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The potent cytotoxic property of Vγ2Vδ2 T cells makes them attractive for adoptive T cell transfer therapy. The transfusing of the expanded Vγ2Vδ2 T cells into cancer patients shows well-tolerated, but the clinical response rates are required to be improved, implying that there is still an unmet efficacy with low toxicity for this novel anti-tumor therapy. In this study, we test the anti-tumor efficacy of a Y-body-based bispecific antibody (bsAb) Vγ2 x PD-L1 that preferentially redirects Vγ2Vδ2 T cells to combat PD-L1 positive tumor cells. With nanomolar affinity levels to Vγ2Vδ2 T cells and PD-L1+ tumor cells, Vγ2 x PD-L1 bridges a Vγ2Vδ2 T cell with a SKOV3 tumor cell to form a cell-to-cell conjugation. In a PD-L1-dependent manner, the bsAb elicits effective activation (CD25+CD69+), IFNγ releasing, degranulation (CD107a+), and cytokine production (IFNγ+ and TNFα+) of expanded Vγ2Vδ2 T cells. The activations of the Vγ2Vδ2 T cells eliminate PD-L1-expressing human cancer cell lines, including H1975, SKOV3, A375, H1299, and H2228 cells, but not PD-L1 negative cells including HEK-293 (293) cells and healthy PBMCs. Finally, we show that combining Vγ2 x PD-L1 with adoptively transferring Vγ2Vδ2 T cells inhibits the growth of existing tumor xenografts and increases the number of Vγ2Vδ2 T cells into the tumor bed. Vγ2 x PD-L1 represents a promising reagent for increasing the efficacy of adoptively transferred Vγ2Vδ2 T cells in the treatment of PD-L1 positive malignant tumors.
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Anticorpos Biespecíficos , Neoplasias , Anticorpos Biespecíficos/farmacologia , Antígeno B7-H1/imunologia , Células HEK293 , Humanos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T , Linfócitos TRESUMO
[This corrects the article DOI: 10.1093/abt/tbaa011.].
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BACKGROUND: A T cell-redirecting bispecific antibody (bsAb) consisting of a tumor-binding unit and a T cell-binding unit is a large group of antibody-based biologics against death-causing cancer diseases. The anti-CD38 × anti-CD3 bsAb (Y150) is potential for treating multiple myeloma (MM). When developing a cell-based reporter gene bioassay to assess the activities of Y150, it was found that the expression of CD38 on the human T lymphocyte cells (Jurkat) caused the nonspecific activation, which interfered with the specific T cells activation of mediated by the Y150 and CD38(+) tumor cells. METHODS: Here, we first knocked-out the CD38 expression on Jurkat T cell line by CRISPR-Cas9 technology, then developed a stable monoclonal CD38(-) Jurkat T cell line with an NFAT-RE driving luciferase expressing system. Further based on the CD38(-) Jurkat cell, we developed a reporter gene method to assess the bioactivity of the anti-CD38 × anti-CD3 bsAb. RESULTS: Knocking out CD38 expression abolished the nonspecific self-activation of the Jurkat cells. The selected stable monoclonal CD38(-) Jurkat T cell line assured the robustness of the report genes assay for the anti-CD38 × anti-CD3 bsAb. The relative potencies of the Y150 measured by the developed reporter gene assay were correlated with those by the flow-cytometry-based cell cytotoxicity assay and by the ELISA-based binding assay. CONCLUSIONS: The developed reporter gene assay was mechanism of action-reflective for the bioactivity of anti-CD38 × anti-CD3 antibody, and suitable for the quality control for the bsAb product.
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The pandemic of COVID-19 caused by SARS-CoV-2 has raised a new challenges to the scientific and industrious fields after over 1-year spread across different countries. The ultimate approach to end the pandemic is the timely application of vaccines to achieve herd immunity. Here, a novel SARS-CoV-2 receptor-binding domain (RBD) homodimer was developed as a SARS-CoV-2 vaccine candidate. Formulated with aluminum adjuvant, RBD dimer elicited strong immune response in both rodents and non-human primates, and protected mice from SARS-CoV-2 challenge with significantly reducing viral load and alleviating pathological injury in the lung. In the non-human primates, the vaccine could prevent majority of the animals from SARS-CoV-2 infection in the respiratory tract and reduce lung damage. In addition, antibodies elicited by this vaccine candidate showed cross-neutralization activities to SARS-CoV-2 variants. Furthermore, with our expression system, we provided a high-yield RBD homodimer vaccine without additional biosafety or special transport device supports. Thus, it may serve as a safe, effective, and low-cost SARS-CoV-2 vaccine candidate.
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The present study aimed to assess the effects of 3,4-dihydroxyacetophenone (DHAP) on human pulmonary artery smooth muscle cells (HPASMCs). HPASMCs were divided into the normoxia group (NG), hypoxia group (HG), and hypoxia and 0.6×10-4 mol/L (HD1), 1.9×10-4 mol/L (HD2) and 6.0×10-4 mol/L (HD3) DHAP treatment groups. Cell cycle was analyzed by flow-cytometrically. HPASMC growth was examined by the proliferating cell nuclear antigen (PCNA) and MTT assays. Intracellular Ca2+ ([Ca2+]i) was measured by laser scanning confocal microscopy. Compared with the NG, the HG showed significantly increased HPASMC proliferation (P<0.05); meanwhile, cells treated with DHAP showed decreased proliferation compared with the HG (P<0.05). Hypoxia enhanced cell cycle progression and DHAP partly restored cell cycle distribution toward the status of NG cells. Furthermore, CDK2 levels were markedly increased in hypoxic cells (P<0.05), while DHAP treatment starkly decreased CDK2 levels in comparison with the HG (P<0.05). Moreover, hypoxia increased intracellular [Ca2+] levels compared with normoxia (P<0.05); meanwhile, DHAP treatment decreased [Ca2+]i compared with the HG (P<0.05). These findings suggested that DHAP inhibits hypoxia-induced proliferation of HPASMCs involving [Ca2+]i reduction. Therefore, DHAP should be considered an ideal candidate for the prevention and/or treatment of hypoxia-associated pulmonary hypertension and pulmonary vascular remodeling.
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Acetofenonas/farmacologia , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Artéria Pulmonar/metabolismo , Adulto , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacosRESUMO
Vγ2Vδ2 T cell-based immunotherapy has benefited some patients in clinical trials, but the overall efficacy is low for solid tumor patients. In this study, a bispecific antibody against both PD-L1 and CD3 (PD-L1 x CD3), Y111, could efficiently bridge T cells and PD-L1 expressing tumor cells. The Y111 prompted fresh CD8+ T cell-mediated lysis of H358 cells, but spared this effect on the fresh Vδ2+ T cells enriched from the same donors, which suggested that Y111 could bypass the anti-tumor capacity of the fresh Vγ2Vδ2 T cells. As the adoptive transfer of the expanded Vγ2Vδ2 T cells was approved to be safe and well-tolerated in clinical trials, we hypothesized that the combination of the expanded Vγ2Vδ2 T cells with the Y111 would provide an alternative approach of immunotherapy. Y111 induced the activation of the expanded Vγ2Vδ2 T cells in a dose-dependent fashion in the presence of PD-L1 positive tumor cells. Moreover, Y111 increased the cytotoxicity of the expanded Vγ2Vδ2 T cells against various NSCLC-derived tumor cell lines with the releases of granzyme B, IFNγ, and TNFα in vitro. Meanwhile, the adoptive transferred Vγ2Vδ2 T cells together with the Y111 inhibited the growth of the established xenografts in NPG mice. Taken together, our data suggested a clinical potential for the adoptive transferring the Vγ2Vδ2 T cells with the Y111 to treat PD-L1 positive solid tumors.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Complexo CD3/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transferência Adotiva , Animais , Anticorpos Biespecíficos/isolamento & purificação , Citocinas , Citotoxicidade Imunológica , Feminino , Humanos , Imunoterapia Adotiva , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Ativação Linfocitária , Camundongos , Ligação Proteica , Proteínas Recombinantes de Fusão , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The present study aimed to assess the effects of 3,4-dihydroxyacetophenone (DHAP) on human pulmonary artery smooth muscle cells (HPASMCs). HPASMCs were divided into the normoxia group (NG), hypoxia group (HG), and hypoxia and 0.6×10-4 mol/L (HD1), 1.9×10-4 mol/L (HD2) and 6.0×10--4 mol/L (HD3) DHAP treatment groups. Cell cycle was analyzed by flow-cytometrically. HPASMC growth was examined by the proliferating cell nuclear antigen (PCNA) and MTT assays. Intracellular Ca2+ ([Ca2+]i) was measured by laser scanning confocal microscopy. Compared with the NG, the HG showed significantly increased HPASMC proliferation (P<0.05); meanwhile, cells treated with DHAP showed decreased proliferation compared with the HG (P<0.05). Hypoxia enhanced cell cycle progression and DHAP partly restored cell cycle distribution toward the status of NG cells. Furthermore, CDK2 levels were markedly increased in hypoxic cells (P<0.05), while DHAP treatment starkly decreased CDK2 levels in comparison with the HG (P<0.05). Moreover, hypoxia increased intracellular [Ca2+] levels compared with normoxia (P<0.05); meanwhile, DHAP treatment decreased [Ca2+]i compared with the HG (P<0.05). These findings suggested that DHAP inhibits hypoxia-induced proliferation of HPASMCs involving [Ca2+]i reduction. Therefore, DHAP should be considered an ideal candidate for the prevention and/or treatment of hypoxia-associated pulmonary hypertension and pulmonary vascular remodeling.
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Acetofenonas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Hipóxia Celular , Células Cultivadas , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Remodelação Vascular/efeitos dos fármacosRESUMO
A bispecific antibody (bsAb) can simultaneously bind two different epitopes or antigens, allowing for multiple mechanistic functions with synergistic effects. BsAbs have attracted significant scientific attentions and efforts towards their development as drugs for cancers. There are 21 bsAbs currently undergoing clinical trials in China. Here, we review their platform technologies, expression and production, and biological activities and bioassay of these bsAbs, and summarize their structural formats and mechanisms of actions. T-cell redirection and checkpoint inhibition are two main mechanisms of the bsAbs that we discuss in detail. Furthermore, we provide our perspective on the future of bsAb development in China, including CD3-bsAbs for solid tumors and related cytokine release syndromes, expression and chemistry, manufacturing and controls, clinical development, and immunogenicity.
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Plasma hormone peptides, including GLP-1, GIP, Glucagon, and OXM, possess multiple physiological roles and potential therapeutic and diagnostic utility as biomarkers in the research of metabolic disorders. These peptides are subject to proteolytic degradation causing preanalytical variations. Stabilization for accurate quantitation of these active peptides in ex vivo blood specimens is essential for drug and biomarker development. We investigated the protease-driven instability of these peptides in conventional serum, plasma, anticoagulated whole blood, as well as whole blood and plasma stabilized with protease inhibitors. The peptide was monitored by both time-course Matrix-Assisted Laser Desorption Ionization Time-to-Flight Mass Spectrometry (MALDI -TOF MS) and Ab-based assay (ELISA or RIA). MS enabled the identification of proteolytic fragments. In non-stabilized blood samples, the results clearly indicated that dipeptidyl peptidase-IV (DPP-IV) removed the N-terminal two amino acid residues from GLP-1, GIP and OXM(1-37) and not-yet identified peptidase(s) cleave(s) the full-length OXM(1-37) and its fragments. DPP-IV also continued to remove two additional N-terminal residues of processed OXM(3-37) to yield OXM(5-37). Importantly, both DPP-IV and other peptidase(s) activities were inhibited efficiently by the protease inhibitors included in the BD P800* tube. There was preservation of GLP-1, GIP, OXM and glucagon in the P800 plasma samples with half-lives > 96, 96, 72, and 45 hours at room temperature (RT), respectively. In the BD P700* plasma samples, the stabilization of GLP-1 was also achieved with half-life > 96 hours at RT. The stabilization of these variable peptides increased their utility in drug and/or biomarker development. While stability results of GLP-1 obtained with Ab-based assay were consistent with those obtained by MS analysis, the Ab-based results of GIP, Glucagon, and OXM did not reflect the time-dependent degradations revealed by MS analysis. Therefore, we recommended characterizing the degradation of the peptide using the MS-based method when investigating the stability of a specific peptide.
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Hormônios Peptídicos/sangue , Sequência de Aminoácidos , Dipeptidil Peptidase 4/sangue , Polipeptídeo Inibidor Gástrico/sangue , Polipeptídeo Inibidor Gástrico/genética , Glucagon/sangue , Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Incretinas/sangue , Dados de Sequência Molecular , Oxintomodulina/sangue , Oxintomodulina/genética , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/genética , Hormônios Peptídicos/genética , Estabilidade Proteica , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Huwentoxin-IV (HWTX-IV), a peptide with 35 amino acid residues, was discovered in the venom of spider Ornithoctonus huwena. The peptide had an inhibitory effect on a tetrodotoxin-sensitive (TTX-S) sodium channel with highly sensitive to Nav1.7, an attractive target for pain release therapy. In this study we further demonstrated the analgesic effects of HWTX-IV using mouse and rat as an inflammatory pain model and/or a neuropathic pain models. In the both cases, the analgesic effects of the peptide were dose-dependent, and statistically significant. In the inflammatory model, 100 µg/kg of HWTX-IV produced an efficient reversal of hyperalgesia up to 63.6% after injection of formalin in rats with the efficiency equivalent to that of morphine at 50 µg/kg, and 200 µg/kg of HWTX-IV produced protective effect up to 55.6% after injection of acetic acid with the efficiency equivalent to that of morphine at 100 µg/kg. In the spinal nerve model, the peptide produced the longer and higher reversal effect on allodynia than Mexiletine. These results demonstrated that HWTX-IV released efficiently the acute inflammatory pain and chronic neuropathic pain in these animals, suggesting that HWTX-IV was a potential and efficient candidate for further clinical drug development against inflammatory and neuropathic pain.
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Analgésicos/farmacologia , Neuralgia/tratamento farmacológico , Venenos de Aranha/farmacologia , Ácido Acético/efeitos adversos , Analgésicos/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Formaldeído/efeitos adversos , Inflamação/tratamento farmacológico , Camundongos , Neuralgia/induzido quimicamente , Ratos , Venenos de Aranha/uso terapêuticoRESUMO
Blood samples collected for proteome studies are subject to a variety of preanalytical instability, among which intrinsic proteolysis activities cause a broad spectrum of protein and peptide degradation. This chapter describes two MALDI MS-based methods for plasma peptidomic analyses; a direct MALDI-TOF MS and an LC MALDI-TOF MS. Using these methods, we compared peptides and their time-dependent changes in traditional serum, four plasma samples with different anticoagulants and additives: EDTA-based, citrate-based, or heparin-based, and EDTA-based with protease inhibitors. For minimizing plasma sample instability and preanalytical variation, we suggest using an optimized blood collection device, minimizing the dwell time during blood collection and handling, controlling centrifugation and handling at room temperature, and saving plasma samples for use at most one freeze/thaw cycle. We have optimized our protocol to achieve reproducibility in peptidomic analyses of plasma samples using MALDI-TOF MS by minimizing preanalytical and analytical variability.
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Coleta de Amostras Sanguíneas/métodos , Plasma/química , Cromatografia Líquida , Humanos , Peptídeos/sangue , Peptídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Peptide biomarkers in plasma or serum are subject to proteolytic degradation caused by intrinsic peptidase activities, resulting in a potential barrier in translating a discovered biomarker into clinical application. This chapter describes a method using time-course MALDI-TOF MS analysis to investigate the stability of a plasma peptide biomarker under a variety of preanalytical situations. A synthesized peptide with the same primary sequence as a potential endogenous biomarker is spiked into a blood sample, and the sample is incubated over time at r.t. (25 ± 1°C) or other preanalytical situations. At a specific period of incubation time, the sample is quenched with the addition of acid with or without an internal control peptide. The spiked peptides in the sample are extracted with one of three procedures for highly soluble, moderately soluble, or essentially insoluble peptides. The peptide samples are then analyzed using MALDI-TOF MS. The abundance changes of the peptide biomarker are monitored by time-course changes of the mass spectra. These changes over-time are measured and fitted to a first-order degradation reaction so that stability of the peptide biomarker (half-life) can be calculated. Kinetics analysis of both parent and shorter (daughter) peptides are also possible by fitting to a sequential multiple-step reaction (SMSR) model. This optimized method facilitates evaluation of biomarker stability, and helps to define sample handling and analytical processing steps that contribute to instability of measured peptide biomarker(s).
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Peptídeos/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Biomarcadores/sangue , Fibrinogênio/análise , Fibrinogênio/química , Peptídeo 1 Semelhante ao Glucagon/sangue , Peptídeo 1 Semelhante ao Glucagon/química , Heparina/sangue , Heparina/química , Humanos , Dados de Sequência Molecular , Estabilidade Proteica , Estatística como Assunto , Fatores de TempoRESUMO
BACKGROUND: During clotting, a thrombin cleaves fibrinogen releasing fibrinopeptide A (FPA). FPA is easily identified in serum using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Using MALDI-TOF MS, we observed multiple, progressively shorter fragments of serum FPA. Following ambient incubation of serum, variations in the content of FPA fragments occur over time. Denaturation of a thrombin by heating the serum sample appears to minimize this variation. These observations suggest that intrinsic proteolytic and peptidolytic activity is elevated in serum and perhaps originates from the coagulation cascade enzymes themselves, especially a thrombin. METHODS: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins was carried out to induce proteolysis and to examine the resultant peptides to reveal a thrombin susceptible parent proteins. One of these identified proteins, hemopexin, was directly digested by a thrombin and the peptides examined to confirm the observations from the initial plasma protein digestion. RESULTS: Extrinsic addition of a thrombin to a subset (3-30 kDa) of plasma proteins results in wide-spread digestion of proteins unrelated to coagulation, revealing a substrate range encompassing more than fibrinogen. Direct digestion of one of these proteins, hemopexin, by a thrombin confirms these observations. CONCLUSIONS: The resulting peptides indicate broad tolerance beyond the consensus R-G cleavage site of fibrinogen; in fact, there appears to be no bias for the amino acid following the R/K residue. These data support our hypothesis that the enzymatic activities inherent to coagulation, or at least to thrombin, contribute to destabilization of the protein and peptide content of serum.
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Fibrinopeptídeo A/análise , Hemopexina/análise , Fragmentos de Peptídeos/sangue , Peptídeo Hidrolases/metabolismo , Proteômica/métodos , Trombina/fisiologia , Sequência de Aminoácidos , Coagulação Sanguínea/fisiologia , Fibrinogênio/metabolismo , Fibrinopeptídeo A/química , Fibrinopeptídeo A/metabolismo , Hemopexina/química , Hemopexina/metabolismo , Temperatura Alta , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Plasma , Inibidores de Proteases/farmacologia , Estabilidade Proteica , Soro , Manejo de Espécimes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Trombina/metabolismoRESUMO
This chapter describes the technology of free flow electrophoresis (FFE) and protocols to separate human plasma for proteome analysis. FFE is a highly versatile technology applied in the field of proteomics because of its continuous processing of sample and high resolution in separation of most kinds of charged or chargeable particles including ions, proteins peptides, organelles, and whole cells. FFE is carried out in an aqueous medium without inducing any solid matrix, such as acrylamide, so that it simplifies complex sample for the downstream analysis. Two FFE protocols are described to separate human plasma proteins under native and denaturing conditions. Plasma separated under native conditions was pooled into acidic-, alkaline-, and albumin- fractions that were furthered for gel-based analysis. Under denaturing condition plasma proteins were separated into 96 fractions. Each fraction can be supplied for in-solution digestion and further LC-MS/MS analysis. From a single FFE fraction 46 different proteins (protein family) have been identified, demonstrating FFE as a high efficient separation tool for human plasma proteome studies.
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Proteínas Sanguíneas/análise , Eletroforese/métodos , Plasma/química , Proteoma/análise , Proteômica/métodos , Eletroforese das Proteínas Sanguíneas/métodos , Proteínas Sanguíneas/química , Humanos , Proteoma/químicaRESUMO
Human plasma and serum samples, including protein and peptide biomarkers, are subjected to preanalytical variations and instability caused by intrinsic proteases. In this study, we directly investigated the stability of peptide biomarkers by spiking an isotopically labeled peptide into human plasma and serum samples and then monitoring its time-dependent change. Fibrinogen peptide A (FPA) was used as a model substrate, and its degradation in a conventional serum and plasma either with citrate, heparin, or EDTA as the anticoagulant, or EDTA plus protease inhibitors (inhibited plasma), was measured using time-course MALDI-TOF MS analysis. The FPA and other peptides tested in this study vary in these samples. However, the peptides are most stable in the inhibited plasma followed by, in general order, EDTA plasma, citrate plasma, heparin plasma and serum, demonstrating the benefit of plasma versus serum, and protease inhibitors for biomarker stabilization. Kinetic analysis indicates that intrinsic peptidases cause an observed first-order Sequential Multiple-Step Reaction (SMSR) in digestion of the peptide. Modeling analysis of the SMSR demonstrates that step reactions differ in their kinetic rate constants, suggesting a significant contribution of the truncated end residue on the substrate specificity of the intrinsic peptidase(s). Our observations further show that synthetic peptides introduced into plasma as internal controls can also be degraded, and thus, their (in)stability as a preanalytical variable should not be overlooked.
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Biomarcadores/química , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Biologia Computacional/métodos , Ácido Edético/química , Humanos , Cinética , Peptídeos/química , Plasma/química , Plasma/metabolismo , Inibidores de Proteases/metabolismo , Proteoma , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome.
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Proteínas Sanguíneas/metabolismo , Peptídeos/metabolismo , Plasma , Sequência de Aminoácidos , Proteínas Sanguíneas/química , Humanos , Dados de Sequência Molecular , Peptídeos/química , Plasma/química , Plasma/metabolismo , Inibidores de Proteases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Rolling-circle amplification (RCA) and ramification amplification (RAM, also known as hyperbranched RCA) are isothermal nucleic acid amplification technologies that have gained a great application in in situ signal amplification, DNA and protein microarray assays, single nucleotide polymorphism detection, as well as clinical diagnosis. Real-time detection of RCA or RAM products has been a challenge because of most real-time detection systems, including Taqman and Molecular Beacon, are designed for thermal cycling-based DNA amplification technology. In the present study, we describe a novel fluorescent probe construct, termed molecular zipper, which is specially designed for quantifying target DNA by real-time monitoring RAM reactions. Our results showed that the molecular zipper has very low background fluorescence due to the strong interaction between two strands. Once it is incorporated into the RAM products its double strand region is opened by displacement, therefore, its fluorophore releases a fluorescent signal. Applying the molecular zipper in RAM assay, we were able to detect as few as 10 molecules within 90 min reaction. A linear relationship was observed between initial input of targets and threshold time (R2 = 0.985). These results indicate that molecular zipper can be applied to real-time monitoring and qualification of RAM reaction, implying an amenable method for automatic RAM-based diagnostic assays.
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Sondas de DNA , DNA/análise , Corantes Fluorescentes , Técnicas de Amplificação de Ácido Nucleico , Replicação do DNA , Temperatura , Fatores de TempoRESUMO
There is a substantial list of pre-analytical variables that can alter the analysis of blood-derived samples. We have undertaken studies on some of these issues including choice of sample type, stability during storage, use of protease inhibitors, and clinical standardization. As there is a wide range of sample variables and a broad spectrum of analytical techniques in the HUPO PPP effort, it is not possible to define a single list of pre-analytical standards for samples or their processing. We present here a compendium of observations, drawing on actual results and sound clinical theories and practices. Based on our data, we find that (1) platelet-depleted plasma is preferable to serum for certain peptidomic studies; (2) samples should be aliquoted and stored preferably in liquid nitrogen; (3) the addition of protease inhibitors is recommended, but should be incorporated early and used judiciously, as some form non specific protein adducts and others interfere with peptide studies. Further, (4) the diligent tracking of pre-analytical variables and (5) the use of reference materials for quality control and quality assurance, are recommended. These findings help provide guidance on sample handling issues, with the overall suggestion being to be conscious of all possible pre-analytical variables as a prerequisite of any proteomic study.
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Proteínas Sanguíneas/química , Coleta de Amostras Sanguíneas/métodos , Proteômica/métodos , Proteômica/normas , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Plaquetas/química , Preservação de Sangue , Biologia Computacional , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/química , Inibidores de Proteases/farmacologia , Análise Serial de Proteínas , Controle de Qualidade , Padrões de Referência , Valores de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Temperatura , Fatores de Tempo , Tripsina/farmacologiaRESUMO
OBJECTIVES: To investigate the anticancer activities of a Chinese herb, Oldenlandia diffusa (Bai Hua She She Cao). METHODS: The water extract of the raw herb Oldenlandia diffusa was used in this study. The in vitro anti-proliferative activities of the extract were tested against eight cancer cell lines and one normal cell line. Microscopic examination and DNA ladder analysis were carried out to determine the pro-apoptotic effect of the extract. In vivo studies were carried out to examine the anticancer activities of the extract using C57BL/6j mice bearing B16-F10 lung metastasis. Oldenlandia diffusa extract was given at the dose level of 5 g raw material/kg on Days 3-12 by oral gavage and the extent of lung metastases were examined on Day 14. RESULTS: The extract exhibited a strong antiproliferative activity against all cancer cell lines tested. The concentrations of growth inhibition at 50% (IC(50)) ranged from 7 to 25 mg raw material/ml after 48-hour treatment. The extract had a very limited cytotoxicity (10% inhibition) on the normal pancreatic cells even at the concentration of 50 mg/mL. Apoptosis in B16-F10 cells after treatment with the extract was observed by microscopic examination and DNA ladder assays. Oral administration of the herbal extract effectively reduced B16-F10 cell growth in the lungs of C57Bl/j mice with a 70% reduction in lung metastases (p < 0.001). CONCLUSIONS: Oldenlandia diffusa extract effectively inhibited the growth of all the eight cancer cell lines and induced significant increase of apoptosis. The extract exhibited minimum toxic effect on normal pancreatic cells. Furthermore, there was a significant inhibition of lung metastases in the animal model with no noticeable adverse effects. The herb extract could be a potential anticancer agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Oldenlandia , Extratos Vegetais/farmacologia , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/uso terapêutico , Técnicas In Vitro , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Extratos Vegetais/uso terapêuticoRESUMO
Genistein, rich in soybean, has been reported to have anti-cancer activity on several cancers. However, the molecular mechanism of its anti-cancer activity still remains unclear. We investigated the effect of genistein on a human oral squamous carcinoma line (SCC-25), and demonstrated that genistein inhibited SCC-25 cell growth via G2/M phase arrest. We observed a significant decrease of proliferating cell nuclear antigen expression in these cells after treatment, but no significant change in the number of apoptotic cells, indicating that the major action of genistein is inhibition of cancer cell proliferation. We also observed a high level of prostaglandin E2 (PGE2) in these cells and PGE2 synthesis in SCC-25 cells was significantly suppressed by genistein. We demonstrated that genistein directly inhibited cycloxygenase-2 (COX-2) activity, an inducible enzyme that converts arachidonic acid to prostaglandins, similar to the action of celecoxib, a selective COX-2 inhibitor. However, the anticancer activity of genistein was much weaker than that of indomethacin (non-selective COX inhibitor), celecoxib and baicalein (flavonoid isolated from Scutellaria baicalensis). These results suggested that genistein might be useful as a chemopreventive agent rather than a chemotherapeutic agent.