL5 secretion in CCR9+ Th cell cocultures. Additionally, effector CCR9+ Th cells rapidly released CCL5 and secreted the highest CCL5 levels upon stimulation., Conclusion: Transcriptomic analysis of circulating CCR9+ Th cells reveals CCR9-specific pathways involved in effector T cell function equally expressed in pSS patients and HC. Given the increased numbers of CCR9+ Th cells in the blood and inflamed glands of pSS patients and presence of inflammatory stimuli to activate these cells this suggests that CCR9-specific functions, such as cell recruitment upon CCL5 secretion, could significantly contribute to immunopathology in pSS., Competing Interests: Currently TR is an employee of Abbvie where he holds stock. TR had no part in the design and interpretation of the study results after he started at Abbvie. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hinrichs, Blokland, Lopes, Wichers, Kruize, Pandit, Radstake and van Roon.)

Guanabara Bay (GB) is an estuary in Brazil, constantly the target of pollutants, such as mercury (Hg). Thus, our study aimed to evaluate (i) total mercury (THg) content in shrimp and squid species from GB; (ii) associate THg content to contamination in swimming crabs; (iii) explore potential differences between species, and size; (iv) correlate abiotic water data to the determined THg contents; (v) verify if Hg concentrations are below acceptable limits. Swimming crabs showed greater Hg contamination compared to other species. For shrimp only biometric variables are related to Hg, while for squid, only abiotic. Only squids did not show a correlation between Hg and animal size. Finally, the detected Hg values are below the tolerable limits established by legislations. Our results indicate that the dynamics of Hg contamination differs between groups and that further studies are needed to elucidate the mechanisms that affect bioaccumulation in different species., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The current study aimed to investigate the effect of exercise combined with undenatured type II collagen (UCII) administration on endurance capacity, lipid metabolism, inflammation, and antioxidant status in rats. Twenty-one male Wistar albino rats were divided into three groups as follows: (1) Sedentary control, (2) Exercise (E), (3) Exercise + UCII (4 mg/kg BW/day; E + UCII). The findings showed that the exhaustive running time in the UCII group was significantly prolonged compared to that of the non-supplemented group ( p < 0.001). When compared to the control group, total serum cholesterol (TC, p < 0.05) and triglyceride (TG, p < 0.05) levels decreased, while creatinine kinase (CK) levels increased in the E group ( p < 0.001). Serum creatinine kinase levels were reduced in the E + UCII group compared to the E group ( p < 0.01). Serum lactate, myoglobin ( p < 0.01), and osteocalcin levels ( p < 0.01) increased significantly in exercised rats compared to sedentary control rats, while serum lactate ( p < 0.01) and myoglobin ( p < 0.0001) levels decreased in the E + UCII group compared to control. Additionally, UCII supplementation caused significant increases in antioxidant enzyme activities [SOD ( p < 0.01) and GSH-Px ( p < 0.05)] and decreases in malondialdehyde (MDA) and tumor necrosis factor (TNF-α) levels ( p < 0.001). Muscle lipogenic protein (SREBP-1c, ACLY, LXR, and FAS) levels were lower in the E + UCII group than in other groups. In addition, UCII supplementation decreased muscle MAFbx, MuRF-1, myostatin and increased MyoD levels in exercised rats. Moreover, the E + UCII group had lower muscle inflammatory markers [TNF-α ( p < 0.0001) and IL-1β ( p < 0.01)] than the control group. These results suggest exercise combined with UCII (4 mg/kg BW/day) modulates lipid, muscle, and antioxidant status in rats.

Angiopoietin-2 (Ang-2), a ligand of the tyrosine kinase receptor Tie2, is essential for vascular development and blood vessel stability and is also involved in monocyte activation. Here, we examined the role of Ang-2 on monocyte activation in patients with systemic sclerosis (SSc). Ang-2 levels were measured in serum and skin of healthy controls (HCs) and SSc patients by ELISA and array profiling, respectively. mRNA expression of ANG2 was analyzed in monocytes, dermal fibroblasts, and human pulmonary arterial endothelial cells (HPAECs) by quantitative PCR. Monocytes were stimulated with Ang-2, or with serum from SSc patients in the presence of a Tie2 inhibitor or an anti-Ang2 neutralizing antibody. Interleukin (IL)-6 and IL-8 production was analyzed by ELISA. Ang-2 levels were elevated in the serum and skin of SSc patients compared to HCs. Importantly, serum Ang-2 levels correlated with clinical disease parameters, such as skin involvement. Lipopolysaccharide (LPS) LPS, R848, and interferon alpha2a (IFN-α) stimulation up-regulated the mRNA expression of ANG2 in monocytes, dermal fibroblasts, and HPAECs. Finally, Ang-2 induced the production of IL-6 and IL-8 in monocytes of SSc patients, while the inhibition of Tie2 or the neutralization of Ang-2 reduced the production of both cytokines in HC monocytes stimulated with the serum of SSc patients. Therefore, Ang-2 induces inflammatory activation of SSc monocytes and neutralization of Ang-2 might be a promising therapeutic target in the treatment of SSc.

Wildlife plays an important role in the epidemiological cycle of Toxoplasma gondii . The European wild rabbit ( Oryctolagus cuniculus ) can be a source of infection to wild and domestic hosts, including human beings. Additionally, as an herbivorous animal, the European wild rabbit may also be a sentinel of environmental contamination with T. gondii and, consequently, an indicator of the potential transmission of this parasite. The purpose of the present work was to detect T. gondii DNA in European wild rabbit from central Portugal, as well as the possible implications for public health. Heart and diaphragm samples were obtained from 28 rabbits hunted in central Portugal. Nested PCR separately amplified the 5' and 3' ends of the surface antigen 2 (SAG2) gene. T. gondii DNA was detected in 19 out of the 28 sampled animals, resulting in a prevalence of 67.9%. These results show that T. gondii infection occurs in European wild rabbit and therefore may pose a potential risk for humans if consumed as raw or undercooked meat. Measures should be taken in order to prevent infection by this zoonotic parasite and for the conservation of wildlife. To the best of our knowledge, this is the first study performed by means of PCR on T. gondii in European wild rabbit meat samples.

Semaphorin (Sema)4A is a transmembrane glycoprotein that is elevated in several autoimmune diseases such as systemic sclerosis, rheumatoid arthritis and multiple sclerosis. Sema4A has a key role in the regulation of Thelper Th1 and Th2 differentiation and we recently demonstrated that CD4 + T cell activation induces the expression of Sema4A. However, the autocrine role of Sema4A on Th cell differentiation remains unknown. Naïve Th cells from healthy controls were cell sorted and differentiated into Th1, Th2 and Th17 in the presence or absence of a neutralizing antibody against the Sema4A receptor PlexinD1. Gene expression was determined by quantitative PCR and protein expression by ELISA and flow cytometry. We found that the expression of Sema4A is induced during Th1, Th2 and Th17 differentiation. PlexinD1 neutralization induced the differentiation of Th1 cells, while reduced the Th2 and Th17 skewing. These effects were associated with an upregulation of the transcription factor T-bet by Th1 cells, and to downregulation of GATA3 and RORγt in Th2 cells and Th17 cells, respectively. Finally, PlexinD1 neutralization regulates the systemic sclerosis patients serum-induced cytokine production by CD4 + T cells. Therefore, the autocrine Sema4A-PlexinD1 signaling acts as a negative regulator of Th1 skewing but is a key mediator on Th2 and Th17 differentiation, suggesting that dysregulation of this axis might be implicated in the pathogenesis of CD4 + T cell-mediated diseases.

Brazilian biodiversity and favourable environmental conditions open up possibilities not yet explored, showing potential to shift the country's monochromatic economy into an emancipated, diversified and sustainable economic environment. This can be made possible through the integral use of its resources, exploring every functional fraction to create novel solutions to modern problems. Biorefineries present an interesting strategy to fully use the potential of agricultural feedstocks and together with green separation methods can contribute to the generation of sustainable processes and products. Passion Fruit ( Passiflora edulis Sims f. flavicarpa Deg species) is produced on a large scale in Brazil and in other tropical countries, and its processing plants generate tons of residues that basically consist of peel, seeds and bagasse, which account for around 75% of its mass. These fractions of P. edulis can contain significant amounts of flavonoids, secondary metabolites that are the main compounds responsible for the fruit's bioactivity (antioxidant, anti-inflammatory, pesticide and biocide, in general). Therefore, this work aims to develop, apply and compare the best conditions for the extraction of isoorientin, orientin and isovitexin from passion fruit applying solid-liquid methodologies, followed by analyte quantification using UHPLC-PDA. Homogenizer-assisted (HAE), ultrasound-assisted (UAE) and microwave-assisted (MAE) extraction techniques were used, as well as a full factorial design to reach optimal parameters concerning the extraction yield and energy and solvent efficiencies. According to the results, the procedure based on HAE presented the best conditions for the extraction of selected flavonoids (1.07, 0.90 and 0.33 mg g -1 of isoorientin, orientin and isovitexin, respectively) and was considered the best method according to the green and sustainable described factors., Competing Interests: Competing interestsThe authors declare that they have no competing interests. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (© The Author(s) 2020.)

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in multiple inflammatory and fibrotic diseases. Here, we used monocyte-derived cells as a model system to study the effects of CXCL4 exposure on dendritic cell development by integrating 65 longitudinal and paired whole genome transcriptional and methylation profiles. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. Importantly, these pro-inflammatory cells directly trigger a fibrotic cascade by producing extracellular matrix molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils that CXCL4 acts as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis., (Copyright © 2020 Silva-Cardoso, Tao, Angiolilli, Lopes, Bekker, Devaprasad, Giovannone, van Laar, Cossu, Marut, Hack, de Boer, Boes, Radstake and Pandit.)

Competing Interests: Competing interests: TR is a principal investigator in the Immunology Catalyst programme at GlaxoSmithKline, which is an independent research programme. He did not receive any financial support other than the research funding for the current project. The other authors have declared no conflicts of interest.

Background: Dirofilaria immitis, Leishmania infantum and Toxoplasma gondii are zoonotic parasites which can affect domestic cats. Considering the lack of published data from the local feline population, this study aimed to assess infection with or exposure to these pathogens in cats from Madeira Island, Portugal., Methods: One hundred and forty-one domestic cats (77 males and 64 females; median age: 2 years) were sampled at a veterinary medical centre in Funchal, from September 2018 to January 2019. Serum samples were tested for D. immitis antigen, with an enzyme-linked immunosorbent assay kit, and for antibodies to Leishmania spp. or to T. gondii, with the direct agglutination test and the modified agglutination test, respectively., Results: Five cats (3.5%; 95% confidence interval, CI: 1.2-8.1) were positive to D. immitis; no cats were seropositive to Leishmania spp. (0%; 95% CI: 0-2.6%); and 43 cats (30.5%; 95% CI: 23.0-38.8%) were seropositive to T. gondii. Prevalence of the D. immitis antigen was significantly different between cats that received ectoparasiticides and those which did not (0 vs 12.2%; P = 0.009). Prevalence of antibodies to T. gondii was significantly different between juvenile and adult cats (12.8 vs 38.0%; P = 0.007). There were two cats concurrently positive to D. immitis and T. gondii, but no statistical association between these two dependent variables was found (P = 0.641)., Conclusions: To our knowledge, this is the first report of the presence of parasites D. immitis and T. gondii in the feline population of Madeira Island. Knowledge on the epidemiological situation of these and other zoonotic pathogens should raise awareness, both at the veterinary medical and public health levels, and contribute to promoting prevention and control.

The development of the Internet of Things (IoT) predicts several new applications, some of which are designed to be incorporated into e-health systems, and some technologies, like cloud computing and device-to-device communication (D2D), are promising for use in the support of resource-constrained devices employed in Mobile-health (m-health) and Telecare Medicine Information Systems (TMIS). In a scenario with billions of devices predicted for the IoT, it is essential to avoid performance and security problems, among others. Security is fundamental for the achievement of optimal performance regarding the sensibility of e-health shared data and, especially, the anonymity of patients and other entities, while it is also essential to consider the scarcity of bandwidth in wireless networks. This paper proposes a new mutual authentication protocol for m-health systems, which supports D2D communication, ensuring security and surpassing the performance and security of other authentication procedures reported in the literature.

Despite the global importance of the zoonotic parasite Toxoplasma gondii, little is known regarding its infection in the Democratic Republic of São Tomé and Príncipe (DRSTP). This is the first report of antibodies to T. gondii in cattle, sheep, goats and pigs from the DRSTP. Antibodies were assessed by the modified agglutination test (MAT), with a cut-off titer of 100 for cattle and 20 for sheep, goats and pigs. The present study revealed an overall seroprevalence of 55.8%; 27.1% in 48 cattle, 68.4% in 98 sheep, 70.1% in 97 goats and 43.7% in 103 pigs. The south geographical area for cattle, the central area for sheep, and adult age and living in the central region for goats were found to be risk factors for seropositivity to T. gondii. These results support the scenario of a considerable presence of sporulated oocysts as well as of infected intermediate hosts in the local environment. Consumption of raw or undercooked meat should be considered as an important potential source of infection for animals and humans in the DRSTP.

Objective: To examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA., Methods: Clinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex., Results: Tie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients., Conclusion: Tie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Cases of thelaziosis by Thelazia callipaeda have been increasing considerably in Europe throughout the 21st century, with recent emphasis on Eastern Europe. A systematic review was conducted using defined search terms across three major databases and, additionally, with the examination of the references of the 56 articles selected. Available information about epidemiological and clinical features of all cases of thelaziosis by T. callipaeda in companion animals, wildlife and humans was extracted, evaluated and subjected to qualitative and quantitative analysis. In all cross-sectional studies about dogs, cats and red foxes, males were more frequently infected than females (dogs: p = 0.0365; cats: p = 0.0164; red foxes: p = 0.0082). Adult dogs seem to be more prone to infection (p < 0.0001), as well as large-sized dogs (p < 0.0001), and companion animals that live exclusively outdoors (p < 0.0001). Dogs and red foxes involved in these cross-sectional studies harboured significantly more female than male nematodes (p < 0.0001). Thelaziosis by T. callipaeda is far from controlled in Europe. Only through updated epidemiological data, knowledge improvement and awareness can correct diagnosis and appropriate treatment and prevention be ensured to tackle this zoonosis., (© B. do Vale et al., published by EDP Sciences, 2020.)

Toxoplasma gondii is a protozoan parasite with worldwide distribution. The accurate detection of this zoonotic agent in cats and other hosts has public health importance. Blood samples from 89 domestic cats were tested for antibodies to T. gondii using 2 commercial agglutination test kits, an indirect (IHAT; Toxo-HAI FUMOUZE; Fumouze Diagnostics) and a modified (MAT; Toxoscreen DA; bioMérieux) agglutination test. Antibodies were found in 16 of 89 (18%) cats by the IHAT and in 23 of 89 (26%) cats by the MAT, with an overall agreement between the 2 serologic tests of 92% (κ = 0.77; i.e., substantial agreement beyond chance). Considering the MAT as the gold standard, the IHAT showed perfect relative specificity (100%) and lower relative sensitivity (70%). The suboptimal sensitivity of the IHAT limits its use in epidemiologic studies in cats.

Objectives: Considering the critical role of microRNAs (miRNAs) in regulation of cell activation, we investigated their role in circulating type-2 conventional dendritic cells (cDC2s) of patients with primary Sjögren's syndrome (pSS) compared to healthy controls (HC). Methods: CD1c-expressing cDC2s were isolated from peripheral blood. A discovery cohort (15 pSS, 6 HC) was used to screen the expression of 758 miRNAs and a replication cohort (15 pSS, 11 HC) was used to confirm differential expression of 18 identified targets. Novel targets for two replicated miRNAs were identified by SILAC in HEK-293T cells and validated in primary cDC2s. Differences in cytokine production between pSS and HC cDC2s were evaluated by intracellular flow-cytometry. cDC2s were cultured in the presence of MSK1-inhibitors to investigate their effect on cytokine production. Results: Expression of miR-130a and miR-708 was significantly decreased in cDC2s from pSS patients compared to HC in both cohorts, and both miRNAs were downregulated upon stimulation via endosomal TLRs. Upstream mediator of cytokine production MSK1 was identified as a novel target of miR-130a and overexpression of miR-130a reduced MSK1 expression in cDC2s. pSS cDC2s showed higher MSK1 expression and an increased fraction of IL-12 and TNF-α-producing cells. MSK1-inhibition reduced cDC2 activation and production of IL-12, TNF-α, and IL-6. Conclusions: The decreased expression of miR-130a and miR-708 in pSS cDC2s seems to reflect cell activation. miR-130a targets MSK1, which regulates pro-inflammatory cytokine production, and we provide proof-of-concept for MSK1-inhibition as a therapeutic avenue to impede cDC2 activity in pSS.

This study evaluated the fatty acid composition and the nutritional profile of Brycon cephalus and Brycon microlepis, fish species from the central Amazon basin, by different methods of quantification. The methods applied were: area normalization (MAN), internal standard (MIS), alternative theoretical (MAT) and alternative experimental (MAE). Significant differences were observed between the methods applied and the species studied. MAN supplied poor information about fatty acids composition and diet formulation, presenting only fatty acid profiles. MIS, MAT and MAE supplied fatty acids composition information on a mass basis. MAT and MAE overestimated results, whereas MIS presented the most accurate results . B. cephalus and B. microlepis showed high contents of approximately 65 mg g -1 of n-3 fatty acids. Eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) content totaled, 104.37 mg 100 g -1 and 117.89 mg 100 g -1 for B. cephalus and B. microlepis, respectively. The nutritional profile of both fish species showed favorable indices for nutritional quality of the lipid fraction, indicating that both Brycon species are healthy dietary choices.

Hydrogels have been studied as promising materials in different biomedical applications such as cell culture in tissue engineering or in wound healing. In this work, we synthesized different nanocellulose-alginate hydrogels containing cellulose nanocrystals, TEMPO-oxidized cellulose nanocrystals (CNCTs), cellulose nanofibers or TEMPO-oxidized cellulose nanofibers (CNFTs). The hydrogels were freeze-dried and named as gels. The nanocelluloses and the gels were characterized by different techniques such as Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and dynamic mechanical thermal analysis (DMTA), while the biological features were characterized by cytotoxicity and cell growth assays. The addition of CNCTs or CNFTs in alginate gels contributed to the formation of porous structure (diameter of pores in the range between 40 and 150 μm). TEMPO-oxidized cellulose nanofibers have proven to play a crucial role in improving the dimensional stability of the samples when compared to the pure alginate gels, mainly after a thermal post-treatment of these gels containing 50 wt % of CNFT, which significantly increased the Ca 2+ crosslinking density in the gel structure. The morphological characteristics, the mechanical properties, and the non-cytotoxic behavior of the CNFT-alginate gels improved bioadhesion, growth, and proliferation of the cells onto the gels. Thus, the alginate-nanocellulose gels might find applications in tissue engineering field, as for instance, in tissue repair or wound healing applications.

Neospora spp. are intracellular protozoa with worldwide distribution and closely related to Toxoplasma gondii, which can infect a variety of mammals including horses. From September 2013 to June 2014, 185 horses from northern, central and southern parts of mainland Portugal were randomly sampled and tested for detection of immunoglobulin (Ig) G antibodies to Neospora spp. using an indirect multi-species enzyme-linked immunosorbent assay (ELISA) commercial test (ID Screen® Neospora caninum Indirect Multi-species; ID.vet Innovative Diagnostics, Grabels, France). Two horses (1.1%; CI: 0.1-3.8%), one male and one female, were found to be seropositive for Neospora spp. Both seropositive animals were horses housed indoors but with access to outdoors, used for leisure activities and were apparently healthy, with good body condition and with no alterations at physical examination. This was the first serologic survey of antibodies to Neospora spp. carried out in horses from Portugal.

Toxoplasma gondii and Leishmania infantum are zoonotic protozoal parasites. Serum samples were obtained from 186 donkeys (Equus africanus asinus) from Portugal and assessed for antibodies to T. gondii by the modified agglutination test (MAT). For titration of antibodies to Leishmania spp. the direct agglutination test was used (DAT). Eleven donkeys were seropositive for T. gondii with titres of 20 (n = 7), 80 (n = 2), 640 (n = 1) and ≥ 2560 (n = 1). One donkey was seropositive for Leishmania spp. (titre of 800). Donkeys in Portugal are exposed to and can be infected with T. gondii and Leishmania spp.

The objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL-4) and IL-10 [IL4-10 fusion protein (FP)] to shift multiple pro-inflammatory pathways towards immune regulation, and to inhibit pro-inflammatory activity in arthritis models. The effects of IL4-10 FP in comparison with IL-4, IL-10 and IL-4 plus IL-10 on pro- and anti-inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4-10 FP to inhibit pro-inflammatory activity in ex-vivo and in-vivo arthritis models was investigated. IL4-10 FP robustly inhibited pro-inflammatory cytokine [IL-1β, tumour necrosis factor (TNF)-α, IL-6 and IL-8] production in whole blood cultures, mediated by both the IL-10 and the IL-4 moiety. IL4-10 fusion protein induced IL-1 receptor antagonist (IL-1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL-1RA/IL-1β and sTNFR/TNF-α ratios. In addition, IL4-10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up-regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4-10 FP robustly inhibited secretion of pro-inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4-10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL-4 and IL-10 stand-alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis., (© 2018 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

Alpha-1-antitrypsin deficiency (AATD) is a genetic autosomal codominant disorder caused by mutations in SERPINA1 gene. It is one of the most prevalent genetic disorders, although it remains underdiagnosed. Whereas at international level there are several areas of consensus on this disorder, in Portugal, inter-hospital heterogeneity in clinical practice and resources available have been adding difficulties in reaching a diagnosis and in making therapeutic decisions in this group of patients. This raised a need to draft a document expressing a national consensus for AATD. To this end, a group of experts in this field was created within the Portuguese Pulmonology Society - Study group on AATD, in order to elaborate the current manuscript. The authors reviewed the existing literature and provide here general guidance and extensive recommendations for the diagnosis and management of AATD that can be adopted by Portuguese clinicians from different areas of Medicine. This article is part of a supplement entitled "Portuguese consensus document for the management of alpha-1-antitrypsin deficiency" which is sponsored by Sociedade Portuguesa de Pneumologia., (Copyright © 2018. Published by Elsevier España, S.L.U.)

Drought-induced wildfires have increased in frequency and extent over the tropics. Yet, the long-term (greater than 10 years) responses of Amazonian lowland forests to fire disturbance are poorly known. To understand post-fire forest biomass dynamics, and to assess the time required for fire-affected forests to recover to pre-disturbance levels, we combined 16 single with 182 multiple forest census into a unique large-scale and long-term dataset across the Brazilian Amazonia. We quantified biomass, mortality and wood productivity of burned plots along a chronosequence of up to 31 years post-fire and compared to surrounding unburned plots measured simultaneously. Stem mortality and growth were assessed among functional groups. At the plot level, we found that fire-affected forests have biomass levels 24.8 ± 6.9% below the biomass value of unburned control plots after 31 years. This lower biomass state results from the elevated levels of biomass loss through mortality, which is not sufficiently compensated for by wood productivity (incremental growth + recruitment). At the stem level, we found major changes in mortality and growth rates up to 11 years post-fire. The post-fire stem mortality rates exceeded unburned control plots by 680% (i.e. greater than 40 cm diameter at breast height (DBH); 5-8 years since last fire) and 315% (i.e. greater than 0.7 g cm -3 wood density; 0.75-4 years since last fire). Our findings indicate that wildfires in humid tropical forests can significantly reduce forest biomass for decades by enhancing mortality rates of all trees, including large and high wood density trees, which store the largest amount of biomass in old-growth forests. This assessment of stem dynamics, therefore, demonstrates that wildfires slow down or stall the post-fire recovery of Amazonian forests.This article is part of a discussion meeting issue 'The impact of the 2015/2016 El Niño on the terrestrial tropical carbon cycle: patterns, mechanisms and implications'., (© 2018 The Author(s).)

Objective: To investigate miRNA expression in relation to transcriptomic changes in plasmacytoid dendritic cells (pDCs) in SLE and APS. pDCs are major producers of IFNα in SLE and APS, and miRNAs are emerging as regulators of pDC activation., Methods: miRNA and mRNA expression were measured by OpenArray and RNA-sequencing in pDCs of SLE, SLE + APS (APS secondary to SLE) and primary APS (PAPS) patients. The miRNA profile of patients was compared with the miRNA pattern of TLR7-activated pDCs., Results: Among 131 miRNAs detected in pDCs, 35, 17 and 21 had a significantly lower level of expression in SLE, SLE + APS and PAPS patients, respectively, as compared with healthy controls (HC). Notably, the miRNA profile did not significantly differ between SLE and APS, but was driven by the presence or absence of an IFN signature. TLR7 stimulation induced a general downregulation of miRNAs, similar to the pattern observed in SLE and APS patients. miR-361-5p, miR-128-3p and miR-181a-2-3p expression was lower in patients with a high IFN signature (false discovery rate <0.05) as compared with patients with a low IFN signature and HCs. Pathway enrichment on the overlap of miRNA targets and upregulated genes from the RNAseq indicated that these miRNAs are involved in pDC activation and apoptosis., Conclusion: Lower miRNA expression in pDCs is shared between SLE, SLE + APS and PAPS and is related to the IFN signature. As pDCs are the alleged source of the IFN signature in these patients, a better understanding of the molecular mechanisms/pathways leading to pDC dysregulation in SLE and APS might open novel pathways for therapeutic intervention.