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1.
Proc Natl Acad Sci U S A ; 116(9): 3443-3448, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808738

RESUMO

Early life exposure to fine particulate matter (PM) in air is associated with infant respiratory disease and childhood asthma, but limited epidemiological data exist concerning the impacts of ultrafine particles (UFPs) on the etiology of childhood respiratory disease. Specifically, the role of UFPs in amplifying Th2- and/or Th17-driven inflammation (asthma promotion) or suppressing effector T cells (increased susceptibility to respiratory infection) remains unclear. Using a mouse model of in utero UFP exposure, we determined early immunological responses to house dust mite (HDM) allergen in offspring challenged from 0 to 4 wk of age. Two mice strains were exposed throughout gestation: C57BL/6 (sensitive to oxidative stress) and BALB/C (sensitive to allergen exposure). Offspring exposed to UFPs in utero exhibited reduced inflammatory response to HDM. Compared with filtered air (FA)-exposed/HDM-challenged mice, UFP-exposed offspring had lower white blood cell counts in bronchoalveolar lavage fluid and less pronounced peribronchiolar inflammation in both strains, albeit more apparent in C57BL/6 mice. In the C57BL/6 strain, offspring exposed in utero to FA and challenged with HDM exhibited a robust response in inflammatory cytokines IL-13 and Il-17. In contrast, this response was lost in offspring exposed in utero to UFPs. Circulating IL-10 was significantly up-regulated in C57BL/6 offspring exposed to UFPs, suggesting increased regulatory T cell expression and suppressed Th2/Th17 response. Our results reveal that in utero UFP exposure at a level close to the WHO recommended PM guideline suppresses an early immune response to HDM allergen, likely predisposing neonates to respiratory infection and altering long-term pulmonary health.


Assuntos
Asma/imunologia , Hipersensibilidade/imunologia , Material Particulado/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/imunologia , Alérgenos/química , Alérgenos/toxicidade , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/genética , Feminino , Hipersensibilidade/genética , Hipersensibilidade/patologia , Imunossupressão , Pulmão/efeitos dos fármacos , Pulmão/patologia , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Pyroglyphidae/química , Células Th17/imunologia , Células Th2/imunologia
2.
Clin Cancer Res ; 23(22): 6893-6903, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28821560

RESUMO

Purpose: The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU).Experimental Design: C2Am was labeled with a NIR fluorophore and injected intravenously into mice bearing human colorectal TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 xenografts that had been treated with a potent agonist of TRAILR2 and in Colo205 tumors treated with 5-FU.Results: Three-dimensional (3D) MSOT images of probe distribution showed development of tumor contrast within 3 hours of probe administration and a signal-to-background ratio in regions containing dead cells of >10 after 24 hours. A site-directed mutant of C2Am that is inactive in PS binding showed negligible binding. Tumor retention of the active probe was strongly correlated (R2 = 0.97, P value < 0.01) with a marker of apoptotic cell death measured in histologic sections obtained post mortem.Conclusions: The rapid development of relatively high levels of contrast suggests that NIR fluorophore-labeled C2Am could be a useful optoacoustic imaging probe for detecting early therapy-induced tumor cell death in the clinic. Clin Cancer Res; 23(22); 6893-903. ©2017 AACR.


Assuntos
Morte Celular , Imagem Molecular , Técnicas Fotoacústicas , Tomografia , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Corantes Fluorescentes , Xenoenxertos , Humanos , Camundongos , Microscopia de Fluorescência , Imagem Molecular/métodos , Tomografia/métodos
3.
Oncotarget ; 8(25): 40115-40131, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28445154

RESUMO

Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key Notch ligands implicated in tumour angiogenesis. They were shown to have opposite effects on mouse retinal and adult regenerative angiogenesis. In tumours, both ligands are upregulated but their relative effects and interactions in tumour biology, particularly in tumour response to therapeutic intervention are unclear. Here we demonstrate that DLL4 and JAG1 displayed equal potency in stimulating Notch target genes in HMEC-1 endothelial cells but had opposing effects on sprouting angiogenesis in vitro. Mouse DLL4 or JAG1 expressed in glioblastoma cells decreased tumour cell proliferation in vitro but promoted tumour growth in vivo. mDLL4-expressing tumours showed fewer but larger vessels whereas mJAG1-tumours produced more vessels. In both tumour types pericyte coverage was decreased but the vessels were more perfused. Both ligands increased tumour resistance towards anti-VEGF therapy but the resistance was higher in mDLL4-tumours versus mJAG1-tumours. However, their sensitivity to the therapy was restored by blocking Notch signalling with dibenzazepine. Importantly, anti-DLL4 antibody blocked the effect of JAG1 on tumour growth and increased vessel branching in vivo. The mechanism behind the differential responsiveness was due to a positive feedback loop for DLL4-Notch signalling, rendering DLL4 more dominant in activating Notch signalling in the tumour microenvironment. We concluded that DLL4 and JAG1 promote tumour growth by modulating tumour angiogenesis via different mechanisms. JAG1 is not antagonistic but utilises DLL4 in tumour angiogenesis. The results suggest that anti-JAG1 therapy should be explored in conjunction with anti-DLL4 treatment in developing anti-Notch therapies in clinics.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Jagged-1/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Animais , Bevacizumab/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Dibenzazepinas/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína Jagged-1/genética , Estimativa de Kaplan-Meier , Proteínas de Membrana/genética , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/prevenção & controle , Interferência de RNA , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Cancer Immunol Res ; 5(1): 29-41, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27923825

RESUMO

Murine syngeneic tumor models are critical to novel immuno-based therapy development, but the molecular and immunologic features of these models are still not clearly defined. The translational relevance of differences between the models is not fully understood, impeding appropriate preclinical model selection for target validation, and ultimately hindering drug development. Across a panel of commonly used murine syngeneic tumor models, we showed variable responsiveness to immunotherapies. We used array comparative genomic hybridization, whole-exome sequencing, exon microarray analysis, and flow cytometry to extensively characterize these models, which revealed striking differences that may underlie these contrasting response profiles. We identified strong differential gene expression in immune-related pathways and changes in immune cell-specific genes that suggested differences in tumor immune infiltrates between models. Further investigation using flow cytometry showed differences in both the composition and magnitude of the tumor immune infiltrates, identifying models that harbor "inflamed" and "non-inflamed" tumor immune infiltrate phenotypes. We also found that immunosuppressive cell types predominated in syngeneic mouse tumor models that did not respond to immune-checkpoint blockade, whereas cytotoxic effector immune cells were enriched in responsive models. A cytotoxic cell-rich tumor immune infiltrate has been correlated with increased efficacy of immunotherapies in the clinic, and these differences could underlie the varying response profiles to immunotherapy between the syngeneic models. This characterization highlighted the importance of extensive profiling and will enable investigators to select appropriate models to interrogate the activity of immunotherapies as well as combinations with targeted therapies in vivo Cancer Immunol Res; 5(1); 29-41. ©2016 AACR.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Sinergismo Farmacológico , Exoma , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Imunomodulação/efeitos dos fármacos , Imunomodulação/genética , Camundongos , Terapia de Alvo Molecular , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcriptoma , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
5.
Forensic Sci Int ; 270: 185-192, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27823839

RESUMO

Dipyrone is an analgesic and antipyretic drug that is sometimes encountered as an adulterant in illicit drug samples, particularly illicit fentanyl containing samples. It undergoes thermal decomposition to aminopyrine and 4-methylaminoantipyrine during analysis via gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). During analysis via high pressure liquid chromatography (HPLC) and high pressure liquid chromatography-mass spectrometry (HPLC-MS), it undergoes hydrolytic decomposition solely to 4-methylaminoantipyrine. Given that mass spectrometry is a widely used confirmatory analytical technique, these instabilities present challenges for the forensic chemist seeking to confirm the presence of dipyrone. Studies were conducted to determine rigorous confirmative protocols for the identification of dipyrone in multicomponent illicit drug samples.


Assuntos
Dipirona/análise , Contaminação de Medicamentos , /química , Cromatografia Líquida de Alta Pressão , Fentanila/química , Toxicologia Forense , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectrometria de Massas por Ionização por Electrospray
6.
Sci Rep ; 6: 25806, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27194388

RESUMO

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.


Assuntos
Vírus Sincicial Respiratório Humano/fisiologia , Transcrição Genética , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Alcaloides/farmacologia , Animais , Linhagem Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Animais de Doenças , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Receptor Smoothened/metabolismo , Transcrição Genética/efeitos dos fármacos , Veratrum/química , Alcaloides de Veratrum/química , Alcaloides de Veratrum/farmacologia , Alcaloides de Veratrum/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
7.
Immunol Lett ; 171: 5-14, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26773232

RESUMO

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.


Assuntos
Anticorpos Monoclonais/imunologia , Células Epiteliais/fisiologia , Epitopos/metabolismo , Intestinos/efeitos dos fármacos , Células de Langerhans/imunologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Animais , Anticorpos Monoclonais/isolamento & purificação , Afinidade de Anticorpos , Diferenciação Celular/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Células Epiteliais/efeitos dos fármacos , Epitopos/imunologia , Células HEK293 , Humanos , Imunização Secundária , Imunomodulação , Intestinos/citologia , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/imunologia , Transdução de Sinais
8.
Nature ; 528(7580): 115-8, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26580020

RESUMO

Large parts of the Antarctic ice sheet lying on bedrock below sea level may be vulnerable to marine-ice-sheet instability (MISI), a self-sustaining retreat of the grounding line triggered by oceanic or atmospheric changes. There is growing evidence that MISI may be underway throughout the Amundsen Sea embayment (ASE), which contains ice equivalent to more than a metre of global sea-level rise. If triggered in other regions, the centennial to millennial contribution could be several metres. Physically plausible projections are challenging: numerical models with sufficient spatial resolution to simulate grounding-line processes have been too computationally expensive to generate large ensembles for uncertainty assessment, and lower-resolution model projections rely on parameterizations that are only loosely constrained by present day changes. Here we project that the Antarctic ice sheet will contribute up to 30 cm sea-level equivalent by 2100 and 72 cm by 2200 (95% quantiles) where the ASE dominates. Our process-based, statistical approach gives skewed and complex probability distributions (single mode, 10 cm, at 2100; two modes, 49 cm and 6 cm, at 2200). The dependence of sliding on basal friction is a key unknown: nonlinear relationships favour higher contributions. Results are conditional on assessments of MISI risk on the basis of projected triggers under the climate scenario A1B (ref. 9), although sensitivity to these is limited by theoretical and topographical constraints on the rate and extent of ice loss. We find that contributions are restricted by a combination of these constraints, calibration with success in simulating observed ASE losses, and low assessed risk in some basins. Our assessment suggests that upper-bound estimates from low-resolution models and physical arguments (up to a metre by 2100 and around one and a half by 2200) are implausible under current understanding of physical mechanisms and potential triggers.


Assuntos
Aquecimento Global , Camada de Gelo , Modelos Teóricos , Observação , Água do Mar/análise , Regiões Antárticas , Teorema de Bayes , Calibragem , Aquecimento Global/estatística & dados numéricos , Oceanos e Mares
9.
Forensic Sci Int ; 242: 135-141, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25062530

RESUMO

The number of analyses of synthetic cannabimimetic drugs of abuse by forensic laboratories in the United States grew rapidly from 2010 to 2012 and then declined somewhat in 2013. In 2010, according to the National Forensic Laboratory Information System (NFLIS), 3,287 reports by federal, state and local forensic laboratories were identified as containing synthetic cannabinoids. In 2011 and 2012, the numbers increased to 23,693 and 42,503, respectively. 27,119 reports were identified in 2013. Several commonly encountered structural sub-classes of these synthetic designer drugs, namely the naphthoylindoles, benzoylindoles, phenylacetylindoles, and cyclopropoylindoles contain a ketone functional group. The Duquenois-Levine color test for the presumptive identification of classical cannabinoids such as Δ(9)-tetrahydrocannabinol is negative for the synthetic cannabimimetics. The van Urk color test for the presumptive identification of indole containing drugs of abuse is also negative for these compounds. The use of 2,4-dinitrophenylhydrazine as an alternative color test reagent (targeting the keto moiety rather than the indole) for presumptive identification of these classes of drugs was investigated.

10.
Semin Cancer Biol ; 25: 69-77, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24406209

RESUMO

The microenvironment of established tumours is often immunosuppressed, and this allows tumours to grow and disseminate without being eliminated by the patient's immune system. The recent FDA approval of immunotherapies such as ipilimumab and sipuleucel-T that directly activate the adaptive and innate immune responses has triggered interest in developing other novel anti-cancer approaches that modulate the immune system. Understanding how the different constituents of the tumour microenvironment influence the immune system is thus crucial and is expected to generate a plethora of factors that can be targeted to boost immunity and trigger long lasting anti-tumour efficacy. Cancer associated fibroblasts (CAFs) are a crucial component of the tumour microenvironment. Through secretion of multiple growth factors, cytokines and proteases, CAFs are known to be key effectors for tumour progression and can promote cancer cell growth, invasiveness and angiogenesis. However, recent publications have also linked CAF biology to innate and adaptive immune cell recruitment and regulation. Here, we review recent findings on how CAFs can influence the immune status of tumours through direct and indirect interaction with immune cells and other key components of the tumour microenvironment.


Assuntos
Fibroblastos/imunologia , Neoplasias/imunologia , Imunidade Adaptativa , Animais , Humanos , Imunidade Celular , Imunidade Inata , Imunoterapia , Inflamação/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fator de Crescimento Transformador beta/fisiologia , Microambiente Tumoral/imunologia
11.
Science ; 342(6162): 1086-9, 2013 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-24200815

RESUMO

Fast-flowing glaciers and ice streams are pathways for ice discharge from the interior of the Antarctic Ice Sheet to ice shelves, at rates controlled by conditions at the ice-bed interface. Using recently compiled high-resolution data sets and a standard inverse method, we computed basal shear stress distributions beneath Pine Island and Thwaites Glaciers, which are currently losing mass at an accelerating rate. The inversions reveal the presence of riblike patterns of very high basal shear stress embedded within much larger areas with zero basal shear stress. Their colocation with highs in the gradient of hydraulic potential suggests that subglacial water may control the evolution of these high-shear-stress ribs, potentially causing migration of the grounding line by changes in basal resistance in its vicinity.

12.
Proc Math Phys Eng Sci ; 468(2147): 3285-3310, 2012 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-23197934

RESUMO

Changes at the grounding line of ice streams have consequences for inland ice dynamics and hence sea level. Despite substantial evidence documenting upstream propagation of frontal change, the mechanisms by which these changes are transmitted inland are not well understood. In this vein, the frequency response of an idealized ice stream to periodic forcing in the downstream strain rate is examined for basally and laterally resisted ice streams using a one-dimensional, linearized membrane stress approximation. This reveals two distinct behavioural branches, which we find to correspond to different mechanisms of upstream velocity and thickness propagation, depending on the forcing frequency. At low frequencies (centennial to millennial periods), slope and thickness covary hundreds of kilometres inland, and the shallow-ice approximation is sufficient to explain upstream propagation, which occurs through changes in grounding-line flow and geometry. At high frequencies (decadal to sub-decadal periods), penetration distances are tens of kilometres; while velocity adjusts rapidly to such forcing, thickness varies little and upstream propagation occurs through the direct transmission of membrane stresses. Propagation properties vary significantly between 29 Antarctic ice streams considered. A square-wave function in frontal stress is explored by summing frequency solutions, simulating some aspects of the dynamical response to sudden ice-shelf change.

13.
Nature ; 489(7414): 141-4, 2012 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-22914090

RESUMO

Rapid warming over the past 50 years on the Antarctic Peninsula is associated with the collapse of a number of ice shelves and accelerating glacier mass loss. In contrast, warming has been comparatively modest over West Antarctica and significant changes have not been observed over most of East Antarctica, suggesting that the ice-core palaeoclimate records available from these areas may not be representative of the climate history of the Antarctic Peninsula. Here we show that the Antarctic Peninsula experienced an early-Holocene warm period followed by stable temperatures, from about 9,200 to 2,500 years ago, that were similar to modern-day levels. Our temperature estimates are based on an ice-core record of deuterium variations from James Ross Island, off the northeastern tip of the Antarctic Peninsula. We find that the late-Holocene development of ice shelves near James Ross Island was coincident with pronounced cooling from 2,500 to 600 years ago. This cooling was part of a millennial-scale climate excursion with opposing anomalies on the eastern and western sides of the Antarctic Peninsula. Although warming of the northeastern Antarctic Peninsula began around 600 years ago, the high rate of warming over the past century is unusual (but not unprecedented) in the context of natural climate variability over the past two millennia. The connection shown here between past temperature and ice-shelf stability suggests that warming for several centuries rendered ice shelves on the northeastern Antarctic Peninsula vulnerable to collapse. Continued warming to temperatures that now exceed the stable conditions of most of the Holocene epoch is likely to cause ice-shelf instability to encroach farther southward along the Antarctic Peninsula.


Assuntos
Aquecimento Global/estatística & dados numéricos , Camada de Gelo , Regiões Antárticas , Geografia , Sedimentos Geológicos/química , Aquecimento Global/história , História do Século XV , História do Século XVI , História do Século XVII , História do Século XVIII , História do Século XIX , História do Século XX , História do Século XXI , História Antiga , História Medieval , Oceanos e Mares , Água do Mar/análise , Temperatura
14.
Cancer Res ; 71(18): 6073-83, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21803743

RESUMO

Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a γ-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic.


Assuntos
Inibidores da Angiogênese/farmacologia , Fibrossarcoma/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Dibenzazepinas/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Fibrossarcoma/irrigação sanguínea , Fibrossarcoma/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Transdução de Sinais , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular/biossíntese
15.
Bioorg Med Chem Lett ; 21(5): 1532-5, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21295466

RESUMO

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.


Assuntos
Anticoagulantes/síntese química , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Desenho de Fármacos , Pirazinas/síntese química , Trombina/antagonistas & inibidores , Administração Oral , Animais , Anticoagulantes/química , Anticoagulantes/farmacologia , Sítios de Ligação , Disponibilidade Biológica , Cães , Estrutura Molecular , Pirazinas/química , Pirazinas/farmacologia , Ratos , Relação Estrutura-Atividade
16.
Bioorg Med Chem Lett ; 21(5): 1536-40, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21295467

RESUMO

A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.


Assuntos
Antitrombinas/síntese química , Benzeno/síntese química , Desenho de Fármacos , Trombina/antagonistas & inibidores , Antitrombinas/química , Antitrombinas/farmacologia , Benzeno/química , Benzeno/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
17.
Blood ; 116(13): 2385-94, 2010 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-20558614

RESUMO

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.


Assuntos
Células Endoteliais/fisiologia , Exossomos/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Receptores Notch/fisiologia , Animais , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Células Endoteliais/ultraestrutura , Exossomos/transplante , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Transplante de Neoplasias , Neovascularização Fisiológica , Transdução de Sinais/fisiologia , Transplante Heterólogo
18.
J Chromatogr A ; 1201(1): 91-9, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18602111

RESUMO

Fipronil, a phenylpyrazole insecticide introduced for pest control on a broad range of crops, undergoes a reinforcement of the regulation within the European Union (2007/52/EC directive) due to its potential effects on environment and human health. In order to assess the plasmatic concentrations of fipronil residues (sulfone, sulfide, fipronil, desulfinyl and amide) in ovine, a methodology based on gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was developed and validated according to the European standard (2002/657/EC). The proposed method allows a large number of samples to be treated concurrently (n=80) using a reduced sample amounts (0.2 mL), and consents to reach a level of quantification of 0.1 pg microL(-1). The sample preparation consisted of a single solid-phase extraction (SPE) purification on a 96-well plate filled with a styrene-divinyl-benzene phase. Linearity was demonstrated all along the investigated range of concentrations, i.e. from 0.25 to 2000 pg microL(-1), with coefficient of determination (R(2)) from 0.977 to 0.994, depending on target analytes. Calculated decision limit (CCalpha) and detection capability (CCbeta) for fipronil, sulfone and sulphide were in the range 0.05-0.16 and 0.28-0.73 pg microL(-1) respectively.


Assuntos
Contaminação de Alimentos/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Resíduos de Praguicidas/química , Plasma/química , Pirazóis/química , Extração em Fase Sólida/métodos , Animais , União Europeia/organização & administração , Cromatografia Gasosa-Espectrometria de Massas/normas , Padrões de Referência , Sensibilidade e Especificidade , Ovinos
19.
Blood ; 111(10): 4997-5007, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18337563

RESUMO

Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial "tip cell" phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFkappaB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFkappaB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.


Assuntos
Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Humanos , Inflamação , NF-kappa B/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas c-sis , Fatores de Tempo , Veias Umbilicais/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
20.
Cancer Res ; 68(6): 1889-95, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18339870

RESUMO

Gene-targeting studies have shown that Delta-like 4 (Dll4) is required for normal embryonic vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are not well defined. We generated primary human umbilical vascular endothelial cells that express Dll4 protein to study Dll4 function and previously showed that Dll4 down-regulates vascular endothelial growth factor (VEGF) receptor 2 and NRP1 expression and inhibits VEGF function. We now report that expression of Dll4 in endothelial cells inhibited attachment and migration to stromal-derived growth factor 1 (SDF1) chemokine. Cell surface, total protein, and mRNA levels of CXCR4, principal signaling receptor for SDF1, were significantly decreased in Dll4-transduced endothelial cells, attributable to a significant reduction of CXCR4 promoter activity. An immobilized recombinant extracellular portion of Dll4 (rhDLL4) was sufficient to down-regulate CXCR4 mRNA and protein, whereas protein levels of SDF1, VEGF, and RDC1 were unchanged. The gamma-secretase inhibitor L-685,458 significantly reconstituted CXCR4 mRNA in rhDLL4-stimulated endothelial cells. CXCR4 mRNA levels were significantly reduced in mouse xenografts of Dll4-transduced human gliomas compared with control gliomas, and vascular CXCR4 was not detected by immunohistochemistry in the enlarged vessels within the Dll4 gliomas. Thus, Dll4 may contribute to vascular differentiation and inhibition of the angiogenic response by regulating multiple receptor pathways.


Assuntos
Células Endoteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Receptores CXCR4/biossíntese , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacologia , Regulação para Baixo , Células Endoteliais/citologia , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Transfecção
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