RESUMEN
BACKGROUND: Many clinical toxicology laboratories receive urine specimens in urine cups that contain point of care (POC) drug testing strips. We conducted this study to evaluate the effect on the stability of commonly measured drugs in the clinical toxicology laboratory when urine is exposed to POC urine drug testing cups. METHODS: Drug free urine was spiked with 85 drugs that were measured by a validated liquid chromatography mass spectrometry (LCMS) method after exposure to POC urine drug testing cups at ambient and 2-6⯰C temperatures. Alterations ≥20% were defined as significant changes in the drugs concentration. RESULTS: Concentrations of amitriptyline, cyclobenzaprine, fentanyl, fluoxetine, flunitrazepam, nortriptyline, paroxetine, and sertraline were significantly reduced when urine specimens were stored inside POC urine drug testing cups for 24â¯h at ambient temperature. Storage of urine in urine chemistry dipsticks reduced the concentration of several drugs. When spiked urine was exposed to an increasing number of POC urine drug testing strips, the concentrations of some drugs were reduced in a dose-dependent manner. The drugs that were absorbed by POC urine drug testing strips were partially back extracted from the strips. CONCLUSION: Exposure of urine specimens to POC urine drug testing strips reduces the concentration of several drugs measured by LCMS method.
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Pruebas en el Punto de Atención , Amitriptilina/análogos & derivados , Amitriptilina/orina , Cromatografía Liquida , Almacenaje de Medicamentos , Fentanilo/orina , Flunitrazepam/orina , Fluoxetina/orina , Humanos , Espectrometría de Masas , Nortriptilina/orina , Paroxetina/orina , Sertralina/orinaRESUMEN
BACKGROUND: Many patients on chronic opioid therapy suffer from constipation, one of the most common side effect of opioids. Movantik™ (naloxegol) is an opioid antagonist that is recently introduced in the market to treat opioid-induced constipation and contains naloxegol as the active ingredient. Naloxegol is a pegylated (polyethylene glycol-modified) derivative of α-naloxol. Detection of naloxone in the patients urine after consumption of naloxegol was not reported by the manufacturer and may mislead the prescribing clinicians. This study was conducted to investigate the presence of naloxone in the urine of patients that consume movnatik in pain management clinics. METHODS: The presence of naloxone and naloxol in the urine of 45 patients that consumed naloxegol and 25 patients that consumed suboxone™ were investigated using a liquid chromatography mass spectrometry (LCMS) method. The urinary concentration of naloxone, naloxol, and their glucuronide conjugates were evaluated in five volunteers that took one pill of naloxegol for one day and one volunteer who took the pill for three days. RESULTS: Naloxone was detected in the urine of 45 individuals that were prescribed naloxegol. Urinary concentration of naloxone showed a distribution with a mean of 25⯱â¯18â¯ng/ml. Consumption of one pill of 25â¯mg naloxegol resulted in the detection of naloxol and naloxone in the urine of 5 volunteers 1â¯h after taking the pill. Evaluation of urine specimens from 25 patients that consumed suboxone™, resulted in the detection of naloxone (180⯱â¯187â¯ng/ml) and naloxol (6.3⯱â¯7.2â¯ng/ml). CONCLUSIONS: This study demonstrated that consumption of naloxegol leads to appearance of naloxone in the urine of patients receiving opioid therapy in pain management clinics.
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Morfinanos , Naloxona , Dolor/tratamiento farmacológico , Dolor/orina , Polietilenglicoles , Detección de Abuso de Sustancias , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Morfinanos/administración & dosificación , Morfinanos/farmacocinética , Naloxona/administración & dosificación , Naloxona/farmacocinética , Polietilenglicoles/administración & dosificación , Polietilenglicoles/farmacocinética , Estudios RetrospectivosRESUMEN
This article was unintentionally published twice in this journal, by the same authors. The following should be considered the version of record and used for citation purposes: "Mehran Haidari, Wei Zhang, James T Willerson and Richard AF Dixon, Disruption of endothelial adherens junctions by high glucose is mediated by protein kinase C-ß-dependent vascular endothelial cadherin tyrosine phosphorylation, Cardiovascular Diabetology, Volume 13, Issue 1, doi: 10.1186/1475-2840-13-112 ". The duplicate "Mehran Haidari, Wei Zhang, James T Willerson and Richard AF Dixon, Disruption of endothelial adherens junctions by high glucose is mediated by protein kinase C-ß-dependent vascular endothelial cadherin tyrosine phosphorylation, Cardiovascular Diabetology, Volume 13, Issue 1, doi: 10.1186/1475-2840-13-105 " is to be ignored. The Publisher apologizes to the readers of the journal for not detecting the duplication during the publication process.
RESUMEN
CONTEXT: -Previous studies suggest that training in pathology residency programs does not adequately prepare pathology residents to become competent in clinical chemistry. OBJECTIVES: -To define the beliefs of pathology residents in the United States regarding their preparation for practicing clinical chemistry in their career, their attitude toward the discipline, and the attractiveness of clinical chemistry as a career. DESIGN: -The residents of all pathology residency programs in the United States were given the opportunity to participate in an online survey. RESULTS: -Three hundred thirty-six pathology residents responded to the survey. Analysis of the survey results indicates that pathology residents are more likely to believe that their income may be lower if they select a career that has a clinical chemistry focus and that their faculty do not value clinical chemistry as much as the anatomic pathology part of the residency. Residents also report that clinical chemistry is not as enjoyable as anatomic pathology rotations during residency or preferable as a sole career path. A large proportion of residents also believe that they will be slightly prepared or not prepared to practice clinical chemistry by the end of their residency and that they do not have enough background and/or time to learn clinical chemistry during their residency programs to be able to practice this specialty effectively post graduation. CONCLUSIONS: -Our survey results suggest that many pathology residents do not have a positive attitude toward clinical chemistry and do not experience a supportive learning environment with an expectation that they will become competent in clinical chemistry with a residency alone.
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Actitud del Personal de Salud , Química Clínica , Química Clínica/educación , Educación de Postgrado en Medicina , Humanos , Internado y Residencia , Patólogos/educación , Patología , Encuestas y CuestionariosRESUMEN
BACKGROUND: We sought to determine the association of plasma level of soluble L-selectin (sL-selectin) and F206L polymorphism of L-selectin with asthma. METHODS: A total of 90 asthmatic patients and 90 sex- and age-matched healthy controls were enrolled. The plasma level of sL-selectin was measured by enzyme-linked immunosorbent assay (ELISA) method. An amplification refractory mutation system polymerase chain reaction was performed to detect F206L polymorphism of L-selectin. RESULTS: The mean plasma levels of sL-selectin was significantly higher in the patients with asthma than the controls (2113 ± 466 vs. 1664 ± 322 ng/ml, P = 0.001). Logistic regression analysis after adjustment for age, sex, and body mass index demonstrated that plasma levels of sL-selectin are an independent biomarkers for asthma (odds ratio [OR], 1.86; 95% confidence interval [95% CI], 1.42-2.24). The area under the receiver operating characteristic (ROC) curve for sL-selectin was 0.792, 95% CI (0.732-0.862), P = 0.0001. Individuals with the minor homozygote of F206L polymorphism of L-selectin demonstrated a higher level of sL-selectin than the major homozygous (2319 ± 732 vs. 1917 ± 453 ng/ml, P = 0.02). No association was found between F206L polymorphism of L-selectin with asthma. CONCLUSION: Our study suggests that plasma level of sL-selectin is an independent biomarker for asthma.
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Asma/sangre , Biomarcadores/sangre , Selectina L/sangre , Adulto , Asma/genética , Femenino , Genotipo , Humanos , Selectina L/genética , Masculino , Persona de Mediana Edad , Polimorfismo Genético/genéticaRESUMEN
BACKGROUND: Hyperglycemia has been recognized as a primary factor in endothelial barrier dysfunction and in the development of micro- and macrovascular diseases associated with diabetes, but the underlying biochemical mechanisms remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) leads to the disruption of endothelial adherens junctions and increases the transendothelial migration (TEM) of leukocytes. METHODS: VE-cad tyrosine phosphorylation, adherens junction integrity and TEM of monocytes in human umbilical vein endothelial cells (HUVECs) treated with high-concentration glucose were evaluated. The role of protein kinase C (PKC) in induction of endothelial cells adherence junction disruption by exposure of HUVECs to high concentration of glucose was explored. RESULTS: The treatment of HUVEC with high-concentration glucose increased VE-cad tyrosine phosphorylation, whereas mannitol or 3-O-methyl-D-glucose had no effect. In addition, high-concentration glucose increased the dissociation of the VE-cad-ß-catenin complex, activation of the Wnt/ß-catenin pathway, and the TEM of monocytes. These alterations were accompanied by the activation of endothelial PKC and increased phosphorylation of ERK and myosin light chain (MLC). High-concentration glucose-induced tyrosine phosphorylation of VE-cad was attenuated by: 1- the inhibition of PKC-ß by overexpression of dominant-negative PKC-ß 2- inhibition of MLC phosphorylation by overexpression of a nonphosphorylatable dominant-negative form of MLC, 3- the inhibition of actin polymerization by cytochalasin D and 4- the treatment of HUVECs with forskolin (an activator of adenylate cyclase). CONCLUSIONS: Our findings show that the high-concentration glucose-induced disruption of endothelial adherens junctions is mediated by tyrosine phosphorylation of VE-cad through PKC-ß and MLC phosphorylation.
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Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Endotelio Vascular/metabolismo , Glucosa/metabolismo , Proteína Quinasa C beta/metabolismo , Movimiento Celular/fisiología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Monocitos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Tirosina/metabolismo , beta Catenina/metabolismoRESUMEN
AIMS: The effect of antioxidants on treatment of cancer is still controversial. Previously, we demonstrated that interaction of breast cancer cells with endothelial cells leads to tyrosine phosphorylation of VE-cadherin and disruption of endothelial adherens junction (EAJ). The molecular mechanism underlying the anti-metastatic effects of mushroom-derived active hexode correlated compound (AHCC) remains elusive. MAIN METHODS: Several cellular and biochemical techniques were used to determine the contribution of oxidative stress in the disruption of EAJ and to test this hypothesis that AHCC inhibits the breast cancer cell-induced disruption of EAJ. KEY FINDINGS: Interaction of breast cancer cells (MDA-MB-231 cells) with human umbilical vein endothelial cells (HUVECs) leads to an increase in generation of reactive oxygen species (ROS). Treatment of HUVECs with H2O2 or phorbol myristate acetate (PMA) led to tyrosine phosphorylation of VE-cadherin, dissociation of ß-catenin from VE-cadherin complex and increased transendothelial migration (TEM) of MDA-MB-231 cells. Induction of VE-cadherin tyrosine phosphorylation by PMA or by interaction of MDA-MB-231 cells with HUVECs was mediated by HRas and protein kinase C-α signaling pathways. Disruption of EAJ and phosphorylation of VE-cadherin induced by interaction of MDA-MB-231 cells with HUVECs were attenuated when HUVECs were pretreated with an antioxidant, N-acetylcysteine (NAC) or AHCC. AHCC inhibited TEM of MDA-MB-231 cells and generation of ROS induced by interaction of MDA-MB-231 cells with HUVECs. SIGNIFICANCE: Our studies suggest that ROS contributes to disruption of EAJ induced by interaction of MDA-MB-231 cells with HUVECs and AHCC attenuates this alteration.
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Uniones Adherentes/efectos de los fármacos , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Extractos Vegetales/farmacología , Acetilcisteína/farmacología , Antígenos CD/metabolismo , Antioxidantes/farmacología , Cadherinas/metabolismo , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Tirosina/metabolismo , beta Catenina/metabolismoRESUMEN
The molecular mechanisms that regulate the endothelial response during transendothelial migration (TEM) of invasive cancer cells remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the disruption of endothelial cell adherens junctions and in the diapedesis of metastatic cancer cells. We sought to determine the signaling mechanisms underlying the disruption of endothelial adherens junctions after the attachment of invasive breast cancer cells. Attachment of invasive breast cancer cells (MDA-MB-231) to human umbilical vein endothelial cells induced tyrosine phosphorylation of VE-cad, dissociation of ß-catenin from VE-cad, and retraction of endothelial cells. Breast cancer cell-induced tyrosine phosphorylation of VE-cad was mediated by activation of the H-Ras/Raf/MEK/ERK signaling cascade and depended on the phosphorylation of endothelial myosin light chain (MLC). The inhibition of H-Ras or MLC in endothelial cells inhibited TEM of MDA-MB-231 cells. VE-cad tyrosine phosphorylation in endothelial cells induced by the attachment of MDA-MB-231 cells was mediated by MDA-MB-231 α(2)ß(1) integrin. Compared with highly invasive MDA-MB-231 breast cancer cells, weakly invasive MCF-7 breast cancer cells expressed lower levels of α(2)ß(1) integrin. TEM of MCF-7 as well as induction of VE-cad tyrosine phosphorylation and dissociation of ß-catenin from the VE-cad complex by MCF-7 cells were lower than in MDA-MB-231 cells. These processes were restored when MCF-7 cells were treated with ß(1)-activating antibody. Moreover, the response of endothelial cells to the attachment of prostatic (PC-3) and ovarian (SKOV3) invasive cancer cells resembled the response to MDA-MB-231 cells. Our study showed that the MDA-MB-231 cell-induced disruption of endothelial adherens junction integrity is triggered by MDA-MB-231 cell α(2)ß(1) integrin and is mediated by H-Ras/MLC-induced tyrosine phosphorylation of VE-cad.
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Uniones Adherentes/metabolismo , Antígenos CD/metabolismo , Neoplasias de la Mama/metabolismo , Cadherinas/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Integrina alfa2beta1/metabolismo , Uniones Adherentes/genética , Uniones Adherentes/patología , Antígenos CD/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Cadherinas/genética , Línea Celular Tumoral , Endotelio Vascular/patología , Femenino , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Integrina alfa2beta1/genética , Sistema de Señalización de MAP Quinasas/genética , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Invasividad Neoplásica , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Fosforilación/genéticaRESUMEN
Vascular endothelial cadherin (VE-cad) tyrosine (Tyr) phosphorylation has been implicated in the disruption of adherens junctions (AJs) induced by inflammatory reactions. The impacts of statins on integrity of AJs and VE-cad Tyr phosphorylation have not been explored. The effects of atorvastatin on IL-1ß and monocyte-induced VE-cad Tyr phosphorylation in human umbilical vein endothelial cells (ECs) were studied. In ECs treated with interleukin (IL)-1ß for 30 min, VE-cad Tyr phosphorylation, dissociation of the VE-cad/ß-catenin complex and transendothelial migration (TEM) of monocytes were increased. These processes were mediated by activation of HRas and RhoA that leads to phosphorylation of myosin light chain (MLC). Atorvastatin inhibited IL-1ß-induced Tyr phosphorylation of VE-cad by inhibiting RhoA and by dephosphorylating MLC. The attenuating effect of atorvastatin on VE-cad Tyr phosphorylation was reversed when RhoA was activated or MLC phosphatase was inhibited. Furthermore, inhibiting farnesyl transferase or geranylgeranyl transferase reproduced the inhibitory effects of atorvastatin on VE-cad Tyr phosphorylation. In addition, atorvastatin inhibited monocyte-induced VE-cad Tyr phosphorylation in ECs and attenuated IL-1ß-induced TEM of monocytes. Our study introduces a novel pleiotropic effect of atorvastatin and suggests that statins protect the integrity of AJs in ECs by inhibiting RhoA-mediated Tyr phosphorylation of VE-cad.
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Uniones Adherentes/efectos de los fármacos , Cadherinas/antagonistas & inhibidores , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Ácidos Heptanoicos/farmacología , Pirroles/farmacología , Tirosina/antagonistas & inhibidores , Atorvastatina , Cadherinas/metabolismo , Endotelio Vascular/citología , Humanos , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Cadenas Ligeras de Miosina/antagonistas & inhibidores , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Tirosina/metabolismoRESUMEN
AIMS: Transendothelial migration (TEM) of monocytes is a crucial step in inflammatory processes such as atherogenesis. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the dissociation of adherens junctions and the increased paracellular permeability of endothelial cells (ECs) that occur during TEM of monocytes. However, the underlying molecular mechanism has not been determined. We tested the hypothesis that the phosphorylation of myosin light chain (MLC) in ECs is crucial for the dissociation of adherens junctions during TEM of monocytes. METHODS AND RESULTS: Using a combination of biochemical and cellular techniques, we provide evidence for the signal transduction pathways that regulate tyrosine phosphorylation of VE-cad in ECs after the attachment of monocytes. Our findings indicate that after interaction of integrins on THP-1 cells with adhesion molecules on ECs, the induction of the HRas\Raf\MEK\ERK signalling cascade leads to the phosphorylation of MLC. This results in the recruitment of Src to the VE-cad complex and tyrosine phosphorylation of VE-cad, which leads to dissociation of ß-catenin from the VE-cad complex, formation of gaps between ECs, and enhancement of THP-1 cell TEM. CONCLUSION: Our studies suggest that monocyte-induced phosphorylation of MLC in ECs enhances TEM of monocytes through dissociation of EC adherens junctions.
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Uniones Adherentes/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Monocitos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Migración Transendotelial y Transepitelial , Antígenos CD/metabolismo , Cadherinas/metabolismo , Línea Celular , Quimiocina CCL2/metabolismo , Técnicas de Cocultivo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/metabolismo , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transfección , Tirosina , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Influenza A viruses are a severe threat worldwide, causing large epidemics that kill thousands every year. Prevention of influenza infection is complicated by continuous viral antigenic changes. Newer anti-influenza agents include MEK/ERK and protein kinase C inhibitors; however, the downstream effectors of these pathways have not been determined. In this study, we identified a common mechanism for the inhibitory effects of a significant group of anti-influenza agents. Our studies showed that influenza infection activates a series of signaling pathways that converge to induce myosin light chain (MLC) phosphorylation and remodeling of the actin cytoskeleton. Inhibiting MLC phosphorylation by blocking RhoA/Rho kinase, phospholipase C/protein kinase C, and HRas/Raf/MEK/ERK pathways with the use of genetic or chemical manipulation leads to the inhibition of influenza proliferation. In contrast, the induction of MLC phosphorylation enhances influenza proliferation, as does activation of the HRas/Raf/MEK/ERK signaling pathway. This effect is attenuated by inhibiting MLC phosphorylation. Additionally, in intracellular trafficking studies, we found that the nuclear export of influenza ribonucleoprotein depends on MLC phosphorylation. Our studies provide evidence that modulation of MLC phosphorylation is an underlying mechanism for the inhibitory effects of many anti-influenza compounds.
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Antivirales/farmacología , Cadenas Ligeras de Miosina/metabolismo , Orthomyxoviridae/efectos de los fármacos , Orthomyxoviridae/fisiología , Replicación Viral/efectos de los fármacos , Actinas/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Adenoviridae/efectos de los fármacos , Adenoviridae/genética , Animales , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Perros , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Células Endoteliales/virología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Gripe Humana/virología , Modelos Biológicos , Ácido N-Acetilneuramínico/metabolismo , Fosforilación/efectos de los fármacos , Plásmidos/metabolismo , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/metabolismo , Ribonucleoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción Genética , Venas Umbilicales/citología , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
AIMS: The localization of atherosclerotic lesions to predictable regions in mammalian arteries has been recognized for over a century. We sought to investigate the association between oxidative stress and regional susceptibility of the mouse aorta to atherosclerosis. MAIN METHODS: En face confocal microscopy was employed to assess oxidative stress in the aortic intima of atherosclerosis-susceptible and protected regions of wild-type C57BL/6 mouse. Expression of reactive oxygen species and antioxidant producing genes were compared in endothelial cells from the susceptible and protected regions. KEY FINDINGS: In vivo administration of redox-sensitive fluorescent dyes revealed an increase in the production of reactive oxygen species (ROS) in the atherosclerosis-susceptible regions relative to the protected regions. In contrast, Hoechst a redox-insensitive dye distributed evenly in the susceptible and protected regions. Accumulation of superoxide in the susceptible regions of the aorta was significantly blocked by the administration of Diphenyleneiodonium, a flavoprotein inhibitor. mRNA levels of superoxide-producing and scavenging enzymes were significantly increased in the regions predisposed to atherosclerosis. The regional difference in oxidative stress was at a lesser magnitude in BALB/c than the atherosclerosis-susceptible mouse (C57BL/6). SIGNIFICANCE: Our study for the first time demonstrated an augmented oxidative stress in atherosclerosis-susceptible regions of the normal mouse aorta.
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Aorta/fisiopatología , Aterosclerosis/fisiopatología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , ARN Mensajero/metabolismo , Receptores de LDL/genética , Superóxidos/metabolismo , Túnica Íntima/patologíaRESUMEN
The emergence of resistance to anti-influenza drugs calls for the search for new antiviral molecules with different resistance profiles. Polyphenolic compounds are found in various plants and have antiviral and antioxidative properties. We tested the hypothesis that oligonol, a lychee fruit-derived low molecular weight polyphenol, possesses anti-influenza effects by inhibiting phosphorylation of extracellular-signal-regulated kinases (ERK). Real time PCR, plaque assay, and immunofluorescence techniques were used to study the effects of oligonol on proliferation of influenza virus. Oligonol inhibits influenza virus proliferation by blocking attachment of the virus to MDCK cells and by suppression of nuclear export of influenza virus ribonucleoprotein (RNP). Infection of MDCK cells with influenza virus leads to an increase in production of reactive oxygen species (ROS) and induction of a ROS-dependent ERK phosphorylation. Inhibition of ERK activation by a dominant negative mutant of ERK or N-acetyl-cysteine (NAC) leads to inhibition of influenza RNP nuclear export. Phorbol 12-myristate 13-acetate (PMA) induces ROS production, ERK phosphorylation and enhances influenza proliferation in MDCK cells. Oligonol and NAC inhibit PMA-induced ERK phosphorylation and ROS production. Our studies suggest that the underlying mechanism for the inhibitory effect of oligonol on influenza virus RNP nuclear export is blocking of ROS-dependent induction of ERK phosphorylation.
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Antivirales/farmacología , Catequina/análogos & derivados , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Virus de la Influenza A/efectos de los fármacos , Litchi/química , Fenoles/farmacología , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Adenoviridae , Animales , Antivirales/uso terapéutico , Transporte Biológico/efectos de los fármacos , Catequina/farmacología , Catequina/uso terapéutico , Línea Celular , Frutas , Técnicas de Transferencia de Gen , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Infecciones por Orthomyxoviridae/metabolismo , Fenoles/uso terapéutico , Fosforilación/efectos de los fármacos , Fitoterapia , Extractos Vegetales/uso terapéutico , Ribonucleoproteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Virales/metabolismoRESUMEN
OBJECTIVE: Influenza can trigger heart attacks, and vaccination against influenza reduces the risk of cardiovascular events. Currently, it is believed that influenza virus in general does not disseminate to extra-pulmonary tissues. We assessed the vascular effects of influenza infection and whether the virus can directly infect atherosclerotic arteries in mice. METHODS/RESULTS: We intranasally infected 4 different types of mice--atherosclerotic apo E-deficient (our primary model), LDL receptor knockout, C57BL/6, and outbred Swiss--with influenza A/HK (H3/N2) virus. On day 7 after infection, we cultured viable virus from lung, aorta, and heart tissue, but not from the blood of apo E-deficient mice. Immunofluorescence studies showed influenza A virus NP1 protein and real time polymerase chain reaction (PCR) assay showed RNA in the aorta of infected apo E-deficient mice. Infected mice had significantly higher blood levels of chemokines and cytokines than control mice. At the local level, gene expression for several chemokines and cytokines was increased and eNOS expression was decreased. Infected mice had a higher density of macrophages in plaque than did control mice. CONCLUSIONS: We have shown for the first time that influenza virus can directly infect and reside in atherosclerotic arteries and that infection was associated with systemic and arterial-level pro-inflammatory changes.
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Aterosclerosis/virología , Orthomyxoviridae/patogenicidad , Animales , Arterias/virología , Inflamación/virología , RatonesRESUMEN
Cytokines gene polymorphisms have been implicated in susceptibility to ischemic stroke. This study aims to determine the influence of the polymorphism within the intron 2 of the interleukin- 1 receptor antagonist (IL-1Ra) gene on the susceptibility to stroke. A variable number of tandem repeats (VNTR) in intron 2 of the IL-1Ra gene was analyzed in 148 patients with stroke and 161 healthy volunteers from the same area. The carriage rate of allele 2 of IL-1Ra gene, low producer, was significantly higher in patients with stroke compared to the controls (29% vs 21% p = 0.02). Frequency of IL1RN1/IL1RN1 genotype in the patients was significantly lower than the controls (49% vs 66% p = 0.003). The distribution of homozygous genotypes of IL1RN2 was not different between the controls and stroke patients while the heterozygous genotype was more frequent among the patients. (39% vs 25%, respectively). Multiple logistic regression analysis demonstrated that individuals who carry allele 2 for IL-1Ra gene had a significantly higher risk for ischemic stroke with an odds ratio of 2.48 (95% CI, 1.67, 3.51, p = 0.006). These data suggest that allele 2 of the IL-1Ra intron 2 gene represents a susceptibility factor in the development of ischemic stroke.
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Predisposición Genética a la Enfermedad , Proteína Antagonista del Receptor de Interleucina 1/genética , Polimorfismo Genético , Accidente Cerebrovascular/genética , Anciano , Femenino , Genotipo , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Secuencias Repetidas en TándemRESUMEN
Influenza epidemics cause numerous deaths and millions of hospitalizations each year. Because of the alarming emergence of resistance to anti-influenza drugs, there is a need to identify new naturally occurring antiviral molecules. We tested the hypothesis that pomegranate polyphenol extract (PPE) has anti-influenza properties. Using real time PCR, plaque assay, and TCID 50% hemagglutination assay, we have shown that PPE suppresses replication of influenza A virus in MDCK cells. PPE inhibits agglutination of chicken red blood cells (cRBC) by influenza virus and is virucidal. The single-cycle growth conditions indicated that independent of the virucidal effect PPE also inhibits viral RNA replication. PPE did not alter virus ribonucleoprotein (RNP) entry into nucleus or translocation of virus RNP from nucleus to cytoplasm in MDCK cells. We evaluated four major Polyphenols in PPE (ellagic acid, caffeic acid, luteolin, and punicalagin) and demonstrated that punicalagin is the effective, anti-influenza component of PPE. Punicalagin blocked replication of the virus RNA, inhibited agglutination of chicken RBC's by the virus and had virucidal effects. Furthermore, the combination of PPE and oseltamivir synergistically increased the anti-influenza effect of oseltamivir. In conclusion, PPE inhibited the replication of human influenza A/Hong Kong (H3N2) in vitro. Pomegranate extracts should be further studied for therapeutic and prophylactic potential especially for influenza epidemics and pandemics.
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Antivirales/uso terapéutico , Taninos Hidrolizables/uso terapéutico , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Lythraceae/química , Oseltamivir/uso terapéutico , Extractos Vegetales/uso terapéutico , Animales , Antivirales/farmacología , Línea Celular , Pollos , Perros , Sinergismo Farmacológico , Eritrocitos/efectos de los fármacos , Hemaglutinación/efectos de los fármacos , Humanos , Taninos Hidrolizables/aislamiento & purificación , Taninos Hidrolizables/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Oseltamivir/farmacología , Fitoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación Viral/efectos de los fármacosRESUMEN
The polymorphism of the E-selectin gene has been implicated in the pathogenesis of atherosclerosis. We sought to explore whether the allelic variants relate to ischemic stroke. We conducted a case-control study of 359 cases of ischemic stroke and 353 community controls. Participants were evaluated for known cerebrovascular risk factors, and the E-selectin S128R and L554F genotypes were established using the polymerase chain reaction (PCR) method. The frequency of minor allele (R) and heterozygous (RS) genotype of E-selectin S128R polymorphism was significantly higher in the stroke patients than in the controls. Evaluation of genetic variation for E-selectin L554F polymorphisms revealed that the frequency of minor allele (F) and its heterozygous genotype (LF) is almost 4 times higher in the stroke patients than the controls (16.7 vs. 4.3 and 33.4 vs. 8.5, respectively). Multivariable logistic regression analysis after adjustment for age, sex and conventional vascular risk factors demonstrated that alleles R of S128R and F of L554F polymorphisms are independent risk factors for ischemic stroke. The combination of 2 minor alleles of E-selectin genes appeared to be the strongest susceptibility factor for ischemic stroke (adjusted OR: 5.89, 95% CI: 2.84-12.21, p = 0.0001). Our study demonstrates that the E-selectin S128R and L554F polymorphisms are associated with susceptibility to ischemic stroke.
Asunto(s)
Selectina E/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo Genético/genética , Accidente Cerebrovascular/genética , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes/genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Análisis de Regresión , Estudios RetrospectivosRESUMEN
A C-T substitution at position 590 in the interleukin-4 (IL-4) gene is associated with increased production of IL-4. We sought to determine the associations between this polymorphism and susceptibility to brucellosis. DNA was extracted from the whole blood of 163 control individuals and 282 patients with brucellosis. A polymorphism in the IL-4 gene at position 590 from the promoter site was determined using an allele-specific PCR method. The prevalence of the T allele of IL-4 polymorphism was significantly higher in the patients group than in controls (28.9% vs 11.4%, p<0.004). Patients with brucellosis had a higher frequency of intermediate producer genotype (CT) (53.5% vs 22.7%, p<0.001) while low producer genotype (CC) was higher in the control group (77.3% vs 44.4%). Multiple logistic regression analysis demonstrated that patients who were heterozygous (CT) for interleukin-4 promoter polymorphism had a significantly higher risk for brucellosis with an odds ratio of 4.2 (95% CI 2.7-6.6, p<0.0001). Our findings demonstrate for the first time an association between IL-4 590 promoter polymorphism and contracting brucellosis in the Iranian population.
Asunto(s)
Brucella/patogenicidad , Brucelosis/inmunología , Predisposición Genética a la Enfermedad , Interleucina-4/genética , Polimorfismo Genético/genética , Regiones Promotoras Genéticas/genética , Adulto , Brucelosis/genética , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Polimorfismo Genético/inmunología , Regiones Promotoras Genéticas/inmunologíaRESUMEN
Atherosclerotic lesions develop in regions of arterial curvature and branch points, which are exposed to disturbed blood flow and have unique gene expression patterns. The cellular and molecular basis for atherosclerosis susceptibility in these regions is not completely understood. In the intima of atherosclerosis-predisposed regions of the wild-type C57BL/6 mouse aorta, we quantified increased expression of several proinflammatory genes that have been implicated in atherogenesis, including vascular cell adhesion molecule-1 (VCAM-1) and a relative abundance of dendritic cells, but only occasional T cells. In contrast, very few intimal leukocytes were detected in regions resistant to atherosclerosis; however, abundant macrophages, including T cells, were found throughout the adventitia (Adv). Considerably lower numbers of intimal CD68+ leukocytes were found in inbred atherosclerosis-resistant C3H and BALB/c mouse strains relative to C57BL/6 and 129; however, leukocyte distribution throughout the Adv of all strains was similar. The predominant mechanism for the accumulation of intimal CD68+ cells was continued recruitment of bone marrow-derived blood monocytes, suggestive of low-grade chronic inflammation. Local proliferation of intimal leukocytes was low. Intimal CD68+ leukocytes were reduced in VCAM-1-deficient mice, suggesting that mechanisms of leukocyte accumulation in the intima of normal aorta are analogous to those in atherosclerosis.
Asunto(s)
Aterosclerosis/inmunología , Susceptibilidad a Enfermedades , Inflamación , Túnica Íntima/inmunología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aorta/anatomía & histología , Aorta/fisiología , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Complejo CD3/inmunología , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Regulación de la Expresión Génica , Leucocitos/inmunología , Masculino , Ratones , Ratones Endogámicos , Células Mieloides/citología , Células Mieloides/fisiología , Linfocitos T/citología , Linfocitos T/fisiología , Túnica Íntima/citología , Túnica Íntima/patología , Túnica Íntima/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
We investigated whether intestinal lipoprotein overproduction in a fructose-fed, insulin-resistant hamster model is prevented with insulin sensitization. Syrian Golden hamsters were fed either chow, 60% fructose for 5 wk, chow for 5 wk with the insulin sensitizer rosiglitazone added for the last 3 wk, or 60% fructose plus rosiglitazone. In vivo Triton studies showed a 2- to 3-fold increase in the large (Svedberg unit > 400) and smaller (Sf 100-400) triglyceride-rich lipoprotein particle apolipoprotein B48 (apoB48) but not triglyceride secretion with fructose feeding in the fasted state (P < 0.01) and partial normalization with rosiglitazone in fructose-fed hamsters. Ex vivo pulse-chase labeling of enterocytes confirmed the oversecretion of apoB48 lipoproteins with fructose feeding. Intestinal lipoprotein oversecretion was associated with increased expression of microsomal triglyceride transfer protein expression. With rosiglitazone treatment of fructose-fed hamsters, there was approximately 50% reduction in apoB48 secretion from primary cultured enterocytes and amelioration of the elevated microsomal triglyceride transfer protein mass and activity in fructose-fed hamsters. In contrast, in the postprandial state, the major differences between nutritional and drug intervention protocols were evident in triglyceride-rich lipoprotein triglyceride and not apoB48 secretion rates. The data suggest that intestinal lipoprotein overproduction can be ameliorated with the insulin sensitizer rosiglitazone.