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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]

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.)

Coxiella burnetii is the etiologic agent of Q fever, a worldwide zoonosis. Cattle, sheep and goats are considered the main reservoirs of the disease. Transmission to humans occurs mainly through the inhalation of infectious aerosols from milk, faeces, urine, and birth products from infected ruminants. In this study, a 2-year longitudinal approach was performed to ascertain the excretion of C. burnetii in bulk tank milk samples of sheep from a mountain plateau in central Portugal, with sampling conducted during the years 2015 and 2016. From a total of 156 bulk tank milk samples tested by qPCR, only one showed to be positive for C. burnetii (1.28% [95%CI: 0.03-6.94]), from 2015, the first year of collection. Bidirectional sequencing and phylogenetic analysis of IS1111 transposase partial region confirmed the presence of C. burnetii DNA. The presence of C. burnetii in raw milk samples highlights the necessity for additional research to determine if raw milk is a potential source for human infection. Animal health surveillance and prevention measures against this zoonotic disease should be considered., (© 2024. The Author(s).)

Ecotoxicological assessments encompass a broad spectrum of biochemical endpoints and ecological factors, allowing for comprehensive assessments concerning pollutant exposure levels and their effects on both fish populations and surrounding ecosystems. While these evaluations offer invaluable insights into the overall health and dynamics of aquatic environments, they often provide an integrated perspective, making it challenging to pinpoint the precise sources and individual-level responses to environmental contaminants. In contrast, biliary pollutant excretion assessments represent a focused approach aimed at understanding how fish at the individual level respond to environmental stressors. In this sense, the analysis of pollutant profiles in fish bile not only serves as a valuable exposure indicator, but also provides critical information concerning the uptake, metabolism, and elimination of specific contaminants. Therefore, by investigating unique and dynamic fish responses to various pollutants, biliary assessments can contribute significantly to the refinement of ecotoxicological studies. This review aims to discuss the multifaceted utility of bile as a potent biomarker for various environmental pollutants in fish in targeted monitoring strategies, such as polycyclic aromatic hydrocarbons, metals, pesticides, pharmaceuticals, estrogenic compounds, resin acids, hepatotoxins and per- and polyfluorinated substances. The main caveats of this type of assessment are also discussed, as well as future directions of fish bile studies., 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 © 2024 Elsevier Inc. All rights reserved.)

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.

Objective: Type 2 conventional dendritic cells (cDC2s) are key orchestrators of inflammatory responses, linking innate and adaptative immunity. Here we explored the regulation of immunological pathways in cDC2s from patients with primary Sjögren's syndrome (pSS)., Methods: RNA sequencing of circulating cDC2s from patients with pSS, patients with non-Sjögren's sicca and healthy controls (HCs) was exploited to establish transcriptional signatures. Phenotypical and functional validation was performed in independent cohorts., Results: Transcriptome of cDC2s from patients with pSS revealed alterations in type I interferon (IFN), toll-like receptor (TLR), antigen processing and presentation pathways. Phenotypical validation showed increased CX3CR1 expression and decreased integrin beta-2 and plexin-B2 on pSS cDC2s. Functional validation confirmed impaired capacity of pSS cDC2s to degrade antigens and increased antigen uptake, including self-antigens derived from salivary gland epithelial cells. These changes in antigen uptake and degradation were linked to anti-SSA/Ro (SSA) autoantibodies and the presence of type I IFNs. In line with this, in vitro IFN-α priming enhanced the uptake of antigens by HC cDC2s, reflecting the pSS cDC2 profile. Finally, pSS cDC2s compared with HC cDC2s increased the proliferation and the expression of CXCR3 and CXCR5 on proliferating CD4 + T cells., Conclusions: pSS cDC2s are transcriptionally altered, and the aberrant antigen uptake and processing, including (auto-)antigens, together with increased proliferation of tissue-homing CD4 + T cells, suggest altered antigen presentation by pSS cDC2s. These functional alterations were strongly linked to anti-SSA positivity and the presence of type I IFNs. Thus, we demonstrate novel molecular and functional pieces of evidence for the role of cDC2s in orchestrating immune response in pSS, which may yield novel avenues for treatment., Competing Interests: Competing interests: TRDJR was the principal investigator in the immune catalyst programme of GlaxoSmithKline, which was an independent research programme. He did not receive any financial support other than the research funding for the current project. Currently, He is an employee of Abbvie, where he holds stock. He 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., (© 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.)

Estuarine environments are highly productive ecosystems exhibiting high fish biodiversity and richness. Estuarine bays yield significant fisheries catches, making them the target of extractive activities by both artisanal and industrial fisheries. These areas, however, are highly vulnerable to chemical contamination, such as polycyclic aromatic hydrocarbons (PAH) associated with oil spills and other anthropogenic activities, which can jeopardize animal health and consumer welfare. In this context, this systematic review aimed to evaluate PAH levels detected in seafood from estuarine bays throughout the entire American continent and assess potential human health risks. The PICO methodology was applied, and 27 experimental papers were evaluated. The findings indicate that naphthalene and pyrene are routinely detected at high concentrations in several species, such as white mullet and catfish, and that biliary metabolites concentrations tend to be higher than tissue levels. Although the effects of PAH on animal health are well defined due to several decades of assessments, food safety evaluations are still not routine, evidencing a significant knowledge gap and the need for legislative measures based on toxicological data., 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 © 2022 Elsevier B.V. All rights reserved.)

Metal and metalloid concentrations in Black Triggerfish, Melichthys niger (Tetraodontiformes, Balistidae), and sediments from Trindade Island, an isolated, pristine, and understudied environment in the Southwestern Atlantic Ocean were determined. Several elements were detected in sediment, including Rare Earth Elements (REE). Hg and the REE Nd were, however, not detected. Elements determined in triggerfish are indicative of systemic circulation and the presence of toxic elements indicates an exposure source. No significant associations were detected between elements in fish organs and total length, suggesting no bioaccumulation with age, nor between organs and sediment, indicating probable dietary origin. Biliary excretion indicated a local chronic source of As and Cd and non-quantifiable REE elimination. Hg concentrations were over thresholds that affect fish biochemistry processes. These baseline data are valuable concerning elemental contamination in remote oceanic islands, with the potential to be applied to future biomonitoring efforts and conservation measures for reef ecosystems worldwide., 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.)

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.

Competing Interests: Declaration of competing interest Vanessa Costa de Sousa Ferreira, Amanda Pimentel Lopes, Nicholas Militão Alves, Fátima Regina Nunes Sousa, Raquel Felipe Vasconcelos, Conceição Silva Martins, Karuza Pereira Alves, Delane Viana Gondim, Vírginia Claúdia Carneiro Girão, Renata Ferreira Carvalho Leitão, Gerly A. C. Brito and Paula Goes have nothing to disclose.

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.)

The zoonotic parasitic nematode Thelazia callipaeda, also known as the oriental eye worm, is endemic in several European countries, including Portugal. Infections may result in ocular disease in domestic and wild animals as well as humans, with more or less severe manifestations. We report the first human case of ocular thelaziosis by T. callipaeda in Portugal, a country where the parasite had already been found to infect dogs, cats, red foxes, wild rabbits and a beech marten. An 80-year-old patient from east-central Portugal, who had been suffering from tearing for a few years, had whitish filiform fragments removed from the left eye. Polymerase chain reaction of partial cytochrome c oxidase subunit 1 and 18S small subunit rRNA genes followed by bidirectional sequencing and BLAST analysis confirmed T. callipaeda haplotype 1, the only haplotype previously reported in Europe. The endemicity of T. callipaeda in domestic and wild animals in east-central Portugal makes it very likely that infection of the human patient had occurred locally. In east-central and other geographical areas of Portugal, veterinarians and physicians, especially ophthalmologists, should regard T. callipaeda as a cause of ocular pathology in animals and humans., (Copyright © 2022. Published by Elsevier B.V.)

Interleukin (IL)-12 and IL-23 are pro-inflammatory cytokines produced by dendritic cells (DCs) and associated with Psoriasis (Pso) and Psoriatic Arthritis (PsA) pathogenesis. Tofacitinib, a Janus kinase inhibitor, effectively suppresses inflammatory cascades downstream the IL-12/IL-23 axis in Pso and PsA patients. Here, we investigated whether Tofacitinib directly regulates IL-12/IL-23 production in DCs, and how this regulation reflects responses to Tofacitinib in Pso patients. We treated monocyte-derived dendritic cells and myeloid dendritic cells with Tofacitinib and stimulated cells with either lipopolysaccharide (LPS) or a combination of LPS and IFN-γ. We assessed gene expression by qPCR, obtained skin microarray and blood Olink data and clinical parameters of Pso patients treated with Tofacitinib from public data sets. Our results indicate that in DCs co-stimulated with LPS and IFN-γ, but not with LPS alone, Tofacitinib leads to the decreased expression of IL-23/IL-12 shared subunit IL12B (p40). In Tofacitinib-treated Pso patients, IL-12 expression and psoriasis area and severity index (PASI) are significantly reduced in patients with higher IFN-γ at baseline. These findings demonstrate for the first time that Tofacitinib suppresses IL-23/IL-12 shared subunit IL12B in DCs upon active IFN-γ signaling, and that Pso patients with higher IFN-γ baseline levels display improved clinical response after Tofacitinib treatment., (© 2022 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

Introduction: Praxelis genus comprises 24 species, however, only two species of this genus have been chemically investigated. Here we investigated Praxelis sanctopaulensis, a native plant from Brazil, that occurs mainly in Cerrado regions., Objective: The goal was to identify the specialised metabolites from P. sanctopaulensis, and compare with those described from Praxelis and Chromolaena species., Methods: The phytochemical study of P. sanctopaulensis was performed through different chromatography techniques, including high-performance liquid chromatography (HPLC), gas chromatography flame ionisation detector (GC-FID), and ultra-high-performance liquid chromatography high-resolution tandem mass spectrometry (UHPLC-HRMS/MS). The structures of the compounds were established based on spectroscopic analysis, total correlated spectroscopy (TOCSY), hydrogen decoupling and computational calculations was used to an unequivocal structural elucidation of a new sesquiterpene. The antioxidant activity was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP), and antimicrobial assay was performed by the microdilution method. Comparison of the flavonoids described P. sanctopaulensis was carried out using principal component analysis., Results: The phytochemical investigation of P. sanctopaulensis led to the isolation of a pair of diastereomers, praxilone A and praxilone B. Seven known compounds were isolated from this species, another 14 fatty acids were detected in hexane fraction, and 26 compounds were identified from ethyl acetate fraction. All these compounds are being described for the first time in this species, with the exception of viridifloric acid. The ethyl acetate fraction showed potent antioxidant activity., Conclusions: Forty-seven compounds are described from P. sanctopaulensis. The combination of different techniques of nuclear magnetic resonance (NMR) spectroscopy and computational calculations allowed the unequivocal structure elucidation of a new cadinene. The clustering analysis showed similarities between the flavonoids identified in P. sanctopaulensis and in Chromolaena species., (© 2021 John Wiley & Sons, Ltd.)

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 ).

The alveolar bone is an important mineralized structure of the periodontal support apparatus, and information about the methylmercury (MeHg) effects on the structural integrity is scarce. Therefore, this study aimed to investigate whether systemic, chronic, and low-dose exposure to MeHg can change the alveolar bone microstructure of rats. Adult Wistar rats (n = 30) were exposed to 0.04 mg/kg/day of MeHg or vehicle through intragastric gavage. The animals were euthanized after 60 days, and blood samples were collected for trolox equivalent antioxidant capacity (TEAC), glutathione (GSH), lipid peroxidation (LPO), and comet assays. The mandible of each animal was collected and separated into hemimandibles that were used to determine the total Hg level in the bone and to analyze microstructural damage and alveolar bone loss in terms of trabecular number (Tb.N), trabecular thickness (Tb.Th), bone volume fraction (BV/TV), and exposed root area of the second molars. MeHg exposure triggered oxidative stress in blood represented by lower levels of GSH and TEAC and the increase in LPO and DNA damage of the blood cells. High total Hg levels were found in the alveolar bone, and the microstructural analyses showed a reduction in Tb.N, Tb.Th, and BV/TV, which resulted in an increase in the exposed root area and a decrease in bone height. Long-term MeHg exposure promotes a systemic redox imbalance associated with microstructural changes and alveolar bone loss and may indicate a potential risk indicator for periodontal diseases., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

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.

In this study, we analyzed the antimicrobial, antibiofilm, and modulatory activities of trans-trans-farnesol (tt-farnesol). The minimum inhibitory concentration (MIC) of this sesquiterpene was evaluated against 31 Gram-positive and Gram-negative bacterial strains and 4 species of the genus Candida. Furthermore, we examined its inhibitory action on biofilm production as well as antibiotic modulation. Only Gram-positive species presented susceptibility to tt-farnesol (MIC ranging from 8 µg/mL to 128 µg/mL). No synergistic or antagonistic effects were observed between tt-farnesol (1/4 and 1/8 of MIC) and first-choice antibiotics against multidrug resistant strains. However, the modulatory action of tt-farnesol (1/2 and 1/4 of the MIC) decreased 8 × MIC of non-inhibitory β-lactam antibiotic against a Methicillin-resistant strain. In the antibiofilm assay, tt-farnesol inhibited biofilm formation, especially in Methicillin-resistant Staphylococcus aureus (MRSA) strains, at concentrations ranging from 2 μg/mL to 128 μg/mL. Additionally, in the molecular docking study, the tt-farnesol molecule demonstrated a remarkable binding affinity with important proteins involved in the biofilm production, such as IcaA and Srt proteins. The antimicrobial action of tt-farnesol on Streptococcus pyogenes and Streptococcus agalactiae strains was evaluated for the first time, presenting an MIC of 16 µg/mL for both strains. Our findings reveal the antibacterial, antibiofilm, and modulatory potential of tt-farnesol to aid in the fight against infectious processes., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.)

Ethnopharmacological Relevance: Miconia albicans (Sw.) Triana has been widely used in Brazilian popular medicine for the treatment of several diseases. Aerial parts are used as an infusion to treat arthrosis and arthritis, to relieve rheumatic and stomach pains, and intestinal disorders due to its anti-inflammatory, anti-mutagenic anti-nociceptive, digestive and hepatoprotective properties., Aim of the Study: This study aimed to characterize the of M. albicans (Sw.) Triana fruits extract (MAFRE) chemical profile and to evaluate its antioxidant, anti-inflammatory and antitumor activities, as well as its toxicity., Materials and Methods: Maceration with methanol as liquid extractor was used to prepare MAFRE. M. albicans (Sw.) Triana fruits chemical composition was characterized by UHPLC-QTOF-MS/MS and GC-FID (fatty acid methyl esters composition from lyophilized fruits). MAFRE antioxidant potential was evaluated in vitro using a combination of assays: Folin-Ciocalteu reducing capacity, DPPH • and ABTS radical scavenging ability and ferric reducing antioxidant power (FRAP). In vitro antiproliferative activity was investigated in four human tumor cell lines (U251, 786-0, HT29 and MDA-MB-231) while the effect on the non-tumor cell viability was assessed in the VERO cell line using the on-step MTT assay. In addition, in vivo anti-inflammatory effect was assessed by Croton oil-induced ear edema in mice followed by myeloperoxidase (MPO) activity evaluation., Results: Thirty-five compounds were identified by UHPLC-QTOF-MS/MS. Among it flavonoids derived from quercetin (8), myricetin (1), kaempferol (2), terpenoids (6) and other compounds (18). GC-FID analysis identified and quantified nine fatty acids: palmitic, stearic, arachidic, behenic, elaidic, oleic, eicosenoic, and linoleic acids. The most abundant fatty acids were polyunsaturated fatty acids (5.33 ± 0.17 mg g -1 ), followed by saturated fatty acids (2.38 ± 0.07 mg g -1 ) and monounsaturated fatty acids (1.74 ± 0.09 mg g -1 ). The extract revealed high content of phenolic compounds (43.68 ± 0.50 mg GAE/g of extract), potent antioxidant, and ferrous chelating capacities. Morever, it proved to be non-toxic to the VERO cells, not affecting cells viability (95% of viable cells). No antiproliferative effect against human tumor cell lines were found. Furthermore, MAFRE significantly (p<0.05) reduced ear edema (≈35%) and MPO activity (84.5%) having a statistical effect similar to traditional steroidal and non-steroidal anti-inflammatory drugs., Conclusions: Taken together, the results evidenced that M. albicans fruit extract has antioxidant properties, a higher concentration of phenolic compounds, flavonoids, fatty acids, and also topical anti-inflammatory activity with low toxicity of extract on VERO cells. Through the ethnomedicinal study, these findings supporting the popular use of M. albicans, but also highlight that not only aerial parts and leaves deserve attention, but the fruits also have anti-inflammatory proprieties and can be a source of phenolic compounds and other substances with potential health benefices., (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.

Coronavirus disease-19 (COVID-19) is caused by the severe acute Respiratory syndrome coronavirus-2 (SARS-CoV-2), which has become unstoppable, spreading rapidly worldwide and, consequently, reaching a pandemic level. This review aims to provide the information available so far on the likely animal origin of SARS-CoV-2 and its possible hosts/reservoirs as well as all natural animal infections and experimental evidence using animal models. Horseshoe bats from the species Rhinolophus affinis seem to be a natural reservoir and pangolins (Manis javanica) appear to be an intermediate host of SARS-CoV-2. Humans remain the most likely spreading source of SARS-CoV-2 to other humans and also to domestic, zoo and farm animals. Indeed, human-to-animal transmission has been reported in cats, dogs, tigers, lions, a puma and minks. Animal-to-human transmission is not a sustained pathway, although mink-to-human transmission remains to be elucidated. Through experimental infections, other animals seem also to be susceptible hosts for SARS-CoV-2, namely ferrets, some non-human primate species, hamsters and transgenic mice, while dogs, pigs and poultry are resistant. A One Health perspective must be implemented in order to develop epidemiological surveillance and establish disease control mechanisms to limit zoonotic transmission. Moreover, research in this field is important to better understand SARS-CoV-2 and to obtain the long-awaited vaccine and specific treatment.

Objective: This work aimed to study the role of inflammation in medication-related osteonecrosis of the jaw (MRONJ) in rats with focus on Wnt signaling., Methods: A total of 36 female Wistar rats (12 weeks ± 200 g) were divided into 2 groups (n = 6) in 3 experiments: saline (SAL) and zoledronic acid (ZOL). For MRONJ induction, rats received 0.1 mg/kg of ZOL (ip) 3×/week for 9 weeks. Animals from the SAL group received 0.1 mg/kg of 0.9% SAL, ip 3×/week for 9 weeks. On the 8th week, 3 left upper molars were extracted, and on the 11th week, they were euthanized. Maxillae were evaluated by macroscopic and histopathological analyses; scanning electron microscopy (SEM); immunohistochemistry for DKK-1, Wnt 10b, and caspase-3; and Raman spectrometry. Gingiva was also collected for TNF-α e IL-1β quantification., Results: Bone necrosis was confirmed by healing impairment, reduced number of viable osteocytes, increased caspase-3 immunoexpression, and increased number of empty lacunae (p < 0.05). ZOL enhanced inflammation and increased gingival levels of IL-1β and TNF-α (p < 0.05). Irregular indentations were seen on bone after ZOL administration. Bone necrosis was marked by reduced amount of total and type I collagen. ZOL reduced the mineral/matrix ratio and increased carbonate/phosphate ratio. It was observed a significant reduction on Wnt10b and beta-catenin immunolabeling in the bone tissue of ZOL group., Conclusion: In summary, MRONJ model caused bone necrosis due to intense inflammation. Wnt signaling seems to play an important role in this process., Clinical Relevance: New therapeutic strategies focusing on Wnt pathway can provide an interesting approach for future treatments.

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.

Introduction/objectives: There are few parameters for assessment of right ventricular (RV) systolic function on echocardiographic examination. Morphofunctional studies are limited by the irregular shape of the RV. Recently, tissue motion annular displacement (TMAD), a technique that evaluates valve annulus displacement toward the cardiac apex, has shown a good correlation with left ventricular global longitudinal strain (GLS) in healthy dogs. Therefore, the aim of this study was to evaluate the longitudinal systolic function of the RV of healthy dogs using TMAD., Animals: A hundred healthy client-owned dogs., Materials and Methods: Cross-sectional study. Physical examination, electrocardiogram, blood pressure measurement, and echocardiography were recorded. The systolic function of the RV was evaluated by GLS free wall and TMAD. Data were compared with those derived from conventional echocardiography., Results: TMAD values varied according to body weight. There was a correlation of TMAD in millimeters with all indices of RV systolic function, including GLS free wall (R:-0.239; p:0.017). TMAD had a correlation with age and heart rate; whereas there was no relationship with sex and blood pressure. The coefficient of variation for the intraobserver evaluation was lower for the TMAD in millimeters (9.9%) compared with the GLS free wall (17.9%). In addition, the mean time to perform TMAD (8.1 s) was lower than that of the GLS free wall (37.7 s)., Conclusions: TMAD is a fast, reproducible, and promising method for assessing RV systolic function in healthy dogs. However, further studies are needed to understand the applicability of this technique in patients with heart disease., Competing Interests: Conflict of Interest Statement The authors do not have any conflicts of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.)