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1.
BMC Palliat Care ; 19(1): 123, 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32795270

RESUMO

BACKGROUND: Palliative and end-of-life care development is hindered by a lack of information about the circumstances surrounding dying in developing and resource-poor countries. Our aims were to develop and obtain face and content validity for a self-administered questionnaire on end-of-life care provision and medical decision-making for use in population-based surveys. METHODS: Modelled on validated questionnaires from research in developed countries, our questionnaire was adapted to the cultural sensitivity and medico-legal context of Trinidad and Tobago. Two sets of semi-structured face-to-face cognitive interviews were done with a sample of physicians, sampling was purposive. Phase 1 assessed interpretation of the questions, terminology and content of the questionnaire. Phase 2 was tested on a heterogeneous group of physicians to identify and fix problematic questions or recurring issues. Adjustments were made incrementally and re-tested in successive interviews. RESULTS: Eighteen physicians were interviewed nationwide. Adaptations to questionnaires used in developed countries included: addition of a definition of palliative care, change of sensitive words like expedited to influenced, adjustments to question formulations, follow-up questions and answer options on medications used were added, the sequence, title and layout were changed and instructions for completion were included at the beginning of the questionnaire. CONCLUSION: A new instrument for assessing and documenting end-of-life care and circumstances of dying in a small, resource-poor Caribbean country was developed and validated, and can be readily used as a mortality follow-back instrument. Our methods and procedures of development can be applied as a guide for similar studies in other small developing countries.


Assuntos
Tomada de Decisões , Inquéritos e Questionários/normas , Assistência Terminal/estatística & dados numéricos , Adulto , Região do Caribe , Feminino , Seguimentos , Recursos em Saúde/provisão & distribuição , Humanos , Entrevistas como Assunto/métodos , Masculino , Pessoa de Meia-Idade , Pesquisa Qualitativa , Reprodutibilidade dos Testes , Inquéritos e Questionários/estatística & dados numéricos , Assistência Terminal/métodos
2.
Ann Palliat Med ; 11(5): 1660-1670, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34930013

RESUMO

BACKGROUND: In resource-poor countries, including in Latin American and the Caribbean, empirical information about the characteristics and incidence of medical end-of-life decisions (MELDs)-withholding or withdrawing potentially life-prolonging medical treatments-is largely absent. METHODS: The aim was to describe the incidence and decision-making characteristics of MELDs taken prior to the death of people who died at home in Trinidad and Tobago (T&T). A mortality follow-back study was used where a representative sample of deaths occurring at home in 2018 was drawn from death certificates at the national death registry. The general practitioners who certified the deaths were sent a questionnaire. RESULTS: The sample consisted of 309 adult deaths and the response rate was 31% (N=96). Physicians were: mostly male (79.2%), practiced medicine for more than twenty-years (63.5%), had no formal palliative care training (69.8%). Non-sudden deaths represented 76% (N=73), of these, medications to alleviate pain and symptoms in the last 7 days of life were administered in 65.8%, including opioids 21%. Potentially life-prolonging treatments were withheld in 9.6% but none withdrawn. No physician/patient discussions about various end-of-life treatment options occurred in 61.6%. Compared to physicians with no formal training in palliative care, those with training more often: prescribed or administered opioids in the last 7 days of life (35.7% vs. 11.1%, P=0.01), had discussions with patients about end-of-life treatment options (60.7% vs. 24.4%, P=0.002), and discussed medication use to alleviate pain and other symptoms with patients (50% vs. 17.8%, P=0.004). CONCLUSIONS: Differences in the care and treatment general practitioners provided to their patients could be associated with them having been formally trained in palliative care. The necessary support to further develop palliative care in T&T is needed.


Assuntos
Tomada de Decisões , Assistência Terminal , Adulto , Analgésicos Opioides/uso terapêutico , Tomada de Decisão Clínica , Morte , Feminino , Humanos , Masculino , Cuidados Paliativos , Inquéritos e Questionários
3.
Assay Drug Dev Technol ; 19(1): 27-37, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33164547

RESUMO

Phenotypic screening is a neoclassical approach for drug discovery. We conducted phenotypic screening for insulin secretion enhancing agents using INS-1E insulinoma cells as a model system for pancreatic beta-cells. A principal regulator of insulin secretion in beta-cells is the metabolically regulated potassium channel Kir6.2/SUR1 complex. To characterize hit compounds, we developed an assay to quantify endogenous potassium channel activity in INS-1E cells. We quantified ligand-regulated potassium channel activity in INS-1E cells using fluorescence imaging and thallium flux. Potassium channel activity was metabolically regulated and coupled to insulin secretion. The pharmacology of channel opening agents (diazoxide) and closing agents (sulfonylureas) was used to validate the applicability of the assay. A precise high-throughput assay was enabled, and phenotypic screening hits were triaged to enable a higher likelihood of discovering chemical matter with novel and useful mechanisms of action.


Assuntos
Diazóxido/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Secretagogos/farmacologia , Compostos de Sulfonilureia/farmacologia , Receptores de Sulfonilureias/metabolismo , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Imagem Óptica , Fenótipo
4.
J Immunol Methods ; 466: 9-16, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30590020

RESUMO

IL-21 is a pleiotropic cytokine that plays a key role in modulating inflammatory responses, including the promotion of autoimmune diseases. Several groups have quantitated circulating levels of IL-21 in plasma and serum samples using various commercial ELISAs. We determined, however, that the most commonly used commercial assays in published literature were not specific or sensitive enough to detect levels of IL-21 in heparin plasma or serum from healthy human individuals. This finding prompted an effort to develop more specific and sensitive methods to quantitate IL-21 in complex biological matrices using proprietary anti-IL-21 antibodies with the Quanterix SiMoA platform and the Meso Scale Discovery (MSD) S-PLEX® format. Assays developed on both technology platforms were characterized in heparin plasma and serum using spike recoveries across a range of concentrations. Each method was able to detect sub-pg/mL levels of IL-21 (predicted Limit of Detection [LOD] of approximately 1.0 fg/mL for both the Quanterix SiMoA and MSD S-PLEX® platforms) which is 200-500 times lower than current commercial assays. Additionally we demonstrated that rheumatoid factor did not interfere with measuring IL-21 in the Quanterix SiMoA assay. Results obtained with the two new ultrasensitive assays showed a strong correlation (r = 0.9428; p < .0001). Additionally, IL-21 levels were significantly increased in samples from patients with Systemic Lupus Erythematosus (mean+/- SD: n = 14, 202.64 +/- 111.47 fg/mL, p = .0001 for Quanterix SiMoA and 275.4 +/- 174.66 fg/mL p = .0001 for MSD S-PLEX®) as well as in samples from patients with Sjögren's Syndrome (mean+/- SD: n = 11, 122.18 +/- 84.50 fg/mL, p = .0029 for Quanterix SiMoA and 183.64 +/- 153.00 fg/mL, p = .0082 for MSD S-PLEX®) when compared to healthy donors (mean+/- SD: n = 11, 38.1 +/- 27.8 fg/mL for Quanterix SiMoA and 58.1 +/- 30.7 fg/mL for MSD S-PLEX®). These ultrasensitive assays, for the first time, allow for the accurate quantitation of human IL-21 in heparin plasma and serum. In addition, these experiments also provide a direct comparison of the MSD S-PLEX® format and Quanterix SiMoA platform technologies, which may have broader implications to future application of these methods to evaluate low abundance proteins in complex biological matrices.


Assuntos
Ensaio de Imunoadsorção Enzimática , Interleucinas/sangue , Voluntários Saudáveis , Humanos , Interleucinas/imunologia
5.
J Biomol Screen ; 11(3): 247-52, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16490779

RESUMO

In this article, the authors compare the assay performance measures, signal window, Z' factor, and assay variability ratio. They examine their mathematical formulae for similarities and differences, describe their statistical sampling properties using the results of a computer simulation, and illustrate their use with example data. Based on these results, the authors recommend the Z' factor as a preferred measure of assay performance for screening assays and point out that none of these measures are adequate for characterizing concentration-response assays.


Assuntos
Modelos Teóricos , Animais , Proliferação de Células , Pulmão/citologia , Pulmão/efeitos dos fármacos , Vison
6.
J Biomol Screen ; 16(6): 588-602, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21521801

RESUMO

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target "agnostic" fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD(2)), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD(2) assay panel. Analysis of PD(2) submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD(2) have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD(2), may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.


Assuntos
Descoberta de Drogas , Fenótipo , Animais , Apolipoproteínas E/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , Insulina/metabolismo , Secreção de Insulina , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Nocodazol/farmacologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Proteínas Wnt/metabolismo
7.
Lipids ; 45(8): 757-64, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20640528

RESUMO

ATP-binding cassette transporter A-1 (ABCA1) mediates the transfer of cellular cholesterol to lipid-poor apolipoproteins. Liver X receptors (LXRs) are regulators of cholesterol homeostasis that increase transcription of ABCA1. Synthetic LXR agonists developed to date have been shown to induce ABCA1 mRNA expression and increase reverse cholesterol transport. Unfortunately, there have been few options for quantitatively measuring ABCA1 protein levels, including a previously described competitive ELISA standardized to an ABCA1 peptide with a sensitivity of 80 ng/ml. To address this unmet need, we developed a novel sandwich ELISA standardized to full-length human recombinant ABCA1 protein with sensitivity of approximately 0.5 ng/ml. To determine if the sandwich ELISA had adequate sensitivity to detect LXR-induced increases in ABCA1, we utilized it to measure ABCA1 levels in untreated and LXR agonist-treated human (THP-1) macrophage cells and human peripheral blood mononuclear cells (PBMC). Data obtained from the ELISA demonstrated an approximately eightfold increase in ABCA1 levels in both macrophages as well as PBMC in response to LXR agonist treatment, and results were highly correlated with those obtained by immunoprecipitation and western blotting. Together, these results suggest that the sandwich ELISA may be a sensitive and effective method for quantitating ABCA1 protein levels.


Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Ensaio de Imunoadsorção Enzimática/métodos , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Humanos , Imunoprecipitação , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo
8.
Brief Funct Genomic Proteomic ; 6(2): 149-58, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17584762

RESUMO

Immunoprecipitation (IP) combined with matrix-assisted laser desorption ionization (MALDI) time of flight (Tof) mass spectrometry has been used to develop quantitative assays for amyloid-beta (Abeta) peptides in cerebrospinal fluid (CSF). Inclusion of (15)N labelled standard peptides allows for absolute quantification of multiple Abeta isoforms in individual samples. Characterization of variability associated with all steps of the assay indicated that the IP step is the single largest contributor to overall variability. Optimization of the assay resulted in overall coefficient of variation

Assuntos
Peptídeos beta-Amiloides/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/metabolismo , Biomarcadores/análise , Biomarcadores/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoprecipitação , Isoformas de Proteínas/líquido cefalorraquidiano
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