RESUMO
Neurogenesis takes place in the adult mammalian brain in three areas: Subgranular zone of the dentate gyrus (DG); subventricular zone of the lateral ventricle; olfactory bulb. Different molecular markers can be used to characterize the cells involved in adult neurogenesis. It has been recently suggested that a population of bone marrow (BM) progenitor cells may migrate to the brain and differentiate into neuronal lineage. To explore this hypothesis, we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells. Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells, then after several months in mature neurons and microglial cells, and thus without central nervous system (CNS) lesion. Most of transgene-expressing cells expressed NeuN, a marker of mature neurons. Thus, BM-derived cells may function as progenitors of CNS cells in adult animals. The mechanism by which the cells from the BM come to be neurons remains to be determined. Although the observed gradual increase in transgene-expressing neurons over 16 mo suggests that the pathway involved differentiation of BM-resident cells into neurons, cell fusion as the principal route cannot be totally ruled out. Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons. Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector. In addition to cells expressing markers of mature neurons, transgene-positive cells were also positive for nestin and doublecortin, molecules expressed by developing neuronal cells. These cells were actively proliferating, as shown by short term BrdU incorporation studies. Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40 vectors migrating to the hippocampus, and these cells were seen at earlier time points in the DG. We show that the cell membrane chemokine receptor, CCR5, and its ligands, enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity. SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells, suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS, both in the basal state and in response to injury. Furthermore, reduction in CCR5 expression in circulating cells provides profound neuroprotection from excitotoxic neuronal injury, reduces neuroinflammation, and increases neuronal regeneration following this type of insult. These results suggest that BM-derived, transgene-expressing, cells can migrate to the brain and that they become neurons, at least in part, by differentiating into neuron precursors and subsequently developing into mature neurons.
RESUMO
UNLABELLED: The cardioprotective effects of moderate ethanol consumption have been known for years and have generally been ascribed to long-term effects of alcohol on blood lipids. However, other mechanisms, particularly ethanol-induced increase in blood vessel density, may also be involved. Our goal was to understand the relationship between ethanol consumption, new blood vessel formation in vivo and protection from injury due to ischemia and ischemia/reperfusion. Using paired ethanol fed and control rats, we assessed capillary density in the heart, brain and skeletal muscle by immunostaining and quantified expression of vascular endothelial growth factor (VEGF) by Western blot analysis and immunocytochemistry. Numbers of vessels were significantly increased in the brain, heart and skeletal muscle of animals fed ethanol-rich diets. VEGF (and its receptors) were upregulated in these organs. These effects were very rapid: highly significantly increased vascularization was seen within 2 weeks of commencing alcohol feeding. A neutralizing VEGF antibody, bevacizumab, inhibited new blood vessel formation induced by moderate doses of ethanol. Ethanol consumption increased vascularization and promoted skeletal muscle regeneration following hindlimb ischemia; these effects were prevented by bevacizumab. Finally, ethanol consumption protected myocardium following experimental ischemia/reperfusion. CONCLUSION: Experimental ethanol ingestion rapidly increases VEGF production, significantly increasing the capillary bed in the heart, brain, and skeletal muscle. Moreover, the ethanol-induced increase of blood vessel density is protective against ischemic events (i.e., hindlimb ischemia and myocardium ischemia/reperfusion) and promotes skeletal muscle regeneration.
Assuntos
Etanol/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab , Encéfalo/irrigação sanguínea , Capilares/crescimento & desenvolvimento , Vasos Coronários/fisiologia , Etanol/administração & dosagem , Masculino , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/fisiologia , Neovascularização Fisiológica/fisiologia , Ratos , Ratos Sprague-Dawley , Regeneração/fisiologia , Regulação para CimaRESUMO
HIV-associated neurocognitive disorder (HAND) is an increasingly common, progressive disease characterized by neuronal loss and progressively deteriorating CNS function. HIV-1 gene products, particularly gp120 and Tat elicit reactive oxygen species (ROS) that lead to oxidant injury and cause neuron apoptosis. Understanding of, and developing therapies for, HAND requires accessible models of the disease. We have devised experimental approaches to studying the acute and chronic effects of Tat on the CNS. We studied acute exposure by injecting recombinant Tat protein into the caudate-putamen (CP). Ongoing Tat expression, which more closely mimics HIV-1 infection of the brain, was studied by delivering Tat-expression over time using an SV40-derived gene delivery vector, SV(Tat). Both acute and chronic Tat exposure induced lipid peroxidation and neuronal apoptosis. Finally, prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/zinc superoxide dismutase (SOD1) or glutathione peroxidase (GPx1), protected from Tat-induced apoptosis and oxidative injury. Thus, injection of recombinant HIV-1 Tat and the expression vector, SV(Tat), into the rat CP cause respectively acute or ongoing apoptosis and oxidative stress in neurons and may represent useful animal models for studying the pathogenesis and, potentially, treatment of HIV-1 Tat-related damage.
Assuntos
Complexo AIDS Demência/terapia , Antioxidantes/administração & dosagem , Modelos Animais de Doenças , Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1 , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidade , Animais , Apoptose/fisiologia , Feminino , Vetores Genéticos , Infecções por HIV/complicações , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Peroxidação de Lipídeos/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/toxicidade , Vírus 40 dos Símios/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/administração & dosagemRESUMO
WE COMPARED LUNG DELIVERY METHODS OF RECOMBINANT ADENOVIRUS (RAD): (1) rAd suspended in saline, (2) rAd suspended in saline followed by a pulse-chase of a perfluorochemical (PFC) liquid mixture, and (3) a PFC-rAd suspension. Cell uptake, distribution, and temporal expression of rAd were examined using A549 cells, a murine model using luciferase bioluminescence, and histological analyses. Relative to saline, a 4X increase in transduction efficiency was observed in A549 cells exposed to PFC-rAd for 2-4 h. rAd transgene expression was improved in alveolar epithelial cells, and the level and distribution of luciferase expression when delivered in PFC-rAd suspensions consistently peaked at 24 h. These results demonstrate that PFC-rAd suspensions improve distribution and enhance rAd-mediated gene expression which has important implications in improving lung function by gene therapy.
RESUMO
Immune-mediated damage to the central nervous system (CNS) is an important contributor to many CNS diseases, including epilepsy. Chemokines play a role in leukocyte recruitment to, and migration across, the blood-brain barrier (BBB) during many such processes. We previously investigated the role of the chemokine receptor CCR5 in a rat model of epilepsy based on intraperitoneal kainic acid (KA) administration. Before KA injection, rats were given intramarrow inoculations of SV(RNAiR5-RevM10.AU1), which carries an interfering RNA (RNAi) that targets CCR5. Decreased CCR5 expression in blood cells after vector administration reduced expression of CCR5 ligands MIP-1α and RANTES in the microvasculature, and strongly protected from BBB leakage, CNS loss and inflammation and facilitated CNS repair. We show here that rSV40-mediated downregulation of CCR5 in lymphocytes decreased cellular adhesion to surfaces carrying CCR5 ligands. These data suggest that reducing CCR5 in peripheral blood mononuclear cells (PBMCs) might alter their adhesion to the microvasculature and their participation in inflammatory processes.
Assuntos
Antagonistas dos Receptores CCR5 , Técnicas de Transferência de Genes , Subpopulações de Linfócitos/metabolismo , Subpopulações de Linfócitos/patologia , Receptores CCR5/genética , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/virologia , Adesão Celular/genética , Adesão Celular/imunologia , Linhagem Celular , Células Cultivadas , Epilepsia/imunologia , Epilepsia/patologia , Epilepsia/virologia , Vetores Genéticos/imunologia , Vetores Genéticos/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/virologia , Ligantes , Subpopulações de Linfócitos/virologia , Interferência de RNA , RNA Viral/genética , RNA Viral/imunologia , RNA Viral/metabolismo , Ratos , Receptores CCR5/metabolismo , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/imunologia , Vírus 40 dos Símios/metabolismoRESUMO
We administered recombinant SV40-derived viral vectors (rSV40s) intravenously to mice with or without prior intraperitoneal injection of mannitol to deliver transgenes to the central nervous system (CNS). We detected transgene-expressing cells (mainly neurons) most prominently in the cortex and spinal cord; prior intraperitoneal mannitol injection increased CNS gene delivery tenfold. Intravenous injection of rSV40s, particularly with mannitol pretreatment, resulted in extensive expression of multiple transgenes throughout the CNS.
Assuntos
Córtex Cerebral/metabolismo , Técnicas de Transferência de Genes , Vírus 40 dos Símios/genética , Medula Espinal/metabolismo , Transgenes , Animais , Células COS , Chlorocebus aethiops , Terapia Genética , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
There are several diseases for which gene transfer therapy to the cerebellum might be practicable. In these studies, we used recombinant Tag-deleted SV40-derived vectors (rSV40s) to study gene delivery targeting the cerebellum. These vectors transduce neurons and microglia very effectively in vitro and in vivo, and so we tested them to evaluate gene transfer to the cerebellum in vivo. Using a rSV40 vector carrying human immunodeficiency virus (HIV)-Nef with a C-terminal FLAG epitope, we characterized the distribution, duration, and cell types transduced. Rats received test and control vectors by stereotaxic injection into the cerebellum. Transgene expression was assessed 1, 2, and 4 weeks later by immunostaining of serial brain sections. FLAG epitope-expressing cells were seen, at all times after vector administration, principally detected in the Purkinje cells of the cerebellum, identified as immunopositive for calbindin. Occasional microglial cells were tranduced; transgene expression was not detected in astrocytes or oligodendrocytes. No inflammatory or other reaction was detected at any time. Thus, SV40-derived vectors can deliver effective, safe, and durable transgene expression to the cerebellum.
Assuntos
Cerebelo/metabolismo , Transdução Genética/métodos , Animais , Contagem de Células/métodos , Cerebelo/citologia , Feminino , Expressão Gênica/fisiologia , Produtos do Gene nef/genética , Produtos do Gene nef/metabolismo , Vetores Genéticos/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Vírus 40 dos Símios/genética , Estatísticas não ParamétricasRESUMO
Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Here we describe a protocol for production and purification of these vectors. These procedures require only packaging cells (e.g., COS-7) and circular vector genome DNA. Amplification involves repeated infection of packaging cells with vector produced by transfection. Cotransfection is not required in any step. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients and resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. These approaches are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. These vectors are best applied to long-term expression of proteins normally encoded by mammalian cells or by viruses that infect mammalian cells, or of untranslated RNAs (e.g., RNA interference). The preparative approaches described facilitate application of these vectors and allow almost any laboratory to exploit their strengths for diverse gene delivery applications.
Assuntos
Técnicas Genéticas , Vetores Genéticos/biossíntese , Vírus 40 dos Símios/genética , Animais , Células COS , Chlorocebus aethiops , Transcrição GênicaRESUMO
Recombinant simian virus 40 (rSV40)-derived vectors are particularly useful for gene delivery to bone marrow progenitor cells and their differentiated derivatives, certain types of epithelial cells (e.g., hepatocytes), and central nervous system neurons and microglia. They integrate rapidly into cellular DNA to provide long-term gene expression in vitro and in vivo in both resting and dividing cells. Techniques used to produce, purify, and quantitate these vectors are simple, give reproducible results, and may be used to generate vectors that are deleted only for large T antigen (Tag), or for all SV40-coding sequences capable of carrying up to 5 kb of foreign DNA. Viruses are purified by centrifugation using discontinuous sucrose or cesium chloride (CsCl) gradients. Resulting vectors are replication-incompetent and contain no detectable wild-type SV40 revertants. Viruses are titered by quantitative polymerase chain reaction (qPCR), described here. qPCR measures the number of rSV40 genomes in purified viral stocks using primers specific for the rSV40, coupled with SYBR Green detection of PCR products. Sample purity is assessed using qPCR via melt (dissociation) curve analysis. The only specialized equipment necessary is a quantitative real-time PCR machine.
Assuntos
Vetores Genéticos/genética , Recombinação Genética/genética , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/fisiologia , Cultura de Vírus/métodos , Replicação Viral/fisiologia , Desoxirribonucleases/metabolismo , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase , Padrões de Referência , Ribonucleases/metabolismoRESUMO
HIV-1 gp120 neurotoxicity and oxidant injury are well documented, but consequent neuroinflammation is less understood. Rat caudate-putamens (CPs) were challenged with 100-500 ng HIV-1BaL gp120, with or without prior rSV40-delivered superoxide dismutase or glutathione peroxidase. CD11b-positive microglia were increased 1 day post-challenge; Iba-1- and ED1-positive cells peaked at 7 days and 14 days. Astrocyte infiltration was maximal at 7-14 days. MIP-1alpha was produced immediately, mainly by neurons. ED1- and GFAP-positive cells correlated with neuron loss and gp120 dose. We also tested the effect of more chronic gp120 exposure on neuroinflammation using an experimental model of continuing gp120 exposure. SV(gp120), a recombinant SV40-derived gene transfer vector was inoculated into the rat CP, leading to chronic expression of gp120, ongoing apoptosis in microglia and neurons, and oxidative stress. Increase in microglia and astrocytes was seen following intra-CP SV(gp120) injection, suggesting that continuing gp120 production increased neuroinflammation. SV(SOD1) or SV(GPx1) significantly reduced MIP-1alpha and limited neuroinflammation following gp120 administration into the CP, as well as microglia and astrocytes proliferation after injection of SV(gp120) in the striatum. Thus, gp120-induced CNS injury, neuron loss and inflammation may be mitigated by antioxidant gene delivery.
Assuntos
Antioxidantes/farmacologia , Encefalite , Terapia Genética/métodos , Glutationa Peroxidase/farmacologia , Proteína gp120 do Envelope de HIV , Superóxido Dismutase/farmacologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aquaporinas/metabolismo , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalite/induzido quimicamente , Encefalite/patologia , Encefalite/prevenção & controle , Proteínas do Olho/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glutationa Peroxidase/genética , Indóis , Proteínas dos Microfilamentos , Microglia/efeitos dos fármacos , Microglia/virologia , Neurônios/metabolismo , Neurônios/virologia , Neurotensina/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Putamen/efeitos dos fármacos , Putamen/virologia , Ratos , Ratos Sprague-Dawley , Vírus 40 dos Símios/genética , Estatísticas não Paramétricas , Superóxido Dismutase/genética , Fatores de Tempo , Transdução Genética/métodosRESUMO
Human immunodeficiency virus 1 (HIV-1) encephalopathy is thought to result in part from the toxicity of HIV-1 envelope glycoprotein gp120 for neurons. Experimental systems for studying the effects of gp120 and other HIV proteins on the brain have been limited to the acute effects of recombinant proteins in vitro or in vivo in simian immunodeficiency virus-infected monkeys. We describe an experimental rodent model of ongoing gp120-induced neurotoxicity in which HIV-1 envelope is expressed in the brain using an SV40-derived gene delivery vector, SV(gp120). When it is inoculated stereotaxically into the rat caudate putamen, SV(gp120) caused a partly hemorrhagic lesion in which neuron and other cell apoptosis continues for at least 12 weeks. Human immunodeficiency virus gp120 is expressed throughout this time, and some apoptotic cells are gp120 positive. Malondialdehyde and 4-hydroxynonenal assays indicated that there was lipid peroxidation in these lesions. Prior administration of recombinant SV40 vectors carrying antioxidant enzymes, copper/ zinc superoxide dismutase or glutathione peroxidase, was protective against SV(gp120)-induced oxidative injury and apoptosis. Thus, in vivo inoculation of SV(gp120) into the rat caudate putamen causes ongoing oxidative stress and apoptosis in neurons and may therefore represent a useful animal model for studying the pathogenesis and treatment of HIV-1 envelope-related brain damage.
Assuntos
Complexo AIDS Demência/etiologia , Modelos Animais de Doenças , Proteína gp120 do Envelope de HIV/efeitos adversos , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/patogenicidade , Complexo AIDS Demência/genética , Adenovirus dos Símios , Aldeídos/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Morte Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Viral da Expressão Gênica/fisiologia , Vetores Genéticos/administração & dosagem , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa Peroxidase/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Indóis , Proteínas dos Microfilamentos , Neurônios/patologia , Neurônios/virologia , Fosfopiruvato Hidratase/metabolismo , Putamen/virologia , Ratos , Ratos Nus , Ratos Sprague-Dawley , Estatísticas não Paramétricas , Fatores de Tempo , Transdução Genética/métodosRESUMO
Toxicity of HIV-1 envelope glycoprotein (gp120) for substantia nigra (SN) neurons may contribute to the Parkinsonian manifestations often seen in HIV-1-associated dementia (HAD). We studied the neurotoxicity of gp120 for dopaminergic neurons and potential neuroprotection by antioxidant gene delivery. Rats were injected stereotaxically into their caudate-putamen (CP); CP and (substantia nigra) SN neuron loss was quantified. The area of neuron loss extended several millimeters from the injection site, approximately 35% of the CP area. SN neurons, outside of this area of direct neurotoxicity, were also severely affected. Dopaminergic SN neurons (expressing tyrosine hydroxylase, TH, in the SN and dopamine transporter, DAT, in the CP) were mostly affected: intra-CP gp120 caused approximately 50% DAT+ SN neuron loss. Prior intra-CP gene delivery of Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) protected SN neurons from intra-CP gp120. Thus, SN dopaminergic neurons are highly sensitive to HIV-1 gp120-induced neurotoxicity, and antioxidant gene delivery, even at a distance, is protective.
Assuntos
Dopamina/metabolismo , Glutationa Peroxidase/metabolismo , Proteína gp120 do Envelope de HIV/toxicidade , Neurônios/fisiologia , Superóxido Dismutase/metabolismo , Animais , Núcleo Caudado/patologia , Núcleo Caudado/fisiopatologia , Morte Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Feminino , Vetores Genéticos , Glutationa Peroxidase/genética , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Vírus 40 dos Símios/genética , Substância Negra/patologia , Substância Negra/fisiopatologia , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
CCR3 has been implicated as a co-receptor for human immunodeficiency virus type 1 (HIV-1), particularly in brain microglia cells. We sought to clarify the comparative roles of CCR3 and CCR5 in the central nervous system (CNS) HIV-1 infection and the potential utility of CCR3 as a target for manipulation via gene transfer. To target CCR3, we developed a single-chain antibody (SFv) and an interfering RNA (RNAi), R3-526. Coding sequences for both were cloned into Tag-deleted SV40-dervied vectors, as these vectors transduce brain microglia and monocyte-derived macrophages (MDM) highly efficiently. These anti-CCR3 transgenes were compared to SFv-CCR5, an SFv against CCR5, and RNAi-R5, an RNAi that targets CCR5, for the ability to protect primary human brain microglia and MDM from infection with peripheral and neurotropic strains of HIV-1. Downregulation of CCR3 and CCR5 by these transgenes was independent from one another. Confocal microscopy showed that CCR3 and CCR5 co-localized at the plasma membrane with each other and with CD4. Targeting either CCR5 or CCR3 largely protected both microglia and MDM from infection by many strains of HIV-1. That is, some HIV-1 strains, isolated from either the CNS or periphery, required both CCR3 and CCR5 for optimal productive infection of microglia and MDM. Some HIV-1 strains were relatively purely CCR5-tropic. None was purely CCR3-tropic. Thus, some CNS-tropic strains of HIV-1 utilize CCR5 as a co-receptor but do not need CCR3, while for other isolates both CCR3 and CCR5 may be required.
Assuntos
Encéfalo/virologia , HIV-1/patogenicidade , Macrófagos/virologia , Microglia/virologia , Receptores CCR3/metabolismo , Receptores CCR5/metabolismo , Animais , Anticorpos/imunologia , Encéfalo/citologia , HIV-1/metabolismo , Humanos , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores CCR3/genética , Receptores CCR5/genética , Receptores de HIV/genética , Receptores de HIV/metabolismo , Replicação ViralRESUMO
INTRODUCTION: Hemophagocytic lymphohistiocytosis is an immune-mediated syndrome that typically has a rapidly progressive course that can result in pancytopenia, coagulopathy, multi-system organ failure and death. CASE PRESENTATION: A 57-year-old Caucasian woman was referred in fulminant hemophagocytic lymphohistiocytosis, with fever, pancytopenia, splenomegaly, mental status changes and respiratory failure. She was found to have stage IV classical Hodgkin lymphoma, in addition to Epstein-Barr virus and cytomegalovirus viremia. Her presentation was preceded by a 3-year prodrome consisting of cytopenia and fever that were partially controlled by steroids and azathioprine. CONCLUSION: Fulminant hemophagocytic lymphohistiocytosis may follow a prodromal phase that possesses features suggestive of a chronic form of hemophagocytic lymphohistiocytosis, but which may also resemble immune cytopenias of other causes. A diagnosis of hemophagocytic lymphohistiocytosis should be considered in the setting of chronic pancytopenia.
RESUMO
BACKGROUND: Gene transfer to the CNS has been approached using various vectors. OBJECTIVE: We illustrate how SV40-derived vectors may be useful to deliver long-term gene expression to the brain, locally or diffusely. RESULTS/CONCLUSION: SV40-derived vectors transduce neurons and microglial cells. The potential utility of both localized and widespread gene delivery in treating neuroAIDS and other CNS diseases characterized by excessive oxidative stress is demonstrated. Finally, direct injection of rSV40 vectors into rat femoral bone marrow (BM) led to transgene expression in CNS neurons and microglia, mostly in the dentate gyrus and in the periventricular subependymal zone, suggesting that BM-derived cells may be progenitors of some CNS cells in adult animals, and that gene delivery to BM may allow transgene expression in newly formed neurons.
Assuntos
Encéfalo/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Vírus 40 dos Símios/genética , Animais , Antioxidantes/metabolismo , Enzimas/genética , Estresse OxidativoRESUMO
We studied the distribution of transgene-expressing cells after direct gene transfer into the bone marrow (BM). Rats received direct injection into the femoral BM of SV(Nef-FLAG), a Tag-deleted recombinant SV40 carrying a marker gene (FLAG epitope). Controls received an unrelated rSV40 or saline. Blood cells (5%) and femoral marrow cells (25%) expressed FLAG throughout. FLAG expression was assessed in different organs at 1, 4 and 16 months. FLAG+ macrophages were seen throughout the body, and were prominent in the spleen. FLAG+ cells were common in pulmonary alveoli. The former included alveolar macrophages and type II pneumocytes. These cells were not detected at 1 month, occasional at 4 months and common at 16 months after intramarrow injection. Rare liver cells were positive for both FLAG and ferritin, indicating that some hepatocytes also expressed this BM-delivered transgene. Control animals were negative. Thus: (a) fixed tissue phagocytes may be accessible to gene delivery by intramarrow transduction of their progenitors; (b) transduced BM-resident cells or their derivatives may migrate to other organs (lungs) and may differentiate into epithelial cells; and (c) intramarrow injection of rSV40s does not detectably transduce parenchymal cells of other organs.
Assuntos
Células da Medula Óssea/metabolismo , Vetores Genéticos/administração & dosagem , Macrófagos Alveolares/metabolismo , Transdução Genética , Transgenes , Animais , Linhagem da Célula , Feminino , Terapia Genética/métodos , Vetores Genéticos/genética , Imuno-Histoquímica , Injeções , Rim/imunologia , Rim/metabolismo , Fígado/imunologia , Fígado/metabolismo , Pulmão/imunologia , Oligopeptídeos , Especificidade de Órgãos , Peptídeos/genética , Peptídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Vírus 40 dos Símios/genética , Baço/imunologiaRESUMO
HIV-1 proteins, especially gp120 and Tat, elicit reactive oxygen species (ROS) and cause neuron apoptosis. We used antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1) and glutathione peroxidase (GPx1) to study signaling and neuroprotection from Tat-induced apoptosis. SOD1 converts superoxide to peroxide; GPx1 converts peroxide to water. Primary human neurons were transduced with SV40-derived vectors carrying SOD1 and GPx1, then HIV-1 Tat protein was added. Both SV(SOD1) and SV(GPx1) delivered substantial transgene expression. Tat decreased endogenous cellular, but not transduced, SOD1 and GPx1. Tat rapidly increased neuron [Ca(2+)](i), which effect was not altered by SV(SOD1) or SV(GPx1). However, both vectors together blocked Tat-induced [Ca(2+)](i) fluxes. Similarly, neither SV(SOD1) nor SV(GPx1) protected neurons from Tat-induced apoptosis, but both vectors together did. Tat therefore activates multiple signaling pathways, in one of which superoxide acts as an intermediate while the other utilizes peroxide. Gene delivery to protect neurons from Tat must therefore target both.
Assuntos
Apoptose/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Produtos do Gene tat/toxicidade , Glutationa Peroxidase/farmacologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Superóxido Dismutase/farmacologia , Cálcio/metabolismo , Células Cultivadas , Sequestradores de Radicais Livres/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Infecções por HIV/fisiopatologia , Infecções por HIV/terapia , HIV-1/química , Humanos , Neurônios/patologia , Neurônios/virologia , Vírus 40 dos Símios/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Virulência , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tagdeleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.
Assuntos
Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos/genética , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/imunologia , Animais , Divisão Celular , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Humanos , Mutagênese Insercional , Integração ViralRESUMO
Using bone marrow-directed gene transfer, we tested whether bone marrow-derived cells may function as progenitors of central nervous system (CNS) cells in adult animals. SV40-derived gene delivery vectors were injected directly into femoral bone marrow, and we examined transgene expression in blood and brain for 0-16 months thereafter by immunostaining for FLAG epitope marker. An average of 5% of peripheral blood cells and 25% of femoral marrow cells were FLAG(+) throughout the study. CNS FLAG-expressing cells were mainly detected in the dentate gyrus (DG) and periventricular subependymal zone (PSZ). Although absent before 1 month and rare at 4 months, DG and PSZ FLAG(+) cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggest that one or more populations of cells resident within adult bone marrow can migrate to the brain and differentiate into CNS-specific cells.
Assuntos
Células da Medula Óssea/citologia , Diferenciação Celular , Linhagem da Célula , Sistema Nervoso Central/citologia , Vírus 40 dos Símios/metabolismo , Células-Tronco/citologia , Transdução Genética/métodos , Animais , DNA Recombinante/metabolismo , Giro Denteado/citologia , Epêndima/citologia , Feminino , Expressão Gênica , Vetores Genéticos/metabolismo , Microglia/citologia , Proteínas do Tecido Nervoso/metabolismo , Oligopeptídeos , Peptídeos , Fenótipo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Sprague-Dawley , TransgenesRESUMO
Antiretroviral chemotherapy penetrates the CNS poorly. CNS HIV, thus sheltered, may injure the brain and complicate control of systemic HIV infection. Microglial cells play a major role in HIV persistence in the CNS but are rarely targeted for gene delivery. Because recombinant SV40 vectors (rSV40s) transduce other phagocytic cells efficiently, we tested rSV40 delivery of anti-HIV genetic therapy to microglial cells. Microglia prepared as enriched cultures from human fetal brain, were transduced with marker vectors, SV(RFP) and SV(Nef/FLAG), respectively, carrying DsRed and HIV-1 Nef bearing a FLAG epitope. By immunostaining and FACS, 95% of unselected cells expressed the transgenes, without detectable toxicity. Microglia were transduced with SV(AT), carrying human alpha1-antitrypsin (alpha1AT), which blocks Env and Gag processing. SV(AT)-treated microglia strongly resisted challenge with HIV-1BaL, even when microglia were transduced with SV(AT) following HIV challenge. Thus, rSV40s effectively transduce microglia and protect them from HIV.