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BACKGROUND: Prostate-specific antigen (PSA) is currently used as a biomarker for diagnosis and management of prostate cancer (CaP). However, PSA typically lacks the sensitivity and specificity desired of a diagnostic marker. OBJECTIVE: The goal of this study was to identify an additional biomarker or a panel of biomarkers that is more sensitive and specific than PSA in differentiating benign versus malignant prostate disease and/or localized CaP versus metastatic CaP. METHODS: Concurrent measurements of circulating interleukin-8 (IL-8), Tumor necrosis factor-α (TNF-α) and soluble tumor necrosis factor-α receptors 1 (sTNFR1) were obtained from four groups of men: (1) Controls (2) with elevated prostate-specific antigen with a negative prostate biopsy (elPSA_negBx) (3) with clinically localized CaP and (4) with castration resistant prostate cancer. RESULTS: TNF-α Area under the receiver operating characteristic curve (AUC = 0.93) and sTNFR1 (AUC = 0.97) were strong predictors of elPSA_negBx (vs. CaP). The best predictor of elPSA_negBx vs CaP was sTNFR1 and IL-8 combined (AUC = 0.997). The strongest single predictors of localized versus metastatic CaP were TNF-α (AUC = 0.992) and PSA (AUC = 0.963) levels. CONCLUSIONS: The specificity and sensitivity of a PSA-based CaP diagnosis can be significantly enhanced by concurrent serum measurements of IL-8, TNF-α and sTNFR1. In view of the concerns about the ability of PSA to distinguish clinically relevant CaP from indolent disease, assessment of these biomarkers in the larger cohort is warranted.
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The advent of highly active antiretroviral therapy (HAART) has significantly improved the prognosis for human immunodeficiency virus (HIV)-infected patients, however the adverse side effects associated with prolonged HAART therapy use continue. Although systemic viral load can be undetectable, the virus remains sequestered in anatomically privileged sites within the body. Nanotechnology-based delivery systems are being developed to target the virus within different tissue compartments and are being evaluated for their safety and efficacy. The current review outlines the various nanomaterials that are becoming increasingly used in biomedical applications by virtue of their robustness, safety, multimodality, and multifunctionality. Nanotechnology can revolutionize the field of HIV medicine by not only improving diagnosis, but also by improving delivery of antiretrovirals to targeted regions in the body and by significantly enhancing the efficacy of the currently available antiretroviral medications.
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Antivirales/administración & dosificación , Predicción , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/terapia , Nanocápsulas/administración & dosificación , Diseño de Fármacos , VIH-1 , HumanosRESUMEN
Nanotechnology offers a new platform for therapeutic delivery of antiretrovirals to the central nervous system (CNS) where human immunodeficiency virus (HIV-1) is sequestered in patients with HIV-1-associated neurological disorders (HAND). HAND is a spectrum of neurocognitive disorders that continue to persist in HIV-1-infected patients in spite of successful highly active antiretroviral therapy (HAART). Nanoformulated antiretroviral drugs offer multifunctionality, that is, the ability to package multiple diagnostic and therapeutic agents within the same nanocomposite, along with the added provisions of site-directed delivery, delivery across the blood-brain barrier (BBB), and controlled release of therapeutics. We have stably incorporated the antiretroviral drug, Amprenavir, within a transferrin (Tf)-conjugated quantum dot (QD), and evaluated the transversing ability of this Tf-QD-Amprenavir nanoplex across an in vitro BBB model and analyzed its antiviral efficacy in HIV-1-infected monocytes. We describe methods for synthesis of the Tf-QD-Amprenavir nanoplex and approaches to evaluate both its BBB transversing capability and antiviral efficacy.
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Fármacos Anti-VIH/metabolismo , Encéfalo/metabolismo , Carbamatos/metabolismo , Nanoconjugados/química , Sulfonamidas/metabolismo , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Encéfalo/irrigación sanguínea , Compuestos de Cadmio/química , Carbamatos/química , Carbamatos/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Impedancia Eléctrica , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Furanos , Expresión Génica , Proteína p24 del Núcleo del VIH/metabolismo , Duplicado del Terminal Largo de VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Microvasos/citología , Microvasos/metabolismo , Tamaño de la Partícula , Cultivo Primario de Células , Puntos Cuánticos , Compuestos de Selenio/química , Sulfatos/química , Sulfonamidas/química , Sulfonamidas/farmacología , Transferrina/química , Sulfato de Zinc/químicaRESUMEN
BACKGROUND: Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS: Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+ . Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS: Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION: Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity.
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Antígeno Prostático Específico/metabolismo , Próstata/enzimología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Catepsina D/biosíntesis , Catepsina D/genética , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Quinasa 1 de Adhesión Focal/biosíntesis , Quinasa 1 de Adhesión Focal/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Masculino , Neovascularización Fisiológica , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Antígeno Prostático Específico/antagonistas & inhibidores , Antígeno Prostático Específico/biosíntesis , Antígeno Prostático Específico/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 Relacionada con Twist/biosíntesis , Proteína 1 Relacionada con Twist/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Zinc/farmacologíaRESUMEN
Matrix metallaprotinase-9 (MMP-9) is zinc-containing proteinase whose expression and trafficking are frequently altered in cancer. MMP-9 in the plasma membrane and the secreted forms are thought to contribute to the invasive and metastatic properties of malignant tumors. We have manipulated the expression of MMP-9 in prostate tumor cell line LNCaP and measured their capacity to invade through a basement membrane matrix. Stable expression of human MMP-9 in a poorly metastatic LNCaP prostate cancer cell line produced a 2-3-fold increase in MMP-9 activity and a comparable increase in invasiveness. Transient transfection of LNCaP stable clone expressing MMP-9 with MMP-9 antisense oligonucleotide (ASODN) produced 55-90% less MMP-9 than control cells and were proportionately less invasive. In contrast, manipulating MMP-9 levels had no effect on cell migration across an uncoated membrane. A standard MMP-9 inhibitor at a concentration ranging from 1-10 nM, caused a nearly quantitative inhibition of extracellular MMP-9 activity and had significant effect on basement membrane invasion. Collectively, these results confirm the role of MMP-9 in tissue remodeling associated with prostate tumor invasion.
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Expresión Génica/genética , Metaloproteinasa 9 de la Matriz/genética , Invasividad Neoplásica/genética , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Membrana Basal/patología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Invasividad Neoplásica/patología , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismoRESUMEN
Allelic variants of the genes for chemokine receptors and their natural ligands, the chemokines, and cytokines can affect HIV-1 disease progression. This study investigates the level of expression of the CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T alleles in two unique HIV-1 infected patient cohorts that represent the two distinct stages of disease progression, namely rapid progressors (RPs) and long term non-progressors (LTNPs) (n=12/group) were recruited. Quantitation of the gene expression of CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T in peripheral blood mononuclear leukocytes (PBML) isolated from patients was performed by real time, quantitative (Q)-PCR using DNA was isolated from PBML. We observed that expression of these HIV-protective alleles was generally greater in the LTNP cohort than the RP cohort. LTNPs expressed more of the protective chemokine, SDF-1alpha than RPs, and no statistically significant difference was observed in RANTES production between the LTNPs and RPs. The LTNPs expressed significantly less amounts of cytokines IL-10 and IL-4 as compared to the RPs. Our results demonstrate that gene polymorphisms for CCR5-Delta32, CCR2b-641, RANTES In1.1C, SDF-1 3'A, IL-10-5'-592A and IL-4-589T may be used as clinical markers to predict progression of HIV-1 infections.
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Quimiocinas/genética , Citocinas/genética , Infecciones por VIH/inmunología , VIH-1 , Progresión de la Enfermedad , Expresión Génica , Frecuencia de los Genes , Marcadores Genéticos , Infecciones por VIH/genética , Humanos , Polimorfismo Genético , Pronóstico , Biosíntesis de ProteínasRESUMEN
Antiretroviral drugs are ineffective at treating viral infection in the brain because they cannot freely diffuse across the blood-brain barrier (BBB). Therefore, HIV-1 viral replication persists in the central nervous system (CNS) and continues to augment the neuropathogenesis process. Nanotechnology can play a pivotal role in HIV-1 therapeutics as it can increase drug solubility, enhance systemic bioavailability, and at the same time offer multifunctionality. Moreover, following conjugation with transferrin (Tf), these drug-loaded nanoformulations can permeate across biological barriers such as the blood brain barrier (BBB) via a receptor mediated transport mechanism. In the current study, we have stably incorporated the antiviral drug, Saquinavir, within Tf-conjugated quantum rods (QRs), which are novel nanoparticles with unique optical properties. We have evaluated the transversing ability of the QR-Tf-Saquinavir nanoformulation across an in vitro model of BBB. In addition, we have analyzed the subsequent antiviral efficacy of this targeted nanoformulation in HIV-1 infected peripheral blood mononuclear cells (PBMCs), which are cultured on the basolateral end of the in vitro BBB model. Our results show a significant uptake of QR-Tf-Saquinavir by brain microvascular endothelial cells (BMVECs), which constitute the BBB. In addition, we observed a significant enhancement in the transversing capability of QR-Tf-Saquinavir across the BBB, along with a marked decrease in HIV-1 viral replication in the PBMCs. These observations indicate that drug-loaded nanoparticles can deliver therapeutics across the BBB. These results highlight the potential of this nanoformulation in the treatment of Neuro-AIDS and other neurological disorders.
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Fármacos Anti-VIH/farmacocinética , Barrera Hematoencefálica , Portadores de Fármacos/farmacocinética , Nanotubos/química , Saquinavir/farmacocinética , Transferrina/farmacocinética , Fármacos Anti-VIH/química , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Portadores de Fármacos/química , Células Endoteliales/metabolismo , VIH-1/efectos de los fármacos , Humanos , Leucocitos Mononucleares/virología , Saquinavir/química , Transferrina/química , Carga ViralRESUMEN
Phytochemicals are dietary phytoestrogens that may play a role in prostate cancer prevention. Forty percent of Americans use complementary and alternative medicines (CAM) for disease prevention and therapy. Ashwagandha (Withania somnifera) contains flavonoids and active ingredients like alkaloids and steroidal lactones which are called 'Withanolides'. We hypothesize that the immunomodulatory and anti-inflammatory properties of Ashwagandha might contribute to its overall effectiveness as an anti-carcinogenic agent. The goal of our study was gain insight into the general biological and molecular functions and immunomodulatory processes that are altered as a result of Ashwagandha treatment in prostate cancer cells, and to identify the key signaling mechanisms that are involved in the regulation of these physiological effects using genomic microarray analysis in conjunction with quantitative real-time PCR and western blot analysis. Ashwagandha treatment significantly downregulated the gene and protein expression of proinflammatory cytokines IL-6, IL-1ß, chemokine IL-8, Hsp70 and STAT-2, while a reciprocal upregulation was observed in gene and protein expression of p38 MAPK, PI3K, caspase 6, Cyclin D and c-myc. Furthermore, Ashwagandha treatment significantly modulated the JAK-STAT pathway which regulates both the apoptosis process as well as the MAP kinase signaling. These studies outline several functionally important classes of genes, which are associated with immune response, signal transduction, cell signaling, transcriptional regulation, apoptosis and cell cycle regulation and provide insight into the molecular signaling mechanisms that are modulated by Ashwagandha, thereby highlighting the use of this bioflavanoid as effective chemopreventive agent relevant to prostate cancer progression.
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The 32-kDa dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32) is recognized to be critical to the pathogenesis of drug addiction. Opiates via the mu-receptor act on the dopaminergic system in the brain and modulates the expression of DARPP-32 phosphoprotein which is an important mediator of the activity of the extracellular signal-regulated kinase (ERK) signaling cascades, the activation of which represents an exciting nexus for drug-induced changes in neural long-term synaptic plasticity. Silencing of DARPP-32 using an siRNA against DARPP-32 may provide a novel gene therapy strategy to overcome drug addiction. In this study, we investigated the effect of the opiate (heroin) on D1 receptor (D1R) and DARPP-32 expression and additionally, evaluated the effects of DARPP-32-siRNA gene silencing on protein phosphatase-1 (PP-1), ERK, and cAMP response element-binding (CREB) gene expression in primary normal human astrocytes (NHA) cells in vitro. Our results indicate that heroin significantly upregulated both D1R and DARPP-32 gene expression, and that DARPP-32 silencing in the NHA cells resulted in the significant modulation of the activity of downstream effector molecules such as PP-1, ERK, and CREB which are known to play an important role in opiate abuse-induced changes in long-term neural plasticity. These findings have the potential to facilitate the development of DARPP32 siRNA-based therapeutics against drug addiction.
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Fosfoproteína 32 Regulada por Dopamina y AMPc/efectos de los fármacos , Heroína/farmacología , Narcóticos/farmacología , Trastornos Relacionados con Opioides/metabolismo , Células Cultivadas , Fosfoproteína 32 Regulada por Dopamina y AMPc/genética , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Procesamiento de Imagen Asistido por Computador , Análisis de Secuencia por Matrices de Oligonucleótidos , Trastornos Relacionados con Opioides/genética , ARN Interferente Pequeño , Receptores de Dopamina D1/biosíntesis , Receptores de Dopamina D1/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
BACKGROUND: Human and animal studies have suggested that diet-derived flavonoids, in particular quercetin may play a beneficial role by preventing or inhibiting oncogenesis, but the underlying mechanism remains unclear. The aim of this study is to evaluate the effect(s) of quercetin on normal and malignant prostate cells and to identify the target(s) of quercetin's action. METHODOLOGY: We addressed this question using cells in culture and investigated whether quercetin affects key biological processes responsible for tumor cell properties such as cell proliferation and apoptosis and also studied the effect of quercetin on the proteome of prostate cancer cells using difference gel electrophoresis (DIGE) to assess changes in the expression of relevant proteins. RESULTS: Our findings demonstrate that quercetin treatment of prostate cancer cells results in decreased cell proliferation and viability. Furthermore, we demonstrate that quercetin promotes cancer cell apoptosis by down-regulating the levels of heat shock protein (Hsp) 90. Depletion of Hsp90 by quercetin results in decreased cell viability, levels of surrogate markers of Hsp90 inhibition (intracellular and secreted), induced apoptosis and activation of caspases in cancer cells but not in normal prostate epithelial cells. Knockdown of Hsp90 by short interfering RNA also resulted in induction apoptosis similar to quercetin in cancer cells as indicated by annexin V staining. CONCLUSION: Our results demonstrate that quercetin down-regulates the expression of Hsp90 which, in turn, induces inhibition of growth and cell death in prostate cancer cells while exerting no quantifiable effect on normal prostate epithelial cells.
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Adenocarcinoma/patología , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Neoplasias de la Próstata/patología , Quercetina/farmacología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Anexina A5/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Flavonoides/farmacología , Proteínas HSP90 de Choque Térmico/genética , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteómica , ARN Interferente Pequeño/genética , TransfecciónRESUMEN
BACKGROUND: Prostate-specific antigen (PSA) is a well-known biomarker for diagnosis and management of prostate cancer. PSA has been shown to have anti-angiogenic activity. We used the emerging proteomic research technology to identify proteins in prostate cancer cells whose expression is regulated by enzymatically active PSA. METHODS: Differentially expressed proteins in PC-3M cells treated with PSA were analyzed by 2D-DIGE analysis and identified by HPLC-MS/MS and SEQUEST data mining. Biological network analysis was carried out using MetaCore integrated software designed for functional analysis of experimental data. Gene expression data for several regulated proteins were confirmed by real-time, quantitative PCR. RESULTS: A total of 41 proteins were significantly (P < 0.05) changed in abundance in PC-3M cells in response to PSA treatment. Proteins from 26 gel-spots were identified. Many of the down-regulated proteins including N8 gene product long isoform, laminin receptor, vimentin, DJ-1 and Hsp60 are known to be involved in tumor progression. DISCUSSION: The relevance of the level of PSA in prostate tissue microenvironment and its relation to tumor progression has not been elucidated. PSA has been shown to down-regulate several proteins that are known to have involvement in tumor progression. This suggests that normal physiological levels of PSA in prostate tissue microenvironment may be promoting non-angiogenic environment and its down-regulation may promote tumor growth.
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Proteínas de Neoplasias/biosíntesis , Antígeno Prostático Específico/farmacología , Neoplasias de la Próstata/metabolismo , Proteómica/métodos , Línea Celular Tumoral , Electroforesis en Gel Bidimensional , Colorantes Fluorescentes/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Proteínas de Neoplasias/genética , Neoplasias de la Próstata/genética , ARN Neoplásico/química , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human immunodeficiency virus (HIV)-1 patients who abuse opiates are at a greater risk of developing neurological complications of AIDS. Alterations in blood-brain barrier (BBB) integrity are associated with cytoskeletal disorganization and disruption of tight junction (TJ) integrity. We hypothesize that opiates in combination with HIV-1 viral proteins can modulate TJ expression in primary brain microvascular endothelial cells (BMVEC), thereby compromising BBB integrity and exacerbating HIV-1 neuropathogenesis. We investigated the effect of morphine and/or tat on the expression of TJ proteins ZO-1, JAM-2, Occludin and P-glycoprotein and the functional effects of TJ modulation in BMVEC. Morphine and/or tat, via the activation of pro-inflammatory cytokines, intracellular Ca(2+) release, and activation of myosin light chain kinase, modulated TJ expression resulting in decreased transendothelial electric resistance and enhanced transendothelial migration across the BBB. These studies may lead to the development of novel anti-HIV-1 therapeutics that target specific TJ proteins, thus preventing TJ disruption in opiate using HIV-1 patients.
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Complejo SIDA Demencia/virología , Síndrome de Inmunodeficiencia Adquirida/virología , Barrera Hematoencefálica/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Morfina/farmacología , Uniones Estrechas/efectos de los fármacos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/farmacología , Complejo SIDA Demencia/sangre , Complejo SIDA Demencia/patología , Síndrome de Inmunodeficiencia Adquirida/sangre , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/patología , Astrocitos/metabolismo , Astrocitos/patología , Astrocitos/virología , Barrera Hematoencefálica/metabolismo , Técnicas de Cultivo de Célula , Citocinas/metabolismo , Fura-2 , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Morfina/sangre , Trastornos Relacionados con Sustancias , Uniones Estrechas/patología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/sangreRESUMEN
The pathogenesis of human immunodeficiency virus (HIV) associated encephalopathy is attributed to infiltration of the central nervous system (CNS) by HIV-1 infected mononuclear cells that transmigrate across the blood brain barrier (BBB). The endothelial tight junctions (TJ) of the blood brain barrier (BBB) play a critical role in controlling cellular traffic into the CNS. Neuropathogenesis of HIV-1 is exacerbated by drugs of abuse such as methamphetamine (Meth) which are capable of dysregulating BBB function. HIV-1 viral proteins like gp120 are both neurotoxic and cytotoxic and have been implicated in the development of HIV-1 dementia (HAD). We hypothesize that gp120 in synergy with Meth can alter BBB permeability via the modulation of tight junction expression. We investigated the effect of Meth and/or gp120 on the basal expression of TJ proteins ZO-1, JAM-2, Occludin, Claudin-3 and Claudin-5, using in vitro cultures of the primary brain microvascular endothelial cells (BMVEC). Further, the functional effects of TJ modulation were assessed using an in vitro BBB model, that allowed measurement of BBB permeability using TEER measurements and transendothelial migration of immunocompetent cells. Our results show that both Meth and gp120 individually and in combination, modulated TJ expression, and these effects involved Rho-A activation. Further, both Meth and gp120 alone and in combination significantly decreased transendothelial resistance across the in vitro BBB and the enhanced transendothelial migration of immunocompetent cells across the BBB. An understanding of the mechanisms of BBB breakdown that lead to neurotoxicity is crucial to the development of therapeutic modalities for Meth abusing HAD patients.
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Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Metanfetamina/farmacología , Uniones Estrechas/efectos de los fármacos , Análisis de Varianza , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Impedancia Eléctrica , Células Endoteliales/efectos de los fármacos , Citometría de Flujo , Regulación de la Expresión Génica/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/farmacología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Permeabilidad/efectos de los fármacos , Uniones Estrechas/metabolismo , Factores de TiempoRESUMEN
Delta-9-tetrahydrocannabinol (Delta(9)-THC), the main psychoactive component of marijuana, is known to dysregulate various immune responses. Cannabinoid (CB)-1 and -2 receptors are expressed mainly on cells of the central nervous system (CNS) and the immune system. The CNS is the primary target of cannabinoids and astrocytes are known to play a role in various immune responses. Thus we undertook this investigation to determine the global molecular effects of cannabinoids on normal human astrocytes (NHA) using genomic and proteomic analyses. NHA were treated with Delta(9)-THC and assayed using gene microarrays and two-dimensional (2D) difference gel electrophoresis (DIGE) coupled with mass spectrometry (MS) to elucidate their genomic and proteomic profiles respectively. Our results show that the expression of more than 20 translated protein gene products from NHA was differentially dysregulated by treatment with Delta(9)-THC compared to untreated, control NHA.
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Astrocitos/efectos de los fármacos , Dronabinol/farmacología , Perfilación de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Psicotrópicos/farmacología , Astrocitos/metabolismo , Células Cultivadas , Chaperonina 60/efectos de los fármacos , Chaperonina 60/metabolismo , Forma BB de la Creatina-Quinasa/efectos de los fármacos , Forma BB de la Creatina-Quinasa/metabolismo , Fructosa-Bifosfato Aldolasa/efectos de los fármacos , Fructosa-Bifosfato Aldolasa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Genómica , Glutatión Peroxidasa/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Humanos , Proteínas del Tejido Nervioso/clasificación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfopiruvato Hidratasa/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , ProteómicaRESUMEN
The US is experiencing an epidemic of cocaine use entangled with HIV-1 infection. Normal human astrocytes (NHA) are susceptible to HIV-1 infection. We utilized LTR-R/U5 amplification, p24 antigen assay and the proteomic method of difference gel electrophoresis (DIGE) combined with protein identification through HPLC-MS/MS to investigate the effect of cocaine on HIV-1 infectivity and the proteomic profile of NHA, respectively. Data demonstrate that cocaine significantly upregulates HIV-1 infection in NHA as measured by LTR-R/U5 amplification and p24 antigen assay. Further, our results show for the first time that cocaine differentially regulates the expression of a number of proteins by NHA that may play a role in the neuropathogenesis of HIV-1 disease.
Asunto(s)
Astrocitos/efectos de los fármacos , Cocaína/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/virología , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/virología , Línea Celular , Femenino , Proteína p24 del Núcleo del VIH/efectos de los fármacos , Proteína p24 del Núcleo del VIH/metabolismo , Duplicado del Terminal Largo de VIH/efectos de los fármacos , VIH-1/genética , VIH-1/metabolismo , Humanos , Drogas Ilícitas/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Proteómica , ARN/análisis , ARN Mensajero/análisis , Valores de Referencia , Estadísticas no Paramétricas , Regulación hacia ArribaRESUMEN
Drug-resistant human immunodeficiency virus (HIV) infections are increasing globally, especially in North America. Therefore, it is logical to develop new therapies directed against HIV binding molecules on susceptible host cells in addition to current treatment modalities against virus functions. Inhibition of the viral genome can be achieved by degrading or silencing posttranslational genes using small interfering (si) ribonucleic acids (RNAs) consisting of double-stranded forms of RNA. These siRNAs usually contain 21-23 base pairs (bp) and are highly specific for the nucleotide sequence of the target messenger RNA (mRNA). These siRNAs form a complex with helicase and nuclease enzymes known as "RNA-induced silencing complex" (RISC) that leads to target RNA degradation. Thus, siRNA has become a method of selective destruction of HIV now used by various investigators around the globe. However, given the sequence diversity of the HIV genomes of infected subjects, it is difficult to target a specific HIV sequence. Therefore, targeting nonvariable HIV binding receptors on susceptible cells or other molecules of host cells that are directly or indirectly involved in HIV infections may be an interesting alternative to targeting the virus itself. Thus, the simultaneous use of siRNAs specific for HIV and host cells may be a unique, new approach to the therapy of HIV infections. In this article, we present evidence that siRNA directed at the CD4 independent attachment receptor (DC-SIGN) significantly inhibits HIV infection of dendritic cells (DCs). This effect may be mediated by modulation of p38 mitogen activated protein kinase (MAPK).
Asunto(s)
Moléculas de Adhesión Celular/antagonistas & inhibidores , Células Dendríticas/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Lectinas Tipo C/antagonistas & inhibidores , ARN Interferente Pequeño/uso terapéutico , Receptores de Superficie Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/biosíntesis , Células Cultivadas , Células Dendríticas/virología , Infecciones por VIH/virología , Humanos , Lectinas Tipo C/biosíntesis , ARN Interferente Pequeño/farmacología , Receptores de Superficie Celular/biosíntesisRESUMEN
HIV-1 affects microglia and astroglia, which subsequently contributes to the neurodegenerative changes. Viral proteins cause neurotoxicity by direct action on the CNS cells or by activating glial cells to cause the release of cytokines, chemokines or neurotoxic substances. Opioid abuse has been postulated as a cofactor in the immunopathogenesis of human immunodeficiency virus (HIV) infection and AIDS. HIV-induced pathogenesis is exacerbated by opiate abuse and that the synergistic neurotoxicity is a direct effect of opiates on the CNS. Chemokines and their receptors have been implicated in the pathogenesis of neuroAIDS. Herein we describe the effects of morphine and/or gp120 on the expression of the genes for the beta-chemokine MIP-1beta and its receptors CCR3 and CCR5 by the U373 cells which are a human brain-derived astrocytoma/glioblastoma cell line. Our results indicate that treatment of U373 cells with morphine significantly downregulated the gene expression of the beta chemokine, MIP-1 beta, while reciprocally upregulating the expression of its specific receptors, CCR3 and CCR5 suggesting that the capacity of mu-opioids to increase HIV-1 co-receptor expression may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression. Additionally, opiates can enhance the cytotoxicity of HIV-1 viral protein gp120 via mechanisms that involve intracellular calcium modulation resulting in direct actions on astroglia, making them an important cellular target for HIV-opiate interactions.
Asunto(s)
Quimiocinas/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/toxicidad , VIH-1/patogenicidad , Morfina/toxicidad , Complejo SIDA Demencia/etiología , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/inmunología , Astrocitos/efectos de los fármacos , Astrocitos/inmunología , Astrocitos/virología , Calcio/metabolismo , Línea Celular Tumoral , Quimiocina CCL4 , Expresión Génica/efectos de los fármacos , Infecciones por VIH/etiología , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Factores Inmunológicos/farmacología , Técnicas In Vitro , Proteínas Inflamatorias de Macrófagos/biosíntesis , Proteínas Inflamatorias de Macrófagos/genética , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Receptores CCR3 , Receptores CCR5/biosíntesis , Receptores CCR5/genética , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genética , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/efectos de los fármacosRESUMEN
Prostate-specific antigen (PSA) is a serine protease that is widely used as a surrogate marker in the early diagnosis and management of prostate cancer. The physiological relevance of tissue PSA levels and their role in prostate tumor growth and metastasis are not known. Free-PSA (f-PSA) was purified to homogeneity from human seminal plasma by column chromatography, eliminating hk2 and all known PSA complexes and retaining its protease activity. Confluent monolayers of prostate cancer cell lines, PC-3M and LNCaP, were treated with f-PSA in a series of in vitro experiments to determine the changes in expression of various genes that are known to regulate tumor growth and metastasis. Gene array, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) results show significant changes in the expression of various cancer-related genes in PC-3M and LNCaP cells treated with f-PSA. In a gene array analysis of PC-3M cells treated with 10 muM f-PSA, 136 genes were upregulated and 137 genes were downregulated. In LNCaP cells treated with an identical concentration of f-PSA, a total of 793 genes was regulated. QPCR analysis reveals that the genes for urokinase-type plasminogen activator (uPA), VEGF, and Pim-1 oncogene, known to promote tumor growth, were significantly downregulated, whereas IFN-gamma, known to be a tumor-suppressor gene, was significantly upregulated in f-PSA-treated PC-3M cells. The effect of f-PSA on VEGF and IFN-gamma gene expression and on protein release in PC-3M cells was distinctly dose-dependent. In vivo studies showed a significant reduction (P = .03) in tumor load when f-PSA was administered in the tumor vicinity of PC-3M tumor-bearing BALB/c nude mice. Our data support the hypothesis that f-PSA plays a significant role in prostate tumor growth by regulating various proangiogenic and antiangiogenic growth factors.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Antígeno Prostático Específico/fisiología , Neoplasias de la Próstata/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Cromatografía , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neovascularización Patológica , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1 , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Dendritic cells are the critical mediators of various immune responses and are the first line of defense against any infection including HIV. They play a major role in harboring HIV and the subsequent infection of T cells and passage of virus through the blood-brain barrier (BBB). The recently discovered DC-specific, CD4-independent HIV attachment receptor, DC-SIGN, and T-cell suppressing factor, indolamine 2,3-dioxygenase (IDO), are known to play a critical role in the immuno-neuropathogenesis of HIV infection. Since brain microvascular cells (BMVEC) express dendritic cell (DC)-specific C type ICAM-3 grabbing nonintegrin (DC-SIGN), it is possible that DC-SIGN may play a critical role in human immunodeficiency virus-type 1 (HIV-1) infection and migration of infected DC across BBB. Matrix metalloproteinases (MMPs) are proteolytic enzymes known to be responsible for maintenance, turnover and integrity of extracellular matrix. Our results show that cocaine upregulates IDO and DC-SIGN expression by DC. Further, cocaine upregulates DC-SIGN and MMPs in BMVEC supporting the hypothesis that cocaine causes membrane permeability facilitating endothelial transmigration of infected DC in to the CNS. Targeting DC-SIGN and IDO with specific monoclonal antibodies, inexpensive synthetic antagonists, antisense oligonucleotides and siRNA may lead to develop novel treatment strategies particularly in high-risk populations such as cocaine users.