RESUMO
The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.
Assuntos
Bioensaio , Relatório de Pesquisa , Citometria de Fluxo/métodos , Ligantes , Biomarcadores/análise , Bioensaio/métodosRESUMO
Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.
Assuntos
Cardiomiopatia Chagásica , Doença de Chagas , Trifosfato de Adenosina/metabolismo , Biomarcadores/metabolismo , Cardiomiopatia Chagásica/diagnóstico , Cardiomiopatia Chagásica/genética , Doença de Chagas/genética , Metilação de DNA , HumanosRESUMO
Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.
Assuntos
Cardiomiopatia Chagásica , Doença de Chagas , Trypanosoma cruzi , Doença de Chagas/genética , Epigênese Genética , Humanos , Fatores de Transcrição/genéticaRESUMO
Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.
Assuntos
Cardiomiopatia Chagásica , Doença de Chagas , Metilação , Doenças Parasitárias , Terapêutica , BiomarcadoresRESUMO
Abstract: Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.
Assuntos
Humanos , Cardiomiopatia Chagásica , Doença de Chagas/genética , Fatores de Transcrição/genética , Trypanosoma cruzi , Epigênese Genética , MetilaçãoRESUMO
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included three Main Workshops and seven Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "context of use" [COU]); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.
Assuntos
Citometria de Fluxo , Biomarcadores/análise , Citometria de Fluxo/métodos , Humanos , Indicadores e Reagentes , Biópsia Líquida , Espectrometria de MassasRESUMO
Chronic Chagas disease (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis compared to other cardiomyopathies. We show the expression and activity of Matrix Metalloproteinases (MMP) and of their inhibitors TIMP (tissue inhibitor of metalloproteinases) in myocardial samples of end stage CCC, idiopathic dilated cardiomyopathy (DCM) patients, and from organ donors. Our results showed significantly increased mRNA expression of several MMPs, several TIMPs and EMMPRIN in CCC and DCM samples. MMP-2 and TIMP-2 protein levels were significantly elevated in both sample groups, while MMP-9 protein level was exclusively increased in CCC. MMPs 2 and 9 activities were also exclusively increased in CCC. Results suggest that the balance between proteins that inhibit the MMP-2 and 9 is shifted toward their activation. Inflammation-induced increases in MMP-2 and 9 activity and expression associated with imbalanced TIMP regulation could be related to a more extensive heart remodeling and poorer prognosis in CCC patients.
Assuntos
Cardiomiopatia Dilatada , Cardiomiopatia Chagásica , Cardiomiopatia Dilatada/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , MiocárdioRESUMO
BACKGROUND: A key step in clinical flow cytometry data analysis is gating, which involves the identification of cell populations. The process of gating produces a set of reportable results, which are typically described by gating definitions. The non-standardized, non-interpreted nature of gating definitions represents a hurdle for data interpretation and data sharing across and within organizations. Interpreting and standardizing gating definitions for subsequent analysis of gating results requires a curation effort from experts. Machine learning approaches have the potential to help in this process by predicting expert annotations associated with gating definitions. METHODS: We created a gold-standard dataset by manually annotating thousands of gating definitions with cell type and functional marker annotations. We used this dataset to train and test a machine learning pipeline able to predict standard cell types and functional marker genes associated with gating definitions. RESULTS: The machine learning pipeline predicted annotations with high accuracy for both cell types and functional marker genes. Accuracy was lower for gating definitions from assays belonging to laboratories from which limited or no prior data was available in the training. Manual error review ensured that resulting predicted annotations could be reused subsequently as additional gold-standard training data. CONCLUSIONS: Machine learning methods are able to consistently predict annotations associated with gating definitions from flow cytometry assays. However, a hybrid automatic and manual annotation workflow would be recommended to achieve optimal results.
Assuntos
Aprendizado de Máquina , Citometria de Fluxo , Humanos , Fluxo de TrabalhoRESUMO
Barcoded flow cytometry is a multiplexing technique allowing for the simultaneous acquisition of cells from different donors or experimental conditions in a high-throughput manner. This approach allows to synchronize acquisition of samples and reduce variance introduced through the operator or technical platform. However, to date, only very few flow cytometry barcoding protocols have been developed, which often suffer from technical limitations. Here, we developed a novel barcoding protocol for a full-spectrum flow cytometry platform. We developed a 21-color immunophenotyping assay for up to 20 different samples analyzed simultaneously with comparable variance between repeated single-tube acquisition and postde-multiplexing. Barcoding offers great potential in parallelizing the analysis of complex cell populations such as peripheral blood mononuclear cells (PBMCs). Consequently, we assessed the performance of our method in situations where PBMCs were challenged with phytohaemagglutinin (PHA), a strong mitogen and broad activator of B cells and T cells, and superantigen Staphylococcus enterotoxin B (SEB) that has been reported to induce polyclonal T cell activation. PBMCs were either barcoded before pooled challenge or challenged individually pre-barcoding. Our final workflow included pooled immunophenotyping followed by machine learning aided single-cell data analysis and enabled us to identify robust PHA and SEB mode of action related phenotypic changes in PBMC immune cell lineages. Conclusively, we present a novel technique allowing the barcoded acquisition and analysis of PBMCs from up to 20 different donors and present a valid basis for the future development of complex immunophenotyping protocols.
Assuntos
Leucócitos Mononucleares , Análise de Célula Única , Imunofenotipagem , Citometria de Fluxo/métodos , Análise de Célula Única/métodos , FenótipoRESUMO
Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy occurring in 30% of the 6 million infected with the protozoan Trypanosoma cruzi in Latin America. Survival is significantly lower in CCC than ischemic (IC) and idiopathic dilated cardiomyopathy (DCM). Previous studies disclosed a selective decrease in mitochondrial ATP synthase alpha expression and creatine kinase activity in CCC myocardium as compared to IDC and IC, as well as decreased in vivo myocardial ATP production. Aiming to identify additional constraints in energy metabolism specific to CCC, we performed a proteomic study in myocardial tissue samples from CCC, IC and DCM obtained at transplantation, in comparison with control myocardial tissue samples from organ donors. Left ventricle free wall myocardial samples were subject to two-dimensional electrophoresis with fluorescent labeling (2D-DIGE) and protein identification by mass spectrometry. We found altered expression of proteins related to mitochondrial energy metabolism, cardiac remodeling, and oxidative stress in the 3 patient groups. Pathways analysis of proteins differentially expressed in CCC disclosed mitochondrial dysfunction, fatty acid metabolism and transmembrane potential of mitochondria. CCC patients' myocardium displayed reduced expression of 22 mitochondrial proteins belonging to energy metabolism pathways, as compared to 17 in DCM and 3 in IC. Significantly, 6 beta-oxidation enzymes were reduced in CCC, while only 2 of them were down-regulated in DCM and 1 in IC. We also observed that the cytokine IFN-gamma, previously described with increased levels in CCC, reduces mitochondrial membrane potential in cardiomyocytes. Results suggest a major reduction of mitochondrial energy metabolism and mitochondrial dysfunction in CCC myocardium which may be in part linked to IFN-gamma. This may partially explain the worse prognosis of CCC as compared to DCM or IC.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/fisiopatologia , Coração/fisiopatologia , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Adolescente , Adulto , Metabolismo Energético/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Miocárdio/patologia , Adulto JovemRESUMO
Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Interferon gama/metabolismo , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Adulto , Idoso , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/fisiopatologia , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia , Miócitos Cardíacos/patologia , Adulto JovemRESUMO
Receptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total-drug-bound RO assay format directly assesses mAb binding to cell surface targets using anti-drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG1 P329GLALA backbone. Using this reagent, we developed a total-drug-bound RO assay format for RG7769, a bi-specific P329GLALA containing mAb targeting PD-1 and TIM3 on T cells. In its fit-for-purpose validated version, this RO assay has been used in the Phase-I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint-inhibitor (CPI) naïve patients and anti-PD-1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre-dosing was roughly twofold lower in patients recently having undergone anti-PD-1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti-PD-1 treatment.
Assuntos
Anticorpos Monoclonais , Bioensaio , Biomarcadores , Citometria de Fluxo , HumanosRESUMO
The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.
Assuntos
Bioensaio , Biotecnologia , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Relatório de Pesquisa , Biomarcadores/análise , HumanosRESUMO
Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes’ mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.
RESUMO
Chronic Chagas disease cardiomyopathy (CCC), an especially aggressive inflammatory dilated cardiomyopathy caused by lifelong infection with the protozoan Trypanosoma cruzi, is a major cause of cardiomyopathy in Latin America. Although chronic myocarditis may play a major pathogenetic role, little is known about the molecular mechanisms responsible for its severity. The aim of this study is to study the genes and microRNAs expression in tissues and their connections in regards to the pathobiological processes. To do so, we integrated for the first time global microRNA and mRNA expression profiling from myocardial tissue of CCC patients employing pathways and network analyses. We observed an enrichment in biological processes and pathways associated with the immune response and metabolism. IFNγ, TNF and NFkB were the top upstream regulators. The intersections between differentially expressed microRNAs and differentially expressed target mRNAs showed an enrichment in biological processes such as Inflammation, inflammation, Th1/IFN-γ-inducible genes, fibrosis, hypertrophy, and mitochondrial/oxidative stress/antioxidant response. MicroRNAs also played a role in the regulation of gene expression involved in the key cardiomyopathy-related processes fibrosis, hypertrophy, myocarditis and arrhythmia. Significantly, a discrete number of differentially expressed microRNAs targeted a high number of differentially expressed mRNAs (>20) in multiple processes. Our results suggest that miRNAs orchestrate expression of multiple genes in the major pathophysiological processes in CCC heart tissue. This may have a bearing on pathogenesis, biomarkers and therapy.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Regulação da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Doença Crônica , Genoma Humano , Humanos , MicroRNAs/genética , Análise de Componente PrincipalRESUMO
BACKGROUND: This phase Ib study evaluated the safety, clinical activity, pharmacokinetics, and pharmacodynamics (PD) of emactuzumab (anti-colony stimulating factor 1 receptor monoclonal antibody (mAb)) in combination with selicrelumab (agonistic cluster of differentiation 40 mAb) in patients with advanced solid tumors. METHODS: Both emactuzumab and selicrelumab were administered intravenously every 3 weeks and doses were concomitantly escalated (emactuzumab: 500 to 1000 mg flat; selicrelumab: 2 to 16 mg flat). Dose escalation was conducted using the product of independent beta probabilities dose-escalation design. PD analyzes were performed on peripheral blood samples and tumor/skin biopsies at baseline and on treatment. Clinical activity was evaluated using investigator-based and Response Evaluation Criteria In Solid Tumors V.1.1-based tumor assessments. RESULTS: Three dose-limiting toxicities (all infusion-related reactions (IRRs)) were observed at 8, 12 and 16 mg of selicrelumab together with 1000 mg of emactuzumab. The maximum tolerated dose was not reached at the predefined top doses of emactuzumab (1000 mg) and selicrelumab (16 mg). The most common adverse events were IRRs (75.7%), fatigue (54.1%), facial edema (37.8%), and increase in aspartate aminotransferase and creatinine phosphokinase (35.1% both). PD analyzes demonstrated an increase of Ki67+-activated CD8+ T cells accompanied by a decrease of B cells and the reduction of CD14Dim CD16bright monocytes in peripheral blood. The best objective clinical response was stable disease in 40.5% of patients. CONCLUSION: Emactuzumab in combination with selicrelumab demonstrated a manageable safety profile and evidence of PD activity but did not translate into objective clinical responses. TRIALREGISTRATION NUMBER: NCT02760797.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Antígenos CD40/metabolismo , Neoplasias/tratamento farmacológico , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Feminino , Humanos , Masculino , Neoplasias/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
OBJECTIVE: To evaluate the levels of engagement of multi-professional health residents of a higher education institution in the northwest of São Paulo. METHOD: A cross-sectional census study in which the Utrecht Work Engagement Scale was used to identify the level of relationship with work (Total score) through 17 questions distributed in the Vigor, Dedication and Absorption dimensions. RESULTS: Participation of 50 professionals, of which 92.0% were female, median age of 24 years, 88.0% were single; 82.0% were satisfied with the program, and 56.0% had thought of giving up. Professionals satisfied with the program had high levels for Total Score (4.0) and Dedication (4.5), and average levels for Absorption (3.9) and Vigor (3.8). Those who reported dissatisfaction had average levels in all dimensions (Vigor: 3.2, Absorption: 3.5, Dedication: 3.5) and in the Total score (3.2), which are considered positive results. CONCLUSION: Professionals presented good levels of engagement in spite of dissatisfactions with the program. The results showed a good relationship between professionals and preceptors and supervisors, which reinforces that support and recognition of professional performance are important for strengthening the engagement, especially at the beginning of the career.
Assuntos
Pessoal de Saúde/psicologia , Internato não Médico , Satisfação Pessoal , Preceptoria , Adulto , Brasil , Estudos Transversais , Feminino , Pessoal de Saúde/educação , Humanos , Relações Interprofissionais , Masculino , Adulto JovemRESUMO
ABSTRACT Objective: To evaluate the levels of engagement of multi-professional health residents of a higher education institution in the northwest of São Paulo. Method: A cross-sectional census study in which the Utrecht Work Engagement Scale was used to identify the level of relationship with work (Total score) through 17 questions distributed in the Vigor, Dedication and Absorption dimensions. Results: Participation of 50 professionals, of which 92.0% were female, median age of 24 years, 88.0% were single; 82.0% were satisfied with the program, and 56.0% had thought of giving up. Professionals satisfied with the program had high levels for Total Score (4.0) and Dedication (4.5), and average levels for Absorption (3.9) and Vigor (3.8). Those who reported dissatisfaction had average levels in all dimensions (Vigor: 3.2, Absorption: 3.5, Dedication: 3.5) and in the Total score (3.2), which are considered positive results. Conclusion: Professionals presented good levels of engagement in spite of dissatisfactions with the program. The results showed a good relationship between professionals and preceptors and supervisors, which reinforces that support and recognition of professional performance are important for strengthening the engagement, especially at the beginning of the career.
RESUMEN Objetivo: Evaluar los niveles de engagement (compromiso) de residentes multiprofesionales en salud de un centro de enseñanza superior del noroeste del Estado de São Paulo. Método: Estudio censal transversal, que utilizó la Utrecht Work Engagement Scale para identificar el nivel de relación con el trabajo (Score general) por medio de 17 cuestiones distribuidas en las dimensiones Vigor, Dedicación y Absorción. Resultados: Participaron 50 profesionales. El 92,0% del sexo femenino, edad mediana de 24 años, el 88,0% solteros; el 82,0% se refirieron satisfechos con el programa; y el 56,0% ya pensaron en desistir. Los profesionales satisfechos con el programa presentaron Score general (4,0) y Dedicación (4,5) altos, y niveles medios de Absorción (3,9) y Vigor (3,8). Los que se refirieron insatisfechos presentaron niveles medios en todas las dimensiones (Vigor: 3,2; Absorción: 3,5; Dedicación: 3,5) y en el Score general (3,2), resultados considerados positivos. Conclusión: Los profesionales presentaron buenos niveles de engagement, aun habiendo insatisfacciones con el programa. Los resultados mostraron buena relación de los profesionales con preceptores y supervisores, reforzando que el apoyo y el reconocimiento del desempeño de los profesionales son importantes para el fortalecimiento del engagement, especialmente en el inicio de la carrera.
RESUMO Objetivo: Avaliar os níveis de engagement de residentes multiprofissionais em saúde de uma instituição de ensino superior do noroeste paulista. Método: Estudo censitário transversal, que utilizou a Utrecht Work Engagement Scale para identificar o nível de relação com o trabalho (Escore geral) por meio de 17 questões distribuídas nas dimensões Vigor, Dedicação e Absorção. Resultados: Participaram 50 profissionais. 92,0% do sexo feminino, idade mediana de 24 anos, 88,0% solteiros; 82,0% referiram-se satisfeitos com o programa, e 56,0% já pensaram em desistir. Os profissionais satisfeitos com o programa apresentaram Escore geral (4,0) e Dedicação (4,5) altos, e níveis médios de Absorção (3,9) e Vigor (3,8). Os que se referiram insatisfeitos apresentaram níveis médios em todas as dimensões (Vigor: 3,2; Absorção: 3,5; Dedicação: 3,5) e no o Escore geral (3,2), resultados considerados positivos. Conclusão: Os profissionais apresentaram bons níveis de engagement, mesmo havendo insatisfações com o programa. Os resultados mostraram boa relação dos profissionais com preceptores e supervisores, reforçando que o apoio e o reconhecimento do desempenho dos profissionais são importantes para o fortalecimento do engagement, principalmente no início da carreira.
Assuntos
Animais , Capacitação de Recursos Humanos em Saúde , Engajamento no Trabalho , Internato e Residência , Relações Interprofissionais , Saúde OcupacionalRESUMO
RESUMO Objetivo: Avaliar os níveis de ansiedade e depressão dos profissionais matriculados em um Programa de Residência Médica em Pediatria de uma instituição de ensino do interior do Estado de São Paulo. Métodos: Estudo transversal descritivo, com todos os médicos residentes matriculados no Programa de Residência Médica em Pediatria. Os dados foram coletados entre os meses de novembro de 2013 e fevereiro de 2014, utilizando-se três instrumentos autoaplicáveis: um instrumento elaborado pelos autores, com dados sociodemográficos; a Escala de Ansiedade de Beck ou Inventário de Ansiedade de Beck e a Escala de Depressão de Beck ou Inventário de Depressão de Beck. Os níveis de ansiedade e depressão foram analisados por uma psicóloga segundo dados dos instrumentos e categorizados em ausência de depressão/ansiedade, depressão/ansiedade leve, depressão/ansiedade moderada e depressão/ansiedade grave. Resultados: Participaram do estudo 36 médicos residentes. Houve predominância do sexo feminino (91,4%), idade mediana de 28 anos (mínimo: 25; máximo: 34), solteiros (86,11%), renda familiar de dez ou mais salários mínimos (47,1%), jornada de trabalho de 12 horas ou mais (55,6%), sem atividade física (55,5%) e de lazer (44,2%), com outro vínculo laboral (71,4%), satisfeitos com o trabalho (88,9%); 52,8% pensaram em desistir do programa. Ansiedade esteve presente em 50,0% dos profissionais e depressão em 44,4%. Houve associação estatística da ansiedade com a faixa etária (p<0,005) e com o desejo de desistir do programa (p=0,038); e da depressão com a faixa etária (p=0,001), prática de atividade física (p=0,016), atividades de lazer (p=0,012) e com o desejo de desistir do programa (p=0,008). Conclusão: Os níveis de ansiedade e depressão foram superiores aos observados em outros programas, havendo associação destes transtornos com a faixa etária e ausência de atividade física e de lazer, evidenciando a necessidade de maior atenção e suporte aos profissionais, de implementação de ações de controle dos fatores estressores entre os residentes de Pediatria ede estratégias de promoção do bem-estar físico e mental desses profissionais.
ABSTRACT Objective: To evaluate the levels of anxiety and depression among professionals enrolled in a Pediatric Residency Program of an educational institution in the State of São Paulo, Brazil. Methods: Cross-sectional and descriptive study with all, registered residents at a pediatric residency program. The data were collected between the months of November 2013 and February 2014, using three instruments: an instrument drawn up by the authors, containing demographic data; the Beck anxiety Scale or Beck anxiety inventory and the Beck Depression scale or Beck Depression inventory. Depression anxiety levels were evaluated by a psychologist, according to data produced by the instruments and categorized in to four levels: absence of depression/anxiety, mild depression/anxiety, moderate depression/anxiety and severe depression/anxiety. Results: The study was based on a sample of 36 medical residents. There was a predominance of females (91.4%), with a median age of 28 years (minimum: 25; maximum: 34), single (86.11%), with a household income of 10 minimum monthly wages or more (47.1%), a working day of 12 hours or more (55.6%), not regularly engaged in physical activity (55.5%) and leisure (44.2%), with another job (71.4%) satisfied with their job (88.9%) and having thought about quitting the program (52.8%). Anxiety was present in 50.0% and depression in 44.4%. Statistical associations were found between anxiety and the age group (p < 0.005) and with a desire to give up the program (p = 0.038); and between depression and age group (p = 0.001), practice of physical activity (p = 0.016), leisure activities (p = 0.012) and a desire to give up the program (p = 0.008). Conclusion: The levels of anxiety and depression were higher than those observed in other programs, with associations observed between these disorders and age, lack of physical activity and leisure, highlighting the need for greater attention and professional support, and the implementation of control measures for stressor factors among the resident pediatricians, and of strategies to promote the physical and mental well-being of these professionals.
RESUMO
Objetivo: Este estudo avaliou os níveis de engagement no trabalho em profissionais de programas de aprimoramento e aperfeiçoamento profissional em saúde. Método: Realizou-se um estudo transversal com 82 profissionais de saúde dos programas de aprimoramento e aperfeiçoamento de uma instituição pública do interior paulista, utilizando-se a Utrech Work Engagement Scale (UWES), um questionário autoaplicável composto de 17 itens de autoavaliação em três dimensões: vigor, dedicação e absorção. Os escores foram calculados conforme modelo estatístico proposto no Manual Preliminar UWES. Resultados: Os níveis de engagement foram muito altos na dimensão vigor, altos na dimensão dedicação e no escore geral e médio na dimensão absorção para 71,61%, 58,03%, 53,75% e 51,22% dos profissionais, respectivamente. Os profissionais apresentaram relação positiva com o trabalho, são responsáveis, motivados e dedicados ao trabalho. Conclusão: O estudo reforça a importância de avaliar os aspectos positivos da relação entre profissionais e ambiente laboral, contribuindo para fortalecer os programas de aprimoramento e aperfeiçoamento profissionais.
Objective: This study evaluated the levels of engagement at work in enhancement programs and professionals training in health. Method: A cross-sectional study with 82 health professionals enhancement programs and improvement of a public institution in the State of São Paulo, using the Utrech Work Engagement Scale (UWES), a self-administered questionnaire composed of seventeen self-assessment items in three dimensions: vigor, dedication and absorption. The scores were calculated according to the statistical model proposed in the Preliminary Manual UWES. Results: Engagement levels were too high on the force, high dedication and dimension in general score, and medium in size to 71.61% absorption, 58.03%, 53.75% and 51.22% of workers, respectively. The professionals present positive relationship with the work; they are responsible, motivated and dedicated to the job and to the patients. Conclusion: Reinforces the importance of studies that evaluate positive aspects of the relationship between professionals and working environment, contributing to strengthen the programs of improvement, advancing the profile of professionals into the labour market.