RESUMO
Erythropoietin (EPO) is a glycoprotein hormone which promotes red cell replenishment and is also a global biotherapeutic medicine widely used to treat anaemia resulting, for example, from chemotherapy. Requirements of the European Pharmacopoeia stipulate that the level of dimer must be quantified in clinical EPO products (with a limit of 2%). Quantification is hampered by the lack of reference preparations containing stable measurable levels of EPO dimer, but the reproducible generation of a stable dimerised EPO preparation is challenging. We describe here the development of a lyophilised, chemically cross-linked EPO preparation, which has good stability and may be used for calibration and system suitability assurance for the size exclusion chromatographic separation of EPO preparations. Graphical abstract.
Assuntos
Reagentes de Ligações Cruzadas/química , Eritropoetina/química , Glutaral/química , Calibragem , Cromatografia em Gel/métodos , Cromatografia em Gel/normas , Eritropoetina/análise , Eritropoetina/uso terapêutico , Liofilização , Humanos , Multimerização Proteica , Estabilidade Proteica , Controle de Qualidade , Padrões de ReferênciaRESUMO
RESEARCH QUESTION: Is formulated and lyophilized, recombinant human Müllerian inhibiting substance, also known as anti-Müllerian hormone (AMH), suitable for the preparation of a WHO international standard to calibrate AMH immunoassays? DESIGN: The AMH content of a trial preparation, coded SS-581, was determined by five laboratories using seven immunoassay methods. Participants were requested to report the content of the preparation in terms of their method calibrators through the measurement of a minimum of five concentrations in the linear part of the dose-response curve. Participants were also asked to measure, concomitantly, a panel of six serum samples containing AMH at concentrations of 0.1-13.0 ng/ml. RESULTS: Across all assays, including two automated assays in development, the geometric mean content was 361.76 ng/ampoule with a geometric coefficient of variation (GCV%) of 39.95%. When measured by immunoassays that were commercially available at the time of the study, the mean content was 423.08 ng/ampoule, with a GCV% of 26.67%. The inter-method geometric means of five serum samples with an AMH concentration >0.3 ng/ml and measured concomitantly with dilutions of SS-581 varied with a range of GCV% of 14.90-22.35%, which may reflect the use of serum sample value transfer to calibrate current immunoassays, some of which use non-human AMH calibrators. The AMH in trial preparation SS-581 was shown to be biologically active in the Müllerian duct regression assay. CONCLUSIONS: A reference material prepared using human recombinant AMH is a promising candidate for the preparation of an international standard for AMH for immunoassays calibrated to recombinant human AMH.
Assuntos
Hormônio Antimülleriano/sangue , Imunoensaio/normas , Calibragem , Feminino , HumanosRESUMO
Structural changes involving tissue remodelling and fibrosis are major features of many pulmonary diseases, including asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Abnormal deposition of extracellular matrix (ECM) proteins is a key factor in the development of tissue remodelling that results in symptoms and impaired lung function in these diseases. Tissue remodelling in the lungs is complex and differs between compartments. Some pathways are common but tissue remodelling around the airways and in the parenchyma have different morphologies. Hence it is critical to evaluate both common fibrotic pathways and those that are specific to different compartments; thereby expanding the understanding of the pathogenesis of fibrosis and remodelling in the airways and parenchyma in asthma, COPD and IPF with a view to developing therapeutic strategies for each. Here we review the current understanding of remodelling features and underlying mechanisms in these major respiratory diseases. The differences and similarities of remodelling are used to highlight potential common therapeutic targets and strategies. One central pathway in remodelling processes involves transforming growth factor (TGF)-ß induced fibroblast activation and myofibroblast differentiation that increases ECM production. The current treatments and clinical trials targeting remodelling are described, as well as potential future directions. These endeavours are indicative of the renewed effort and optimism for drug discovery targeting tissue remodelling and fibrosis.
Assuntos
Pneumopatias/tratamento farmacológico , Pneumopatias/fisiopatologia , Remodelação das Vias Aéreas/fisiologia , Asma/tratamento farmacológico , Asma/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos , Fibrose/fisiopatologia , Glicoproteínas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Metaloproteinases da Matriz/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fator de Crescimento Transformador betaRESUMO
Recent clinical trials are evaluating induced pluripotent stem cells (iPSCs) as a cellular therapy in the field of regenerative medicine. The widespread clinical utility of iPSCs is expected to be realized using allogeneic cells that have undergone thorough safety evaluations, including assessment of their immunogenicity. IPSC-derived neural crest stem cells (NCSCs) have significant potential in regenerative medicine; however, their application in cellular therapy has not been widely studied to date, and no reports on their potential immunogenicity have been published so far. In this study, we have assessed the expression of immune-related antigens in iPSC-NCSCs, including human leukocyte antigen (HLA) class I and II and co-stimulatory molecules. To investigate functional immunogenicity, we used iPSC-NCSCs as stimulator cells in a one-way mixed lymphocyte reaction. In these experiments, iPSC-NCSCs did not stimulate detectable proliferation of CD3+ and CD3+CD8+ T cells or induce cytokine production. We show that this was not a result of any immunosuppressive features of iPSC-NCSCs, but rather more consistent with their non-immunogenic molecular phenotype. These results are encouraging for the potential future use of iPSC-NCSCs as a cellular therapy.
RESUMO
The roles played by arachidonic acid and its cyclooxygenase (COX)-generated and lipoxygenase (LOX)-generated metabolites have been studied using rodent islets and insulin-secreting cell lines, but very little is known about COX and LOX isoform expression and the effects of modulation of arachidonic acid generation and metabolism in human islets. We have used RT-PCR to identify mRNAs for cytosolic phospholipase A(2) (cPLA(2)), COX-1, COX-2, 5-LOX, and 12-LOX in isolated human islets. COX-3 and 15-LOX were not expressed by human islets. Perifusion experiments with human islets indicated that PLA(2) inhibition inhibited glucose-stimulated insulin secretion, whereas inhibitors of COX-2 and 12-LOX enzymes enhanced basal insulin secretion and also secretory responses induced by 20 mmol/l glucose or by 50 mumol/l arachidonic acid. Inhibition of COX-1 with 100 mumol/l acetaminophen did not significantly affect glucose-stimulated insulin secretion. These data indicate that the stimulation of insulin secretion from human islets in response to arachidonic acid does not require its metabolism through COX-2 and 5-/12-LOX pathways. The products of COX-2 and LOX activities have been implicated in cytokine-mediated damage of beta-cells, so selective inhibitors of these enzymes would be expected to have a dual protective role in diabetes: they would minimize beta-cell dysfunction while maintaining insulin secretion through enhancing endogenous arachidonic acid levels.
Assuntos
Ácido Araquidônico/fisiologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ácido Araquidônico/metabolismo , Sequência de Bases , Cadáver , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Primers do DNA , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/enzimologia , Lipoxigenase/genética , Fosfolipases A/genética , RNA Mensageiro/genética , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.
Assuntos
Bioensaio/métodos , Produtos Biológicos/química , Interferon-alfa/análise , Produtos Biológicos/metabolismo , Proliferação de Células , Cromatografia/métodos , Desenho de Fármacos , Eletroforese em Gel de Poliacrilamida , Humanos , Interferon alfa-2 , Interferon-alfa/metabolismo , Metionina/química , Oxigênio/química , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Manejo de Espécimes , TemperaturaRESUMO
We have investigated the use of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as an alternative to a selection of late-stage functional bioassays for determination of the potency of preparations of vascular endothelial growth factor (VEGF). Responses were measured in cultures of human umbilical vein endothelial cells (HUVECs). Late-stage responses measured were cell survival and proliferation, and production of interleukin-8 (IL-8), interleukin-6 (IL-6), and tissue factor. The dose-response range was similar across the assays, increasing from 2 ng/mL VEGF and reaching a maximum between 30 ng/mL and 125 ng/mL VEGF. A number of VEGF-induced mRNA species demonstrated dose-response curves suitable for VEGF potency determination. IL-8 mRNA induction after 45 min incubation with VEGF, which showed maximal responses between 15.6 ng/mL and 62.5 ng/mL VEGF, was selected for further characterization. This gene-expression bioassay was robust across a range of cell seeding densities and could be used for samples processed immediately following incubation with VEGF and for cell lysates stored at -80 degrees C for 3 months. We also compared this gene-expression bioassay and the assays of late-stage responses in the potency measurement of the inhibitors of VEGF activity, anti-VEGF monoclonal antibody MAB293, and a VEGF soluble receptor VEGFsR1 preparation. We present a critical evaluation of the use of qRT-PCR in assaying the potency of VEGF and its inhibitors, and of the potential of this platform for measuring the potency of other biological therapeutics.
Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Anticorpos Monoclonais/farmacologia , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Interleucina-8/genética , RNA Mensageiro/análise , Tromboplastina/biossíntese , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
We have generated MIN6 beta-cells that stably overexpress cytosolic phospholipase A(2) (cPLA(2)) and show a ninefold increase in cPLA(2) activity. Overexpression of cPLA(2) did not affect the capacity of MIN6 cells to show elevations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to tolbutamide and KCl, and these depolarizing stimuli produced insulin secretion profiles in cPLA(2)-overexpressing cells similar to those they produced in passage-matched nontransfected MIN6 cells. However, cPLA(2)-overexpressing MIN6 cells did not respond to elevations in extracellular glucose with increases in ATP, [Ca(2+)](i), or insulin secretion. Nontransfected MIN6 cells showed a rapid and sustained increase in NAD(P)H autofluorescence in response to 25 mmol/l glucose, and this was reduced by approximately 95% in MIN6 cells overexpressing cPLA(2). This effect was mimicked in nontransfected MIN6 cells by p-(trifluoromethoxy) phenylylhydrazone, a mitochondrial uncoupler. Quantitative RT-PCR indicated that mRNA for uncoupling protein-2 (UCP-2) was increased in the cPLA(2)-overexpressing MIN6 cells, and this could be prevented by exposure to 100 mumol/l methyl arachidonyl fluorophosphate, a cPLA(2) inhibitor. Glucose caused a decrease in rhodamine 123 fluorescence in control cells, but not in those overexpressing cPLA(2), consistent with the transfected cells being unable to maintain mitochondrial proton gradients as a consequence of UCP-2 upregulation. Our data indicate that overexpression of cPLA(2) results in severe impairment of the calcium and secretory responses of beta-cells to glucose through upregulation of UCP-2 and uncoupling of mitochondrial metabolism from ATP generation.
Assuntos
Insulina/metabolismo , Fosfolipases A/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Glucose/farmacologia , Secreção de Insulina , Canais Iônicos , Ilhotas Pancreáticas , Proteínas de Membrana Transportadoras/genética , Proteínas Mitocondriais/genética , NAD/metabolismo , NADP/metabolismo , Fosfolipases A/genética , Cloreto de Potássio/farmacologia , RNA Mensageiro/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolbutamida/farmacologia , Transfecção , Proteína Desacopladora 2RESUMO
Cyclo-oxygenase (COX), the enzyme responsible for conversion of arachidonic acid to prostanoids, exists as two isoforms. In most tissues, COX-1 is a constitutive enzyme involved in prostaglandin-mediated physiological processes, whereas COX-2 is thought to be induced by inflammatory stimuli. However, it has previously been reported that COX-2 is the dominant isoform in islets and an insulin-secreting beta-cell line under basal conditions. We have investigated the relative abundance of COX-1 and COX-2 mRNAs in MIN6 cells, a mouse insulin-secreting cell line, and in primary mouse and human islets. We found that COX-2 was the dominant isoform in MIN6 cells, but that COX-1 mRNA was more abundant than that of COX-2 in freshly isolated mouse islets. Furthermore, COX-2 expression was induced by maintenance of mouse islets in culture, and experiments with human islets indicated that exposure of the islets to hyperglycemic conditions was sufficient to upregulate COX-2 mRNA levels. Given that hyperglycemia has been reported to increase human beta-cell production of interleukin-1beta and that this cytokine can induce COX-2 expression, our observations of glucose-induced induction of COX-2 in human islets suggest that this is one route through which hyperglycemia may contribute to beta-cell dysfunction.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Ilhotas Pancreáticas/enzimologia , Isoenzimas/genética , Prostaglandina-Endoperóxido Sintases/genética , Animais , Sequência de Bases , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Primers do DNA , Humanos , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas de Membrana , Camundongos , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
When formulating a biopharmaceutical protein, its stability in the liquid state is critical. In addition, when preparing biological reference materials the stability, both when lyophilised and after reconstitution, needs to be determined. In order to optimise the stability in aqueous conditions (as indicated by Tmelt or denaturation point) the impact of different excipient choices should be evaluated. Micro differential scanning calorimetry is a well established method for these applications but can be time consuming even when an autosampler is used. Differential scanning fluorimetry (DSF) is a novel technique which measures the fluorescence of a dye when bound to the hydrophobic regions of a denatured protein. We have investigated these techniques for their suitability using alpha-1-protease inhibitor (A1PI) as a model system and found similar trends in terms of the impact of different excipients by both methods. DSF is a promising method and has advantages in terms of speed and quantities of biological material required and can be performed using a PCR instrument.
Assuntos
Biofarmácia/métodos , Varredura Diferencial de Calorimetria/métodos , Fluorometria/métodos , Proteínas/química , Química Farmacêutica/métodos , Estabilidade de Medicamentos , Excipientes/química , Liofilização/métodos , Desnaturação Proteica , Padrões de Referência , alfa 1-Antitripsina/químicaRESUMO
Human embryonic stem (hES) cells are pluripotent cells isolated from early human embryos. They can be grown in vitro and made to differentiate into many different cell types. These properties have suggested that they may be useful in cell replacement therapy for many degenerative diseases. However, if hES cells could also be manufactured with mutations significant in human disease, they could provide a powerful in-vitro tool for modelling disease processes and progression in a number of different cell types, as well as providing an ideal system for studying in-vitro toxicity and efficacy of drugs and other therapeutic systems such as gene therapy. Embryos with such mutations are generated as part of routine genetic testing during preimplantation genetic diagnosis, providing the opportunity to generate cell lines with significant mutations. A human embryonic stem cell line homozygous for the most common mutation leading to cystic fibrosis in humans (delta F508) has been generated and characterized. This cell line has the same morphology and expresses proteins typical of other unaffected hES cell lines. This cell line represents an important in-vitro tool for understanding the pathophysiology of cystic fibrosis, and presents exciting opportunities to test the efficacy and toxicity of new therapies relevant to CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Células-Tronco Pluripotentes , Técnicas de Cultura de Células , Linhagem Celular , Separação Celular , Humanos , Cariotipagem , Células-Tronco Pluripotentes/metabolismo , Diagnóstico Pré-Implantação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de SequênciaRESUMO
Mature beta-cells and nerve cells share many functional similarities despite originating from different embryonic germ layers. The aim of this study was to investigate the potential of neural stem cells (NSCs), isolated from foetal rat brain, as a starting material from which to generate functionally responsive, insulin-containing cells. Our results demonstrated that NSCs can be significantly expanded in vitro and can be induced to express increased preproinsulin mRNA levels. In addition, these NSC-derived cells expressed transcriptional and functional elements associated with a mature beta-cell phenotype. The differentiated cells showed functional responses typical of pancreatic beta-cells, including glucose-dependent increases in metabolism and rapid elevations in intracellular Ca(2+) in response to the sulphonylurea tolbutamide or to increased glucose concentration. These results suggest that NSCs may have potential as a starting material from which to generate beta-cell surrogates for the treatment of patients with Type 1 diabetes mellitus.
Assuntos
Diferenciação Celular/fisiologia , Insulina/metabolismo , Células-Tronco Multipotentes/metabolismo , Animais , Cálcio/metabolismo , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Células-Tronco Multipotentes/citologia , Proinsulina/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
The generation of human embryonic stem (hES) cells has captured the public and professional imagination, largely due their potential as a means of overcoming many debilitating and degenerative diseases by cell replacement therapy. Despite this potential, few well-characterized hES cell lines have been derived. Indeed, in the UK, despite several centres having been active in this area for more than 2 years, there are as yet no published reports of human embryonic stem cells having been generated. Part of the reason for this lack of progress may relate to the quality of embryos available for research. Embryos surplus to therapeutic requirements following routine assisted reproduction treatment are often of poor quality and a large proportion may be aneuploid. This study reports a new approach to hES cell derivation. Embryos surplus to therapeutic requirements following preimplantation genetic diagnosis were used. Although unsuitable for embryo transfer due to the high risk of genetic disease, these embryos are from fertile couples and thus may be of better quality than fresh embryos surplus to assisted reproduction treatment cycles. Embryos donated after cryopreservation were also used, and putative hES lines were derived from both sources of embryos. The cell lines described here are thought to be the first reported hES cell lines to have been derived in the UK.