RESUMEN
Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs.
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Analgésicos Opioides/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/fisiología , Metadona/farmacología , Replicación Viral/fisiología , Animales , Técnicas de Cultivo de Célula , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICRRESUMEN
Saposin and its precursor prosaposin are endogenous proteins with neurotrophic and anti-apoptotic properties. Prosaposin or its analog prosaposin-derived 18-mer peptide (PS18) reduced neuronal damage in hippocampus and apoptosis in stroke brain. Its role in Parkinson's disease (PD) has not been well characterized. This study aimed to examine the physiological role of PS18 in 6-hydroxydopamine (6-OHDA) cellular and animal models of PD. We found that PS18 significantly antagonized 6-OHDA -mediated dopaminergic neuronal loss and TUNEL in rat primary dopaminergic neuronal culture. In SH-SY5Y cells overexpressing the secreted ER calcium-monitoring proteins, we found that PS18 significantly reduced thapsigargin and 6-OHDA-mediated ER stress. The expression of prosaposin and the protective effect of PS18 were next examined in hemiparkinsonian rats. 6-OHDA was unilaterally administered to striatum. The expression of prosaposin was transiently upregulated in striatum on D3 (day 3) after lesioning and returned below the basal level on D29. The 6-OHDA-lesioned rats developed bradykinesia and an increase in methamphetamine-mediated rotation, which was antagonized by PS18. Brain tissues were collected for Western blot, immunohistochemistry, and qRTPCR analysis. Tyrosine hydroxylase immunoreactivity was significantly reduced while the expressions of PERK, ATF6, CHOP, and BiP were upregulated in the lesioned nigra; these responses were significantly antagonized by PS18. Taken together, our data support that PS18 is neuroprotective in cellular and animal models of PD. The mechanisms of protection may involve anti-ER stress.
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Neuroblastoma , Fármacos Neuroprotectores , Enfermedad de Parkinson , Saposinas , Animales , Humanos , Ratas , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Neuroblastoma/metabolismo , Fármacos Neuroprotectores/farmacología , Oxidopamina/toxicidad , Enfermedad de Parkinson/metabolismo , Saposinas/genética , Saposinas/metabolismo , Sustancia Negra/metabolismoRESUMEN
Stroke is a major cause of long-term disability world-wide. Limited pharmacological therapy has been used in stroke patients. Previous studies indicated that herb formula PM012 is neuroprotective against neurotoxin trimethyltin in rat brain, and improved learning and memory in animal models of Alzheimer's disease. Its action in stroke has not been reported. This study aims to determine PM012-mediated neural protection in cellular and animal models of stroke. Glutamate-mediated neuronal loss and apoptosis were examined in rat primary cortical neuronal cultures. Cultured cells were overexpressed with a Ca++ probe (gCaMP5) by AAV1 and were used to examine Ca++ influx (Ca++i). Adult rats received PM012 before transient middle cerebral artery occlusion (MCAo). Brain tissues were collected for infarction and qRTPCR analysis. In rat primary cortical neuronal cultures, PM012 significantly antagonized glutamate-mediated TUNEL and neuronal loss, as well as NMDA-mediated Ca++i. PM012 significantly reduced brain infarction and improved locomotor activity in stroke rats. PM012 attenuated the expression of IBA1, IL6, and CD86, while upregulated CD206 in the infarcted cortex. ATF6, Bip, CHOP, IRE1, and PERK were significantly down-regulated by PM012. Using HPLC, two potential bioactive molecules, paeoniflorin and 5-hydroxymethylfurfural, were identified in the PM012 extract. Taken together, our data suggest that PM012 is neuroprotective against stroke. The mechanisms of action involve inhibition of Ca++i, inflammation, and apoptosis.
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Fármacos Neuroprotectores , Accidente Cerebrovascular , Ratas , Animales , Neuroprotección , Ratas Sprague-Dawley , Modelos Animales de Enfermedad , Fármacos Neuroprotectores/farmacología , Accidente Cerebrovascular/etiología , Encéfalo/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , GlutamatosRESUMEN
Abnormal accumulation of alpha-synuclein (αSyn) in the remaining nigra dopaminergic neurons is a common neuropathological feature found in patients with Parkinson's disease (PD). Antibody-based immunotherapy has been considered a potential approach for PD treatment. This study aims to investigate the effectiveness of active immunization against αSyn in a mouse model of PD. Adult mice were immunized with or without a synthetic peptide containing the C-terminal residues of human αSyn and activation epitopes, followed by an intranigral injection of adeno-associated virus vectors for overexpressing human αSyn. Upon the peptide injection, αSyn-specific antibodies were raised, accompanied by degeneration of dopaminergic neurons and motor deficits. Furthermore, the induction of neuroinflammation was postulated by the elevation of astroglial and microglial markers in the immunized mice. Instead of lessening αSyn toxicity, this peptide vaccine caused an increase in the pathogenic species of αSyn. Our data demonstrated the potential adverse effects of active immunization to raise antibodies against the C-terminal fragment of αSyn. This drawback highlights the need for further investigation to weigh the pros and cons of immunotherapy in PD. Applying the αSyn C-terminal peptide vaccine for PD treatment should be cautiously exercised. This study provides valuable insights into the intricate interplay among immune intervention, αSyn accumulation, and neurodegeneration.
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Enfermedad de Parkinson , alfa-Sinucleína , Adulto , Humanos , Animales , Ratones , alfa-Sinucleína/genética , Enfermedad de Parkinson/terapia , Locomoción , Inmunoterapia , Anticuerpos , InmunizaciónRESUMEN
Immunogenic cell death is characterized by damage-associated molecular patterns, which can enhance the maturation and antigen uptake of dendritic cells. Shikonin, an anti-inflammatory and antitumor phytochemical, was exploited here as an adjuvant for dendritic cell-based cancer vaccines via induction of immunogenic cell death. Shikonin can effectively activate both receptor- and mitochondria-mediated apoptosis and increase the expression of all five tested damage-associated molecular patterns in the resultant tumor cell lysates. The combination treatment with damage-associated molecular patterns and LPS activates dendritic cells to a high maturation status and enhances the priming of Th1/Th17 effector cells. Shikonin-tumor cell lysate-loaded mature dendritic cells exhibit a high level of CD86 and MHC class II and activate Th1 cells. The shikonin-tumor cell lysate-loaded dendritic cell vaccines result in a strong induction of cytotoxic activity of splenocytes against target tumor cells, a retardation in tumor growth, and an increase in the survival of test mice. The much enhanced immunogenicity and efficacy of the current cancer vaccine formulation, that is, the use of shikonin-treated tumor cells as cell lysates for the pulse of dendritic cells in culture, may suggest a new ex vivo approach for developing individualized, dendritic cells-based anticancer vaccines.
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Apoptosis/efectos de los fármacos , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/trasplante , Melanoma Experimental/terapia , Naftoquinonas/administración & dosificación , Neoplasias Cutáneas/terapia , Animales , Apoptosis/inmunología , Antígeno B7-2/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Femenino , Genes MHC Clase II/inmunología , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th17/efectos de los fármacos , Células Th17/inmunologíaRESUMEN
BACKGROUND: Shikonin, a phytochemical purified from Lithospermum erythrorhizon, has been shown to confer diverse pharmacological activities, including accelerating granuloma formation, wound healing, anti-inflammation and others, and is explored for immune-modifier activities for vaccination in this study. Transdermal gene-based vaccine is an attractive approach for delivery of DNA transgenes encoding specific tumor antigens to host skin tissues. Skin dendritic cells (DCs), a potent antigen-presenting cell type, is known to play a critical role in transmitting and orchestrating tumor antigen-specific immunities against cancers. The present study hence employs these various components for experimentation. METHOD: The mRNA and protein expression of RANTES were detected by RT-PCR and ELISA, respectively. The regional expression of RANTES and tissue damage in test skin were evaluated via immunohistochemistry assay. Fluorescein isothiocyanate sensitization assay was performed to trace the trafficking of DCs from the skin vaccination site to draining lymph nodes. Adjuvantic effect of shikonin on gene gun-delivered human gp100 (hgp100) DNA cancer vaccine was studied in a human gp100-transfected B16 (B16/hgp100) tumor model. RESULTS: Among various phytochemicals tested, shikonin induced the highest level of expression of RANTES in normal skin tissues. In comparison, mouse RANTES cDNA gene transfection induced a higher level of mRANTES expression for a longer period, but caused more extensive skin damage. Topical application of shikonin onto the immunization site before gene gun-mediated vaccination augmented the population of skin DCs migrating into the draining lymph nodes. A hgp100 cDNA gene vaccination regimen with shikonin pretreatment as an adjuvant in a B16/hgp100 tumor model increased cytotoxic T lymphocyte activities in splenocytes and lymph node cells on target tumor cells. CONCLUSION: Together, our findings suggest that shikonin can effectively enhance anti-tumor potency of a gene-based cancer vaccine via the induction of RANTES expression at the skin immunization site.
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Antiinflamatorios no Esteroideos/farmacología , Vacunas contra el Cáncer/inmunología , Quimiocina CCL5/inmunología , Naftoquinonas/farmacología , Vacunas de ADN/inmunología , Antígeno gp100 del Melanoma/inmunología , Animales , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/metabolismo , Línea Celular Tumoral , Quimiocina CCL5/biosíntesis , Quimiocina CCL5/genética , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Humanos , Masculino , Ratones , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/terapia , Vacunas de ADN/genética , Vacunas de ADN/metabolismo , Antígeno gp100 del Melanoma/biosíntesis , Antígeno gp100 del Melanoma/genéticaRESUMEN
Blinatumomab, a bispecific T cell engager (BiTE) antibody targeting CD19 and CD3ε, can redirect T cells toward CD19-positive tumor cells and has been approved to treat relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL). However, chemotherapeutic regimens can severely reduce T cells' number and cytotoxic function, leading to an inadequate response to blinatumomab treatment in patients. In addition, it was reported that a substantial portion of R/R B-ALL patients failing blinatumomab treatment had the extramedullary disease, indicating the poor ability of blinatumomab in treating extramedullary disease. In this study, we investigated whether the adoptive transfer of ex vivo expanded γ9δ2 T cells could act as the effector of blinatumomab to enhance blinatumomab's antitumor activity against B-cell malignancies in vivo. Repeated infusion of blinatumomab and human γ9δ2 T cells led to more prolonged survival than that of blinatumomab or human γ9δ2 T cells alone in the mice xenografted with Raji cells. Furthermore, adoptive transfer of γ9δ2 T cells reduced tumor mass outside the bone marrow, indicating the potential of γ9δ2 T cells to eradicate the extramedullary disease. Our results suggest that the addition of γ9δ2 T cells to the blinatumomab treatment regimens could be an effective approach to enhancing blinatumomab's therapeutic efficacy. The concept of this strategy may also be applied to other antigen-specific BiTE therapies for other malignancies.
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Traslado Adoptivo/métodos , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T/inmunología , Animales , Células Cultivadas , Terapia Combinada , Células HEK293 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
Amphetamine-type stimulants have become important and popular abused drugs worldwide. Methamphetamine (Meth) sensitization, characterized by a progressive increase in behavioral responses after repeated administration, has been reported in rodents and patients. This behavioral effect has been used as a laboratory model to study drug addiction and schizophrenia. The mesolimbic dopaminergic pathway plays a significant role in the development of Meth behavioral sensitization. Previous studies have reported that the ablation of nucleus accumbens (NAc) by electrolytic or thermal lesioning attenuates addictive behavior to opioids in animals. However, these studies were only conducted in opioid addictive rodents. Furthermore, these ablation procedures also damaged the non-dopaminergic neurons and fibers passing through the NAc. The purpose of this study was to examine the therapeutic effect of NAc lesioning by a selective dopaminergic toxin in Meth-sensitized animals. Adult mice received repeated administration of Meth for 7 days. Open-field locomotor activity and stereotype behavior were significantly increased after Meth treatment, suggesting behavior sensitization. A partial lesion of dopaminergic terminals was made through stereotaxic administration of dopaminergic toxin 6-hydroxydopamine (6-OHDA) to the NAc in the Meth -sensitized mice. Meth behavioral sensitization was significantly antagonized after the lesioning. Brain tissue was collected for qRT-PCR analysis. Repeated administration of Meth increased the expression of tyrosine hydroxylase (TH), BDNF, and Shati, a marker for Meth sensitization, in the NAc. Treatment with 6-OHDA significantly antagonized the upregulation of TH and Shati. Taken together, these data suggest that local administration of 6-OHDA mitigated Meth sensitization in chronic Meth-treated animals. Our data support a new surgical treatment strategy for Meth abuse.
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Estimulantes del Sistema Nervioso Central/administración & dosificación , Dopamina/metabolismo , Metanfetamina/administración & dosificación , Núcleo Accumbens/fisiopatología , Oxidopamina/uso terapéutico , Animales , Humanos , Masculino , Ratones , Oxidopamina/farmacologíaRESUMEN
Accumulation of α-Synuclein (αSyn) in nigral dopaminergic neurons is commonly seen in patients with Parkinson's disease (PD). We recently reported that transduction of intracellular single-chain intrabody targeting the 53-87 amino acid residues of human αSyn by recombinant adeno associated viral vector (AAV-NAC32) downregulated αSyn protein in SH-SY5Y cells and rat brain. This study characterizes the behavioral phenotype and dopaminergic protection in animals receiving AAV-NAC32. Our results show that adult DAT-Cre rats selectively overexpress αSyn in nigra dopaminergic neurons after local administration of AAV-DIO-αSyn. These animals develop PD-like phenotype, including bradykinesia and loss of tyrosine hydroxylase (TH) immunoreactivity in substantia nigra pars compacta dorsal tier (SNcd). An injection of AAV-NAC32 to nigra produces a selective antibody against αSyn and normalizes the behavior. AAV-NAC32 significantly increases TH, while reduces αSyn immunoreactivity in SNcd. Altogether, our data suggest that an AAV-mediated gene transfer of NAC32 antibody effectively antagonizes αSyn-mediated dopaminergic degeneration in nigra, which may be a promising therapeutic candidate for synucleinopathy or PD.
Asunto(s)
Anticuerpos/uso terapéutico , Inmunoterapia/métodos , Locomoción , Enfermedad de Parkinson/terapia , alfa-Sinucleína/inmunología , Animales , Anticuerpos/inmunología , Células CHO , Cricetinae , Cricetulus , Dependovirus/genética , Neuronas Dopaminérgicas/metabolismo , Vectores Genéticos/genética , Masculino , Enfermedad de Parkinson/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Ratas , Ratas Long-Evans , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
2'-Fucosyllactose (2'-FL) is a major oligosaccharide in human milk and is present at trace levels in cow milk. 2'-FL reduces inflammation in the gastrointestinal tract. Its action in the central nervous system has not been well characterized. The purpose of this study is to determine 2'-FL-mediated neural protection and repair in culture and stroke brain. In rat primary cortical neuronal cultures, 2'-FL significantly antagonized N-methyl-D-aspartate (NMDA) or glutamate-mediated changes in ATP production, MAP2 immunoreactivity, and TUNEL. The influx of Ca++ (Ca++i) was examined in primary cortical neurons expressing GCaMP5, an endogenous calcium probe. NMDA increased Ca++i; 2'-FL significantly attenuated this reaction. In a rat middle cerebral artery occlusion model of stroke, we found that intracerebroventricular pretreatment or oral posttreatment with 2'-FL significantly reduced brain infarction, mitigated microglial activation, improved locomotor activity, and upregulated brain-derived neurotrophic factor (BDNF) expression. Post-stroke delivery of 2'-FL increased bromodeoxyuridine (BrdU) labeling in the perilesioned area. These BrdU cells co-expressed NeuN, or nestin, or GFAP. Using subventricular Matrigel cultures, we demonstrated that 2'-FL increased cell migration from subventricular zone explant. This response was reduced by anti-BDNF blocking antibody. In conclusion, our data suggest that 2'-FL has neuroprotective action through inhibition of Ca++i, inflammation, and apoptosis. Posttreatment with 2'-FL facilitates neural repair in stroke brain.
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Encéfalo/efectos de los fármacos , Leche Humana/metabolismo , Neuronas/efectos de los fármacos , Oligosacáridos/farmacología , Trisacáridos/farmacología , Animales , Encéfalo/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Neuronas/metabolismo , Oligosacáridos/metabolismo , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Accumulation of α-synuclein (αSyn) in the dopaminergic neurons is a common pathology seen in patients with Parkinson's disease (PD). Overproduction of αSyn potentiates the formation of oligomeric αSyn aggregates and enhances dopaminergic neuron degeneration. Downregulating intracellular monomeric αSyn prevents the formation of αSyn oligomers and is a potential therapeutic strategy to attenuate the progression of PD. OBJECTIVE: The purpose of this study is to investigate the efficacy of gene delivery of αSyn-specific single-chain antibodies in vitro and in vivo. METHODS AND RESULTS: The plasmids for αSyn and selective antibodies (NAC32, D10, and VH14) were constructed and were transfected to HEK293 and SH-SY5Y cells. Co-expression of αSyn with NAC32, but not D10 or VH14, profoundly downregulated αSyn protein, but not αSyn mRNA levels in these cells. The interaction of αSyn and NAC32 antibody was next examined in vivo. Adeno-associated virus (AAV)-αSyn combined with AAV-NAC32 or AAV-sc6H4 (a negative control virus) were stereotactically injected into the substantia nigra of adult rats. AAV-NAC32 significantly reduced AAV-encoded αSyn levels in the substantia nigra and striatum and increased tyrosine hydroxylase immunoreactivity in the striatum. Also, in the animals injected with AAV-NAC32 alone, endogenous αSyn protein levels were significantly downregulated in the substantia nigra. CONCLUSION: Our data suggest that AAV-mediated gene transfer of NAC32 is a feasible approach for reducing the expression of target αSyn protein in brain.
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Cuerpo Estriado/metabolismo , Enfermedad de Parkinson/metabolismo , Anticuerpos de Cadena Única/metabolismo , Sustancia Negra/metabolismo , alfa-Sinucleína/metabolismo , Animales , Células Cultivadas , Dependovirus , Modelos Animales de Enfermedad , Regulación hacia Abajo , Técnicas de Transferencia de Gen , Células HEK293 , Humanos , RatasRESUMEN
The purpose of this study was to examine the influence of adenovirus-carried VEGF165 transgene at 5 x 10(10) pfu (Ad-VEGF) on vascular formation, cardiac geometry and ventricular function in infarcted hearts of the rat and to explore the mechanism of Ad-VEGF-mediated actions on ventricular function by quantitative proteomic analysis. Seven days after coronary occlusion, intramyocardial injection with normal saline (vehicle control), adenovirus-carried beta-galactosidase gene (Ad-LacZ, vector control) or Ad-VEGF to infarcted hearts was conducted. Seven days after intramyocardial injection, ventricular function, cardiac morphology and vascular density were assessed after echocardiographic analysis and immunohistological staining. One dimensional gel electrophoresis coupled with stable isotope dimethyl labeling and LC/MS/MS was used to quantify the abundance ratio of each protein pair in Ad-VEGF- and Ad-LacZ-treated hearts. Our data indicated that both Ad-VEGF and Ad-LacZ increased arteriolar densities. However, the former increased arterial densities but the latter did not. Compared with the vehicle control, Ad-LacZ reversed occlusion-induced wall thinning and functional impairment but Ad-VEGF did not. Quantitative proteomic analysis showed increased ratios of plasma proteins (such as albumin) and oxygen carriers (such as myoglobin) by Ad-VEGF and decreased ratios of proteins involved in glycolysis, calcium homeostasis and lipolysis by Ad-VEGF. Taken together, our functional, morphological and proteomic data suggest that intramuscular delivery of Ad-LacZ at higher doses may improve ventricular function and wall thinning with arteriolar formation. Excessive amounts of VEGF by Ad-VEGF may offset Ad-LacZ-induced improvement in ventricular functions by interfering with calcium homeostasis and lipolysis in infarcted hearts.
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Vasos Coronarios/crecimiento & desarrollo , Corazón/fisiopatología , Infarto del Miocardio/fisiopatología , Factores de Crecimiento Endotelial Vascular/fisiología , Función Ventricular/fisiología , Adenoviridae/genética , Animales , Vectores Genéticos , Masculino , Miocardio/metabolismo , Proteómica , Ratas , Ratas Wistar , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Transgenes , Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Methamphetamine (Meth) is a widely abused stimulant. High-dose Meth induces degeneration of dopaminergic neurons through p53-mediated apoptosis. A recent study indicated that treatment with the p53 inhibitor, pifithrin-alpha (PFT-α), antagonized Meth-mediated behavioral deficits in mice. The mechanisms underpinning the protective action of PFT-α against Meth have not been identified, and hence, their investigation is the focus of this study. Primary dopaminergic neuronal cultures were prepared from rat embryonic ventral mesencephalic tissue. High-dose Meth challenge reduced tyrosine hydroxylase immunoreactivity and increased terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling (TUNEL) labeling. PFT-α significantly antagonized these responses. PFT-α also reduced Meth-activated translocation of p53 to the nucleus, an initial step before transcription. Previous studies have indicated that p53 can also activate cell death through transcription-independent pathways. We found that PFT-α attenuated endoplasmic reticulum (ER) stressor thapsigargin (Tg)-mediated loss of dopaminergic neurons. ER stress was further monitored through the release of Gaussia luciferase (GLuc) from SH-SY5Y cells overexpressing GLuc-based Secreted ER Calcium-Modulated Protein (GLuc-SERCaMP). Meth or Tg significantly increased GLuc release in to the media, with PFT-α significantly reducing GLuc release. Additionally, PFT-α significantly attenuated Meth-induced CHOP expression. In conclusion, our data indicate that PFT-α is neuroprotective against Meth-mediated neurodegeneration via transcription-dependent nuclear and -independent cytosolic ER stress pathways.
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Benzotiazoles/farmacología , Estimulantes del Sistema Nervioso Central/toxicidad , Neuronas Dopaminérgicas/efectos de los fármacos , Metanfetamina/toxicidad , Tolueno/análogos & derivados , Animales , Línea Celular Tumoral , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Femenino , Humanos , Embarazo , Ratas , Tolueno/farmacologíaRESUMEN
The cytomegalovirus (CMV) immediate early promoter has been extensively developed and exploited for transgene expression in vitro and in vivo, including human clinical trials. The CMV promoter has long been considered a stable, constitutive, and ubiquitous promoter for transgene expression. Using two different CMV-based promoters, we found an increase in CMV-driven transgene expression in the rodent brain and in primary neuronal cultures in response to methamphetamine, glutamate, kainic acid, and activation of G protein-coupled receptor signaling using designer receptors exclusively activated by designer drugs (DREADDs). In contrast, promoters derived from human synapsin 1 (hSYN1) gene or elongation factor 1α (EF1α) did not exhibit altered transgene expression in response to the same neuronal stimulations. Overall, our results suggest that the long-standing assertion that the CMV promoter confers constitutive expression in neurons should be reevaluated, and future studies should empirically determine the activity of the CMV promoter in a given application.
RESUMEN
Methamphetamine (Meth) is one of the most frequently abused drugs worldwide. Recent studies have indicated that antibodies with high affinity for Meth reduce its pharmacological effects. The purpose of this study was to develop a technique for virus-based passive immunization against Meth effects. We generated a recombinant adeno-associated virus serotype-8 vector (AAV-MethAb) carrying the gene for a Meth-specific monoclonal antibody (MethAb). Infection of 293 cells with AAV-MethAb resulted in the expression and secretion of antibodies which bind to Meth. The viral vector was then examined in adult ICR mice. Systemic administration of AAV-MethAb resulted in long-term expression of MethAb in the serum for up to 29 weeks. Serum collected from the animals receiving AAV-MethAb retained a high specificity for (+)-Meth. Animals were challenged with Meth five weeks after viral injection. Meth levels in the brain and serum were reduced while Meth-induced locomotor activity was significantly attenuated. In conclusion, AAV-MethAb administration effectively depletes Meth from brain and serum while reducing the behavioral response to Meth, and thus is a potential therapeutic approach for Meth abuse.
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Trastornos Relacionados con Anfetaminas/terapia , Anticuerpos Neutralizantes/inmunología , Hipercinesia/terapia , Inmunización Pasiva/métodos , Metanfetamina/inmunología , Trastornos Relacionados con Anfetaminas/complicaciones , Animales , Anticuerpos Neutralizantes/genética , Dependovirus/genética , Células HEK293 , Humanos , Hipercinesia/etiología , Masculino , Metanfetamina/toxicidad , Ratones , Ratones Endogámicos ICRRESUMEN
Synapsin III is a new synapsin family gene with the putative function of synaptogenesis regulation and neurotransmitter release in the brain. The gene was mapped to 22q12-q13, a schizophrenia susceptible region gene as suggested by several linkage studies. Hence, the synapsin III gene is considered a candidate gene of schizophrenia. We systematically sequenced the protein coding and 5'-promoter regions of the synapsin III gene to look for mutations in 62 Han Chinese schizophrenic patients from Taiwan with positive family history. Further case-control association study was performed among 163 patients and 151 controls using the genetic polymorphic markers identified from these 62 patients. Three single nucleotide polymorphisms (SNPs) were identified: g.-631C > G and g.-196G>A at 5'-promoter region, and g.69G>A at exon 1. Besides, no other mutations were identified in these patients. The g.69G>A polymorphism does not alter the amino acid threonine at codon 23 (ACG>ACA). Further case-control association studies also did not find significant differences of genotype or allele frequency distributions of these three polymorphisms between 163 patients and 151 non-psychotic comparison individuals. Hence, our data are not in favor of a large effect of synapsin III gene in the pathogenesis of schizophrenia.
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Neuropéptidos/genética , Fosfoproteínas/genética , Esquizofrenia/genética , Pueblo Asiatico/genética , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Sinapsinas , TaiwánRESUMEN
Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1ß (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Neuritas/metabolismo , Neurogénesis , Neuronas/metabolismo , Medicina Regenerativa/métodos , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/genética , Biomarcadores/metabolismo , Forma de la Célula , Células Cultivadas , Reprogramación Celular , Genotipo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Fenotipo , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Methamphetamine is a highly addictive psychostimulant that has been seriously abused worldwide, and currently there are no approved medications for the treatment of its abuse. Conventional treatments for drug addiction mainly seek to use small molecule agonists or antagonists to target the drug receptors in the brain, but unfortunately it is difficult to find a similar small molecule for the treatment of methamphetamine dependence. Alternatively, anti-methamphetamine antibodies can sequester the drug in the bloodstream and reduce the amount of drug available to the central nervous system, acting as peripheral pharmacokinetic antagonists. This review describes the development of antibody-based immunotherapies, classified into active and passive immunizations, for the treatment of methamphetamine addiction. Furthermore, an alternative therapeutic approach, using a recombinant adeno-associated virus-mediated gene transfer technique to achieve in vivo expression of characterized anti-methamphetamine monoclonal antibodies, is proposed in this article.
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Anticuerpos Monoclonales , Inmunoterapia , Metanfetamina/toxicidad , Trastornos Relacionados con Sustancias/tratamiento farmacológico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/patología , Dependovirus , Humanos , Metanfetamina/antagonistas & inhibidores , Metanfetamina/inmunología , Trastornos Relacionados con Sustancias/inmunología , VacunaciónRESUMEN
BACKGROUND: Narcolepsy is a neuropsychiatric disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and abnormal rapid eye movement (REM) sleep. Tumor necrosis factor α (TNF α) and its cognate receptors have been reported to be involved in the pathophysiology of narcolepsy in addition to the HLA antigen system. Our study aimed to determine if the TNF-α system was associated with narcolepsy in our patients. METHODS: We first measured the plasma level of TNF α in 56 narcoleptic patients and 53 control subjects using a highly sensitive enzyme-linked immunosorbent assay. We next determined the genotype of three single nucleotide polymorphisms (SNPs) (T-1031C, C-863A, and C-857T) at the promoter region of the TNF-α gene and one missense SNP (T587G, M196R) at the exon 6 of the tumor necrosis factor receptor 2 gene, TNFR2, in a sample of 75 narcoleptic patients and 201 control subjects by direct sequencing analysis. RESULTS: We found a significant elevation of plasma level of TNF α in patients with narcolepsy compared with the control subjects (4.64pg/mL vs 1.06pg/mL; P=.0013). However, we did not find significant differences between these two groups in the allelic and genotypic distributions of the investigated polymorphisms. CONCLUSIONS: Our study suggests that an increased TNF-α level was associated with narcolepsy in our patients, and that chronic inflammation due to various factors might have led to the increased TNF-α levels found in our patients.
Asunto(s)
Narcolepsia/genética , Narcolepsia/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genética , Adolescente , Adulto , Cataplejía/genética , Cataplejía/metabolismo , Niño , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Taiwán , Adulto JovenRESUMEN
BACKGROUND: Both alcoholism and heroin dependence are common substance use disorders with a high genetic basis. A recent genetic study reported that the autism susceptibility candidate 2 gene (AUTS2) was involved in regulating the alcohol drinking behavior. In our previous total gene expression profiling study, we found that the AUTS2 transcript was significantly down-regulated in lymphoblastoid cell lines (LCL) in heroin dependent individuals compared with control subjects, which prompted us to investigate whether AUTS2 is associated with heroin dependence. METHODS: We compared the AUTS2 transcript level of LCL between 124 heroin dependent males and 116 control males using real-time quantitative PCR, and conducted a genetic association study of the rs6943555 of AUTS2 with heroin dependence using a sample of 546 heroin dependent males and 373 control males. RESULTS: We first verified that the average transcript level of AUTS2 in the heroin dependent group was significantly lower than that in the control group (p=0.017). In the genetic association analysis, we found that AA homozygotes of rs6943555 were significantly over-represented in the heroin dependent subjects compared with the control subjects (odds ratio=1.7, 95% confidence interval: 1.08-2.74, p=0.017). Analyzing the sample from the AUTS2 transcript experiment, we found that AA carriers (n=19) had significantly lower AUTS2 mRNA levels in their LCL compared to TT carriers (n=97, p=0.002) and AT carriers (n=91, p=0.005). CONCLUSIONS: Our data indicate that the AUTS2 gene might be associated with heroin dependence, and reduced AUTS2 gene expression might confer increased susceptibility to heroin dependence.