Coupling of atherosclerosis progression and bone disturbances in inflammatory rheumatic diseases

Cardiovascular diseases (CV) and osteoporosis (OP) are among the most prevalent health problems, significantly contributing to increased morbidity and mortality of the population. The incidence of these conditions increases with age and they frequently coexist in the same patient. Epidemiological studies have demonstrated that patients with CV diseases have an increased risk for osteoporotic fractures and that patients with low bone mass have an increased risk for CV events. The inflammatory process has been implicated in the pathogenesis of both diseases, particularly in patients with immune-mediated rheumatic diseases like Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). In this work we hypothesize that bone and vascular disturbances share common pathophysiological mechanisms and that a connection between the progression of both conditions exists. Therefore, the aim of this thesis was to understand the interaction between vessels and bones in the context of inflammatory rheumatic diseases through the study of the effect of inflammation on tissues. In the first part of this study we evaluated how, in chronic inflammatory rheumatic diseases, namely RA and SLE, the vessels are disturbed, leading to vascular alterations and CV disease. For that we have assessed the early vascular alterations observed in these patients without previous CV events, through the determination of serum levels of vascular biomarkers and the study of endothelial function. We have then assessed the incidence of CV events in the RA cohort after 5 years and identified the contribution of traditional CV disease risk factors and RA-related parameters to CV events. We found that RA and SLE show distinct patterns of early vascular changes, more pronounced in SLE patients, and that the inflammatory activity was central to the increased endothelial activation biomarkers and to endothelial dysfunction. Additionally, patients with RA have a much higher incidence of CV events than the incidence reported for the general Portuguese population, for which the inflammatory and endothelial activation markers contribute significantly. In the SLE cohort, we evaluated how bone metabolism biomarkers, specifically OPG (osteoprotegerin) and RANKL (receptor activator of nuclear factor-ƙB ligand), are affected by the disease. We have found that these patients have an increased sRANKL/OPG ratio, due to reduced OPG levels. Also, both OPG and sRANKL levels were associated with SLE specific antibodies, but not with corticosteroids therapy, suggesting that the increased osteoclastic stimuli are driven by SLE disease mechanisms. Finally, we intended to comprehend the role of inflammation as common contributor to the interplay between bones and vessels. For that we have first used a mouse model of atherosclerosis (ApoE-/-) and evaluated bones and vessels disturbances and verified their association with inflammatory markers. Our results suggest that inflammation is not the principal driver for atherosclerosis progression and bone disturbances in this animal model, but that altered lipid transport and high-fat diet, associated with mice aging, may play a more significative role. Secondly, using bone and vessel samples from organ donors and atheroma plaques from endarterectomized patients with advanced atherosclerosis, we have evaluated the role of inflammation in the relationship between bone changes and vascular pathology. Our results suggest that the relationship between the changes observed in bones and vessels in the context of atherosclerotic disease and osteoporosis, may rely on the intrinsic connection between the tissues involved, independently of the progression of both diseases. Overall, our results suggest that the connection between atherosclerosis and osteoporosis rely on an intrinsic connection between the tissues and that the contribution of the risk factors for the diseases progression, including inflammation, affect more pronouncedly patients with immune-mediated rheumatic diseases

Ocorrência de fungos e bactérias em águas não tratadas destinadas a consumo humano

Dissertação apresentada à Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para cumprimento dos requisitos necessários à obtenção do grau de Mestre em BiotecnologiaApesar da sua vasta ocorrência, tem sido prestada pouca atenção à presença de fungos em ambientes aquáticos. São, portanto, necessários mais estudos nesta área uma vez que os fungos aquáticos poderão estar associados a várias doenças, problemas com o sabor e o cheiro e contaminação de bebidas e alimentos. Por comparação de métodos de detecção de fungos em três tipos de água (água superficial,água de nascente e água subterrânea) com composições muito diferentes, concluiu-se que a filtração por membrana, usando Dichloran Rose-Bengal Chloramphenicol como meio de cultura será uma técnica adequada para a análise de fungos nestas três matrizes. Os três tipos de águas estudadas apresentaram variações evidentes no que respeita à ocorrência de bactérias indicadoras de contaminação fecal e fungos. Foi detectada a presença ubíqua de fungos, embora a sua presença seja mais demarcada nas fontes de água sujeitas a factores ambientais – superficial e de nascente. Os resultados obtidos na análise temporal dos perfis de populações de fungos nas diferentes matrizes demonstraram que diferentes espécies deverão ser encontradas nos diferentes tipos de águas. Foram, por isso, isolados e identificados alguns dos géneros e espécies de fungos filamentosos e leveduras mais frequentemente encontrados em cada tipo de água

Atherosclerosis and Bone Loss in Humans–Results From Deceased Donors and From Patients Submitted to Carotid Endarterectomy

Funding: We wish to thank all the collaborators (administrative staff, nurses, etc.) of the surgery block, as well as the doctors of the vascular surgery and transplantation departments of the Hospital of Santa Maria for the availability and assistance in the collection of the samples. We also thank Sociedade Portuguesa de Reumatologia for funding with two fellowships: Fundo de Apoio à Investigação 2014 and SPR/MSD 2015. DC-F received funding from a PhD grant from Fundação para a Ciência e a Tecnologia (SFRH/BD/80940/2011).Background and Aims: Atherosclerosis and osteoporosis share common risk factors, as well as inflammatory mechanisms. Our aim was to understand how atherosclerotic lesions are related with disturbances in bone. Methods: Gene expression of pro-inflammatory and bone metabolism related proteins (IL-1β, IL-6, IL-17A, TNF, RANKL, OPG, COL1, CTSK, OCL, TRAP, CBFA1, DKK1, SOST, ADIPOQ, and ADIPOR1) were analyzed in arteries and bones from 45 deceased donors and adipose tissue was used as control. Additionally, in 139 patients with advanced atherosclerosis submitted to carotid endarterectomy we compared calcium content (Alizarin red) and plaque inflammatory scores (CD3+, CD68+, and adiponectin) of patients with normal bone mineral density (BMD) with those with low BMD and explored the associations between gene expression in atherosclerotic plaques and BMD. Serum levels of pro-inflammatory and bone related proteins were measured both in donors and patients. Associations were investigated by the Pearson or Spearman correlation tests, and multivariate regression analyzes were performed when justified. Results: Gene expression of bone remodeling and pro-inflammatory proteins correlated positively in bone and aorta, independently of age and sex of donors, but not in adipose tissue. The expression of bone formation genes was significantly higher in atheroma plaques from endarterectomized patients with normal vs. low BMD as well as inflammatory CD68+ scores, regardless of patients' age and sex, but not of body mass index. No relationship was observed between serum levels and gene expression levels of pro-inflammatory or bone remodeling proteins. Conclusions: Our results suggest that the relationship between bones and vessels in the context of atherosclerotic disease and osteoporosis may rely on the intrinsic connection between the tissues involved, independently of disease stage. Serum measurements of pro-inflammatory and bone-remodeling proteins do not accurately translate tissue pathologic processes.publishersversionpublishe

TRAF1/C5 but Not PTPRC Variants Are Potential Predictors of Rheumatoid Arthritis Response to Anti-Tumor Necrosis Factor Therapy

Background. The aim of our work was to replicate, in a Southern European population, the association reported in Northern populations between PTPRC locus and response to anti-tumor necrosis factor (anti-TNF) treatment in rheumatoid arthritis (RA). We also looked at associations between five RA risk alleles and treatment response. Methods. We evaluated associations between anti-TNF treatment responses assessed by DAS28 change and by EULAR response at six months in 383 Portuguese patients. Univariate and multivariate linear and logistic regression analyses were performed. In a second step to confirm our findings, we pooled our population with 265 Spanish patients. Results. No association was found between PTPRC rs10919563 allele and anti-TNF treatment response, neither in Portuguese modeling for several clinical variables nor in the overall population combining Portuguese and Spanish patients. The minor allele for RA susceptibility, rs3761847 SNP in TRAF1/C5 region, was associated with a poor response in linear and logistic univariate and multivariate regression analyses. No association was observed with the other allellic variants. Results were confirmed in the pooled analysis. Conclusion. This study did not replicate the association between PTPRC and the response to anti-TNF treatment in our Southern European population. We found that TRAF1/C5 risk RA variants potentially influence anti-TNF treatment response.This work was supported by a grant from Harvard-Portugal Program HMSP-ICS/SAU-ICT/0002/2010. Daniel H. Solomon received support for this work from the NIH (K24-AR-055989). Elizabeth W. Karlson received support for this work from NIH (K24-AR-AR0524). Reuma.pt received unrestricted grants from Abbott, Bristol-Myers Squibb, Merck Sharp and Dohme, Pfizer, Roche, and UCB Pharma

Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis

ABSTARCT: Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available.

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Early vascular alterations in SLE and RA patients--a step towards understanding the associated cardiovascular risk.

Accelerated atherosclerosis represents a major problem in both systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients, and endothelial damage is a key feature of atherogenesis. We aimed to assess early endothelial changes in SLE and RA female patients (127 SLE and 107 RA) without previous CV events. Biomarkers of endothelial cell activation (intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), thrombomodulin (TM), and tissue factor (TF)) were measured and endothelial function was assessed using peripheral artery tonometry. Reactive hyperemia index (RHI), an indicator of microvascular reactivity, and augmentation index (AIx), a measure of arterial stiffness, were obtained. In addition, traditional CV risk factors, disease activity and medication were determined. Women with SLE displayed higher sICAM-1 and TM and lower TF levels than women with RA (p = 0.001, p<0.001 and p<0.001, respectively). These differences remained significant after controlling for CV risk factors and medication. Serum levels of vascular biomarkers were increased in active disease and a moderate correlation was observed between sVCAM-1 levels and lupus disease activity (rho = 0.246) and between TF levels and RA disease activity (rho = 0.301). Although RHI was similar across the groups, AIx was higher in lupus as compared to RA (p = 0.04). Also in active SLE, a trend towards poorer vasodilation was observed (p = 0.06). In conclusion, women with SLE and RA present with distinct patterns of endothelial cell activation biomarkers not explained by differences in traditional CV risk factors. Early vascular alterations are more pronounced in SLE which is in line with the higher CV risk of these patients