Purificação e caracterização de moléculas com atividade antibacteriana produzidas por bactérias do género Bacillus

Dissertação de Mestrado em Ciências Biomédicas.As bactérias produzem várias substâncias antibacterianas desde antibióticos, lisozimas, exotoxinas e também bacteriocinas. As bacteriocinas são péptidos antimicrobianos de síntese ribossomal que têm suscitado um interesse crescente no combate a estirpes resistentes a antibióticos. Algumas espécies de Bacillus produzem bacteriocinas/BLIS que possuem ação contra MRSA e outras estirpes resistentes. As aplicações das bacteriocinas vão desde a Saúde Humana até à utilização como bioconservantes. O objetivo deste trabalho foi o estudo e caracterização da atividade antibacteriana de 2 isolados de Bacillus (S54c e S150c) da Coleção Açoriana de Bactérias Esporulantes, bem como das suas moléculas ativas. Os dois isolados mostraram maior crescimento e maior atividade antibacteriana em meio Brain Heart Infusion após 18 horas de incubação. Para o sobrenadante filtrado destes isolados e para as frações ativas do sobrenadante do isolado S54c foi determinada a estabilidade térmica e a sensibilidade a enzimas hidrolíticas. O resultado destes testes foi avaliado pela determinação da atividade antibacteriana pelo método de difusão em agar contra a bactéria Micrococcus luteus. A atividade dos isolados S54c e S150C contra Staphylococcus aureus e MRSA foi avaliada pelo método descrito para bactérias esporulantes e o espectro de atividade do sobrenadante do isolado S54c foi determinado. O sobrenadante liofilizado do isolado S54c foi fracionado por cromatografia de exclusão molecular, coluna Sephacryl S-200 40 ml, e as frações positivas visualizadas em SDS-PAGE. Os sobrenadantes dos isolados S54c, S150c e a fração>30kDa do sobrenadante do isolado S54c possuem estabilidade térmica até aos 100ºC, a fração entre os 3-30kDa do isolado S54c é estável até aos 121ºC. Mostrou-se que a atividade antibacteriana dos isolados S54c, S150c e a fração>30kDa (S54c) é hidrolisada pela proteinase K e quimiotripsina, enquanto a fração entre os 3-30 kDa(S54c) apenas é hidrolisada pela proteinase K. Os resultados obtidos indicam que a atividade antibacteriana é de origem proteica e permitem classificar estas moléculas como BLIS. Os isolados S54c e S150c mostraram atividade contra S.aureus e MRSA, contudo o S150c apresentou uma atividade mais reduzida. O sobrenadante filtrado do isolado S54c mostrou atividade contra M.luteus e B.cereus e o fracionamento em cromatografia de exclusão molecular resultou na identificação de uma fração ativa. A análise da sequência parcial do gene 16S mostrou que o isolado S54c é uma estirpe de B.cereus. Este trabalho permitiu identificar parcialmente BLIS produzidas por B.cereus S54c e que podem vir a ser purificadas por técnicas cromatográficas.ABSTRACT: Bacteria produce a high number of antibacterial molecules, including antibiotics, lisozimas, exotoxinas and bacteriocins. Bacteriocins are antimicrobial peptides of ribosomal synthesis that are raising a growing interest in the fighting against bacteria resistant strains. Some species of Bacillus produce bacteriocins/BLIS active against MRSA and other resistant strains. Bacteriocin applications are vast, from human health to biopreservatives. The objective of this work was the study and characterization of antibacterial activity of two Bacillus isolates (S54c and S150c) from the Azorean Sporulated Bacteria Collection. The two isolates showed improved growth and higher antibacterial activity in Brain Heart Infusion broth after 18 hours incubation. The antibacterial activity, of the cell free supernatant of both isolates and active fractions of S54c, were evaluated for thermal stability and degradation by hydrolytic enzymes. The results of these testes were evaluated by the agar well diffusion assay against Micrococcus luteus. S54c and S150c were tested against Staphylococcus aureus and MRSA by the method used for sporulating bacteria and the activity range of S54c cell free supernatant was determined. The freeze dried cell free supernatant of S54c was fractioned by Gel Filtration Chromatography (Sephacryl S-200 40 ml) and positive fractions were separated by SDS-PAGE. The antibacterial activity of S54c and S150c cell free supernatants and the S54c >30kDa fraction showed thermal stability until 100ºC. The S54c 3-30kDa fraction proved to be stable at 121ºC. The activity of S54c, S150c and S54c >30kDa fraction, is hydrolysed by proteinase K, chymotripsin while the 3-30 kDa(S54c) fraction is hydrolysed only by proteinase K. The results show that antibacterial activity is of protein nature and allow classifying these molecules as BLIS. The S54c and S150c isolates showed activity against S. aureus and MRSA, but S150s showed lower activity. The cell free supernatant of S54c showed activity against M. luteus and B. cereus and the gel filtration chromatography of the freeze dried cell free supernatant resulted in the identification of an active fraction. The analysis of the partial sequence of the 16S gene showed that S54c is a strain of B. cereus. This work allowed the partial fractioning and identification of BLIS produced by B. cereus S54c that can be purified by applying chromatographic techniques

How did COVID-19 pandemic changed the Portuguese rheumatology?

© 2001-2021 Sociedade Portuguesa de Reumatologia.We have been facing a tremendous challenge motivated by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic. With a few days of warning Rheumatologists had to rethink their established conventional management of patients based on presential appointments, to ensure ongoing adequate care to rheumatic patients. The first operating principles were to maintain rheumatic diseasesstability, treatment access and protection from the potentialrisk of infection by SARS-CoV-2.info:eu-repo/semantics/publishedVersio

Histopathology of psoriatic arthritis synovium: a narrative review

Copyright © 2022 Tenazinha, Barros, Fonseca and Vieira-Sousa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Psoriatic arthritis (PsA) is a phenotypically heterogeneous chronic inflammatory disease associated to type I major histocompatibility complex alleles whose complex pathogenesis is still not completely understood. The psoriatic synovium shares general features of chronic inflammation with rheumatoid arthritis (RA) and other arthritis, such as hyperplasia of the intimal lining layer, sublining influx of inflammatory cells and neoangiogenesis, but recognizing disease-specific histopathologic findings may help in diagnosis and definition of therapeutic targets. Available literature reports conflicting data regarding the extension of lining hyperplasia, that does not allow depiction from RA. Sublining inflammatory cells consist of T and B cells and macrophages, plasma cells, mast cells and follicular dendritic cells, with a higher amount of overall T, mast cell and IL-17 producing CD8+ T lymphocytes and lower proportion of plasma cells when compared to the rheumatoid synovium. The amount of synovium IL17+ CD8+ T cells correlates positively to measures of disease activity. Lymphoid follicles with characteristics of germinal centers have been identified, similar to the ones described in RA. Neoangiogenesis is more prominent in PsA but can also be an outstanding feature in some RA samples, and different molecules involved in the process appear to have different influence in each disease. IL-17 and IL-22 expression in the synovium does not allow depiction between diseases. Among other cytokines and molecules likely implicated in disease physiopathology, only IL-35 is demonstrated to be reduced in PsA when compared to RA.info:eu-repo/semantics/publishedVersio

Arthroscopic guided synovial biopsies

Copyright © 2021 Orr, Vieira-Sousa, Fonseca and Veale. This is an open-accessarticle distributed under the terms of the Creative Commons Attribution License (CC-BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.No use, distribution or reproduction is permitted which does not comply with these terms.Synovial tissue can be safely and reliably collected for research and clinical purposes using arthroscopy. This technique offers the obvious advantage of allowing direct visualization, and targeted biopsy of specific areas of interest within the joint, as well as for the collection of tissue which will include a lining layer. Much has been learnt by studying the synovium retrieved using this technique concerning the pathobiology of inflammatory arthritis. Furthermore, recent evidence suggests that the tissue retrieved may enable the identification of unique pathotypes that will allow for a precise approach to treatment selection in individual patients. Although ultrasound guided techniques for sampling synovial tissue have gained in popularity over the last decade, both methodologies are expected to compliment each other, each having unique benefits and drawbacks. We present here a detailed description of the arthroscopy technique reporting on our collective experience at two centers in Europe.info:eu-repo/semantics/publishedVersio

Determinants of health-related quality of life in spondyloarthritis and rheumatoid arthritis: data from the COMOSPA and COMORA studies

© 2022 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Objectives: To assess the hierarchy of outcomes contributing to health-related quality of life (HRQoL) in spondyloarthritis (SpA) and rheumatoid arthritis (RA). Methods: Data from the international cross-sectional COMOSPA and COMORA studies were used. HRQoL was assessed using the EuroQOL 5-dimension 3-level (EQ-5D-3 L). First, multivariable linear regression models were used to identify associations between EQ-5D-3 L (dependent variable) and several demographic and clinical variables (independent variables). Second, a decision tree was built using Chi-square Automatic Interaction Detector, a method of unbiased hierarchical multivariable analysis (dependent variable: EQ-5D-3 L). Results: In total, 3984 patients with SpA and 3920 patients with RA were included. In SpA, HRQoL was associated with BASFI (adjusted B=-0.006; 95%CI=-0.007 to -0.005), ASDAS (-0.052; -0.071 to -0.033), work productivity loss score (-0.002; -0.003 to -0.002), NSAID treatment (-0.052; -0.083 to -0.020), bDMARD treatment (-0.051; -0.082 to -0.021), university education (-0.051; -0.075 to -0.027) and radiographic sacroiliitis (0.035; 0.004 to 0.030). In RA, HRQoL was associated with modified Health Assessment Questionnaire (MHAQ) (-0.220, -0.253 to -0.188), DAS28-CRP-3v (-0.027, -0.036 to -0.018), work productivity loss score (-0.003, -0.003 to -0.002), presence of erosions (-0.042, -0.065 to -0.020), alcohol consumption ≥3 units/day (0.012, 0.001 to 0.024)) and csDMARD treatment (0.034, 0.001 to 0.066). The decision tree revealed BASFI and MHAQ as first variables with the most discriminative power on EQ-5D-3 L, followed by work productivity loss and disease activity, in both SpA and RA cohorts. Conclusion: In SpA and RA, physical function is the main contributor to HRQoL measured by EQ-5D-3 L, followed by disease activity and work productivity loss.info:eu-repo/semantics/publishedVersio

Rheumatology practice amidst the COVID-19 pandemic : a pragmatic view

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.The coronavirus disease 2019 (COVID-19) pandemic has come with many challenges for healthcare providers and patients alike. In addition to the direct burden it has placed on societies and health systems, it had a significant impact in the care of patients with chronic diseases, as healthcare resources were deployed to fight the crisis, and major travel and social restrictions were adopted. In the field of rheumatology, this has required notable efforts from departments and clinicians to adapt to the novel status quo and assure the follow-up of patients with rheumatic and musculoskeletal diseases. In the present viewpoint, we provide a practical approach to tackle this reality. Key measures include setting up preventive team management strategies, optimising communication with patients and reorganising patient care in all its dimensions. We then anticipate the nuances of rheumatology practice as restrictive measures are progressively lifted, while an effective vaccine is still pending. This includes the need to reimpose the same strategy as further waves unfold. Finally, we look ahead and address the lessons we can incorporate into post-COVID-19 rheumatology.info:eu-repo/semantics/publishedVersio

The GO-DACT protocol : a multicentre, randomized, double-blind, parallel-group study to compare the efficacy of golimumab in combination with methotrexate (MTX) versus MTX monotherapy

© 2001-2020 Sociedade Portuguesa de ReumatologiaThe GO-DACT is an investigator-initiated, national, multicentric randomized placebo-controlled double-blinded trial, that assesses dactylitis as primary endpoint. Psoriatic arthritis patients naïve to methotrexate and biologic disease modifying anti-rheumatic drugs, with at least one active dactylitis, were assigned to golimumab in combination with methotrexate or placebo in combination with methotrexate, for 24 weeks. Both clinical (dactylitis severity score and the Leeds dactylitis index) and imaging (high resolution magnetic resonance imaging), among others, were assessed as outcomes. The main objective of GO-DACT is to provide evidence to improve the treatment algorithm and care of psoriatic arthritis patients with active dactylitis. In this manuscript we describe the GO-DACT protocol and general concepts of the methodology of this trial.info:eu-repo/semantics/publishedVersio

GO-DACT : a phase 3b randomised, double-blind, placebo-controlled trial of GOlimumab plus methotrexate (MTX) versus placebo plus MTX in improving DACTylitis in MTX-naive patients with psoriatic arthritis

© author(s) (or their employer(s)) 2020. Re-use permitted under CC BY- nC. no commercial re-use. see rights and permissions. Published by BMJ. This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-­NC 4.0) license.Objectives: To assess the efficacy of golimumab in combination with methotrexate (MTX) versus MTX monotherapy in psoriatic arthritis (PsA) dactylitis. Methods: Multicentre, investigator-initiated, randomised, double-blind, placebo-controlled, parallel-design phase 3b trial in 11 Portuguese rheumatology centres. Patients with PsA along with active dactylitis and naive to MTX and biologic disease-modifying antirheumatic drugs (bDMARDs) were randomly assigned to golimumab or placebo, both in combination with MTX. The primary endpoint was Dactylitis Severity Score (DSS) change from baseline to week 24. Key secondary endpoints included DSS and Leeds Dactylitis Index (LDI) response, and changes from baseline in the LDI and MRI dactylitis score. Analysis was by intention-to-treat for the primary endpoint. Results: Twenty-one patients received golimumab plus MTX and 23 MTX monotherapy for 24 weeks. One patient from each arm discontinued. Patient inclusion was halted at 50% planned recruitment due to a favourable interim analysis. Median baseline DSS was 6 in both arms. By week 24, patients treated with golimumab plus MTX exhibited significantly greater improvements in DSS relative to MTX monotherapy (median change of 5 vs 2 points, respectively; p=0.026). In the golimumab plus MTX arm, significantly higher proportions of patients achieved at least 50% or 70% improvement in DSS and 20%, 50% or 70% improvement in LDI in comparison to MTX monotherapy. Conclusions: The combination of golimumab and MTX as first-line bDMARD therapy is superior to MTX monotherapy for the treatment of PsA dactylitis.info:eu-repo/semantics/publishedVersio

Predictors of DAPSA28 remission in patients with psoriatic arthritis initiating a first TNF-inhibitor : results from 13 European registries

The EuroSpA Research Collaboration Network was financially supported by Novartis Pharma AG. Novartis had no influence on the data collection, statistical analyses, manuscript preparation or decision to submit the manuscript.Peer reviewe

Predictors of DAPSA28 remission in patients with psoriatic arthritis initiating a first TNF-inhibitor: results from 13 European registries.

OBJECTIVES In bio-naïve patients with Psoriatic arthritis (PsA) initiating a Tumour Necrosis Factor inhibitor (TNFi), we aimed to identify baseline predictors of Disease Activity index for PsA in 28 joints (DAPSA28) remission (primary objective) and DAPSA28 moderate response at 6 months, as well as drug retention at 12 months across 13 European registries. METHODS Baseline demographic and clinical characteristics were retrieved and the three outcomes investigated per registry and in pooled data, using logistic regression analyses on multiply imputed data. In the pooled cohort, selected predictors that were either consistently positive or negative across all three outcomes, were defined as common predictors. RESULTS In the pooled cohort (n = 13 369), six-month proportions of remission, moderate response and 12-month drug retention were 25%, 34% and 63% in patients with available data (n = 6,954, n = 5,275 and n = 13 369, respectively). Baseline predictors of remission, moderate response and 12-month drug retention were identified, five common across all three outcomes. Odds ratios (95% confidence interval) for DAPSA28 remission were: age, per year: 0.97 (0.96-0.98); disease duration, years (10 vs ≤ 10 mg/l: 1.52 (1.22-1.89) and one mm increase in patient fatigue score: 0.99 (0.98-0.99). CONCLUSION Baseline predictors of remission, response and adherence to TNFi were identified, of which five were common for all three outcomes, indicating that the predictors emerging from our pooled cohort may be considered generalisable from the country- to disease-level