RESUMO
PURPOSE: To compare student performance, examiner perceptions and cost of GPT-assisted (generative pretrained transformer-assisted) clinical and professional skills assessment (CPSAs) items against items created using standard methods. METHODS: We conducted a prospective, controlled, double-blinded comparison of CPSA items developed using GPT-assistance with those created through standard methods. Two sets of six practical cases were developed for a formative assessment sat by final year medical students. One clinical case in each set was created with GPT-assistance. Students were assigned to one of the two sets. RESULTS: The results of 239 participants were analysed in the study. There was no statistically significant difference in item difficulty, or discriminative ability between GPT-assisted and standard items. One hundred percent (n = 15) of respondents to an examiner feedback questionnaire felt GPT-assisted cases were appropriately difficult and realistic. GPT-assistance resulted in significant labour cost savings, with a mean reduction of 57% (880 GBP) in labour cost per case when compared to standard case drafting methods. CONCLUSIONS: GPT-assistance can create CPSA items of comparable quality with significantly less cost when compared to standard methods. Future studies could evaluate GPT's ability to create CPSA material in other areas of clinical practice, aiming to validate the generalisability of these findings.
RESUMO
The molecular and cellular pathways that support the maintenance and stability of tumor neovessels are not well defined. The efficacy of microtubule-disrupting agents, such as combretastatin A4 phosphate (CA4P), in inducing rapid regression of specific subsets of tumor neovessels has opened up new avenues of research to identify factors that support tumor neoangiogenesis. Herein, we show that CA4P selectively targeted endothelial cells, but not smooth muscle cells, and induced regression of unstable nascent tumor neovessels by rapidly disrupting the molecular engagement of the endothelial cell-specific junctional molecule vascular endothelial-cadherin (VE-cadherin) in vitro and in vivo in mice. CA4P increases endothelial cell permeability, while inhibiting endothelial cell migration and capillary tube formation predominantly through disruption of VE-cadherin/beta-catenin/Akt signaling pathway, thereby leading to rapid vascular collapse and tumor necrosis. Remarkably, stabilization of VE-cadherin signaling in endothelial cells with adenovirus E4 gene or ensheathment with smooth muscle cells confers resistance to CA4P. CA4P synergizes with low and nontoxic doses of neutralizing mAbs to VE-cadherin by blocking assembly of neovessels, thereby inhibiting tumor growth. These data suggest that the microtubule-targeting agent CA4P selectively induces regression of unstable tumor neovessels, in part through disruption of VE-cadherin signaling. Combined treatment with anti-VE-cadherin agents in conjunction with microtubule-disrupting agents provides a novel synergistic strategy to selectively disrupt assembly and induce regression of nascent tumor neovessels, with minimal toxicity and without affecting normal stabilized vasculature.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Caderinas/fisiologia , Endotélio Vascular/efeitos dos fármacos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Capilares/crescimento & desenvolvimento , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Humanos , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , beta Catenina/fisiologiaRESUMO
Connexins (Cxs) provide a means for intercellular communication and play important roles in the pathophysiology of vascular cardiac diseases. Infection of endothelial cells (ECs) with first-generation E1/E3-deleted E4+ adenovirus (AdE4+) selectively modulates the survival and angiogenic potential of ECs by as of yet unrecognized mechanisms. We show here that AdE4+ vectors potentiate Cx expression in ECs in vitro and in mouse heart tissue. Infection of ECs with AdE4+, but not AdE4-, resulted in a time- and dose-dependent induction of junctional Cx40 expression and suppression of Cx43 protein and mRNA expression. Treatment of ECs with PKA inhibitor H89 or PI3K inhibitor LY294002 prevented the AdE4+-mediated regulation of Cx40 and Cx43 that was associated with diminished AdE4+-mediated survival of ECs. Moreover, both PKA activity and cAMP-response element (CRE)-binding activity were enhanced by treatment of ECs with AdE4+. However, there is no causal evidence of a cross-talk between the 2 modulatory pathways, PKA and PI3K. Remarkably, Cx40 immunostaining was markedly increased and Cx43 was decreased in the heart tissue of mice treated with intra-tracheal AdE4+. Taken together, these results suggest that AdE4+ may play an important role in the regulation of Cx expression in ECs, and that these effects are mediated by both the PKA/CREB and PI3K signaling pathways.
Assuntos
Adenoviridae/genética , Proteínas E4 de Adenovirus/genética , Conexina 43/metabolismo , Conexinas/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endotélio Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Conexina 43/análise , Conexina 43/genética , Conexinas/análise , Conexinas/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Células Endoteliais/química , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Camundongos , Miocárdio/metabolismo , Toxina Pertussis/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteína alfa-5 de Junções ComunicantesRESUMO
5,6,7,8-Tetrahydrobiopterin (BH(4)) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH(4), in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH(4) insufficiency, but rather results from a diminished ratio of BH(4) vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH(2)). In support of this hypothesis, [(3)H]BH(4) binding studies revealed that BH(4) and BH(2) bind eNOS with equal affinity (K(d) approximately 80 nM) and BH(2) can rapidly and efficiently replace BH(4) in preformed eNOS-BH(4) complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH(2) levels increased from undetectable to 40% of total biopterin. This BH(2) accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH(4) supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH(4):BH(2) and not absolute intracellular levels of BH(4). Reciprocally, superoxide production was found to negatively correlate with intracellular BH(4):BH(2). Hyperglycemia-associated BH(4) oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH(4):BH(2), rather than BH(4) depletion per se, as the molecular trigger for NO insufficiency in diabetes.
Assuntos
Biopterinas/análogos & derivados , Células Endoteliais/metabolismo , Glucose/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animais , Biopterinas/metabolismo , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Camundongos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III , Oxirredução , Ligação Proteica , Ratos , Ratos Zucker , Fatores de Tempo , TrítioRESUMO
UNLABELLED: We report a case of hypotension and bradycardia associated with intravenous fomepizole infusion. CASE REPORT: A 59-year-old man presented to hospital 10 hours after ethylene glycol ingestion with ataxia, slurred speech, metabolic acidosis, heart rate 70/min, blood pressure 160/100 mmHg. Treatment with hemodialysis and fomepizole began 7.5 hours after admission. Severe bradycardia (29/min) and hypotension (69 mmHg systolic) occurred immediately following a 30 minute intravenous infusion of the first (19 mg/kg) fomepizole dose, but rapidly corrected with 1 mg atropine. Transient bradycardia (48/min) and hypotension (89/57 mmHg) recurred immediately after the second (10 mg/kg) fomepizole dose, also given during dialysis. DISCUSSION: Hemodialysis may cause a drop in blood pressure and heart rate; however, the close temporal relationship with fomepizole infusions, dose-related symptom intensity and recurrence with rechallenge suggest a causal relationship with fomepizole. Hemodialysis, acidosis and high initial fomepizole dose may have enhanced patient susceptibility, as a post-dialysis fomepizole dose was well tolerated. CONCLUSION: Fomepizole may precipitate bradycardia and/or hypotension during hemodialysis. Monitor vital signs closely during and immediately after infusion.
Assuntos
Antídotos/efeitos adversos , Pirazóis/efeitos adversos , Diálise Renal , Acidose/complicações , Antídotos/administração & dosagem , Antídotos/uso terapêutico , Pressão Sanguínea/efeitos dos fármacos , Bradicardia/induzido quimicamente , Relação Dose-Resposta a Droga , Etilenoglicol/intoxicação , Fomepizol , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipotensão/induzido quimicamente , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Pirazóis/administração & dosagem , Pirazóis/uso terapêuticoRESUMO
Similar to endothelial cells (ECs), vascular endothelial growth factor (VEGF) induces Bcl-2 expression on VEGF receptor-positive (VEGFR(+)) primary leukemias and cell lines, promoting survival. We investigated the molecular pathways activated by VEGF on such leukemias, by performing a gene expression analysis of VEGF-treated and untreated HL-60 leukemic cells. One gene to increase after VEGF stimulation was heat shock protein 90 (Hsp90). This was subsequently confirmed at the protein level, on primary leukemias and leukemic cell lines. VEGF increased the expression of Hsp90 by interacting with KDR and activating the mitogen-activated protein kinase cascade. In turn, Hsp90 modulated Bcl-2 expression, as shown by a complete blockage of VEGF-induced Bcl-2 expression and binding to Hsp90 by the Hsp90-specific inhibitor geldanamycin (GA). GA also blocked the VEGF-induced Hsp90 binding to APAF-1 on leukemic cells, a mechanism shown to inhibit apoptosis. Notably, VEGF blocked the proapoptotic effects of GA, correlating with its effects at the molecular level. Earlier, we showed that in some leukemias, a VEGF/KDR autocrine loop is essential for cell survival, whereas here we identified the molecular correlates for such an effect. We also demonstrate that the generation of a VEGF/VEGFR autocrine loop on VEGFR(+) cells such as ECs, also protected them from apoptosis. Infection of ECs with adenovirus-expressing VEGF resulted in elevated Hsp90 levels, increased Bcl-2 expression, and resistance to serum-free or GA-induced apoptosis. In summary, we demonstrate that Hsp90 mediates antiapoptotic and survival-promoting effects of VEGF, which may contribute to the survival advantage of VEGFR(+) cells such as subsets of leukemias.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Genes bcl-2/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/fisiologia , Leucemia/patologia , Linfocinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Benzoquinonas , Meios de Cultura Livres de Soro , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Lactamas Macrocíclicas , Leucemia/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Isoformas de Proteínas/farmacologia , Quinonas/farmacologia , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento/efeitos dos fármacos , Receptores de Fatores de Crescimento/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Especially when exposed to inflammatory stimuli, endothelial cells (EC) have been shown to promote the maturation of monocytes into dendritic cells (DC) and the long-term proliferation of CD34+ cells by constitutive cytokine production and direct cellular contact. We therefore hypothesized that cytokine-stimulated EC would induce hematopoietic progenitor cells to develop into mature dendritic cells. To test this theory, human CD34+ cells derived from cord blood or leukapheresis products were cultured with a monolayer of either interleukin (IL)-1beta, IL-4, or tumor necrosis factor (TNF)-alpha-stimulated human umbilical cord EC. The cells in suspension were analyzed weekly over a period of 6 weeks. IL-1beta supported cell expansion, whereas IL-4 had no effect on cell expansion or DC differentiation. Only TNF-alpha-stimulated EC induced the development of mature, allostimulatory DC with a high expression of CD83, HLA-DR, CD1a, and costimulatory molecules like CD80 and CD86. Acute myeloid leukemia cells from the cell line Kasumi-1 also developed DC-like features when cocultured with TNF-alpha-stimulated EC. Direct contact between endothelial and progenitor cells increased the number of developing DC. Cell cycle analysis and apoptosis studies demonstrated a reduced G2M fraction, an increased S fraction, and a decrease in TNF-alpha-dependent apoptosis of DC developing in the presence of endothelial cells. As shown by electron and confocal microscopic studies, intimate interactions between EC and DC occurred, resulting in the internalization of the developing DC within the EC monolayer and a bidirectional exchange of proteins. We conclude that, via the action of TNF-alpha, inflamed human endothelium can induce CD34+ and leukemic cells to differentiate into dendritic cells.
Assuntos
Citocinas/metabolismo , Células Dendríticas/citologia , Células Endoteliais/metabolismo , Sangue Fetal/citologia , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD , Antígenos CD1/metabolismo , Antígenos CD34/imunologia , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Antígenos HLA-DR/metabolismo , Humanos , Imunoglobulinas/metabolismo , Interleucina-4/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Tumorais Cultivadas , Antígeno CD83RESUMO
The early 4 region (E4) of the adenoviral vectors (AdE4(+)) prolongs human endothelial cell (EC) survival and alters the angiogenic response, although the mechanisms for the EC-specific, AdE4(+)-mediated effects remain unknown. We hypothesized that AdE4(+) modulates EC survival through activation of the vascular endothelial (VE)-cadherin/Akt pathway. Here, we showed that AdE4(+), but not AdE4(-) vectors, selectively stimulated phosphorylation of both Akt at Ser(473) and Src kinase in ECs. The phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin abrogated AdE4(+) induction of both phospho-Akt expression and prolonged EC survival. Regulation of phospho-Akt was found to be under the control of various factors, namely VE-cadherin activation, Src kinase, tyrosine kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Downstream targets of Akt signaling resulted in glycogen synthase kinase-3alpha/beta phosphorylation, beta-catenin up-regulation, and caspase-3 suppression, all of which led to AdE4(+)-mediated EC survival. Furthermore, infection with AdE4(+) vectors increased the angiogenic potential of ECs by promoting EC migration and capillary tube formation in Matrigel plugs. This selective AdE4(+)-mediated enhanced motility of ECs was also blocked by PI3K inhibitors. Taken together, these results suggest that activation of the VE-cadherin/Akt pathway is critical for AdE4(+)-mediated survival of ECs and angiogenic potential.
Assuntos
Proteínas E4 de Adenovirus/genética , Caderinas/metabolismo , Sobrevivência Celular/genética , Endotélio Vascular/citologia , Neovascularização Fisiológica/genética , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Androstadienos/farmacologia , Apoptose/genética , Células Cultivadas , Cromonas/farmacologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Humanos , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt , WortmaninaRESUMO
Reporter genes, including green fluorescent protein (GFP), have been used to monitor the expression of transgenes introduced into vascular cells by gene transfer vectors. Here, we demonstrate that GFP by itself can selectively induce expression of certain genes in endothelial cells. Elevation of the cytoplasmic concentration of GFP in endothelial cells, specifically, resulted in a robust upregulation of heat shock protein 70 (HSP70). GFP induced both mRNA and protein expression of HSP70 in a dose-dependent manner. GFP-mediated up-regulation of HSP70 resulted in induction of cyclooxygenase-2 (COX-2) followed by prostaglandin E2 (PGE2) production. GFP-mediated up-regulation of HSP70 is independent of mitogen-activated protein kinase and phosphatidylinositol-3-kinase signaling cascades because inhibition of these pathways had no effect on HSP70 increases. Adenoviral delivery of GFP into murine vasculature significantly enhanced blood flow, suggesting that sufficient PGE2 is produced to induce vasodilation. Identification of the molecular partners that interact with GFP will increase our understanding of the vascular-specific factors that regulate stress angiogenesis and hemostasis.