RESUMO
An unusual case of purely osteoblastic metastasis in the spine from adenocarcinoma of the pancreas was reported. Severe low back pain with osteoblastic lesions pictured in a lumbar X-ray study were the initial manifestations. A percutaneous transpedicular vertebral bodies biopsy was performed and showed a metastatic adenocarcinoma. This clinical presentation is unusual and the diagnosis of pancreatic carcinoma should be considered when radiographs or bone scans show osteoblastic bone lesions.
Assuntos
Adenocarcinoma/secundário , Neoplasias Pancreáticas/patologia , Neoplasias da Coluna Vertebral/secundário , Adenocarcinoma/complicações , Adenocarcinoma/diagnóstico , Humanos , Dor Lombar/etiologia , Masculino , Pessoa de Meia-Idade , Osteoblastos , Radiografia , Cintilografia , Neoplasias da Coluna Vertebral/complicações , Neoplasias da Coluna Vertebral/diagnóstico , Coluna Vertebral/diagnóstico por imagemRESUMO
Direct injection of double-stranded adeno-associated virus type 2 (dsAAV2) with a mu-opioid receptor (MOR) mutant [S4.45(196)A], and a reporter protein (enhanced green fluorescent protein) into the spinal cord (S2/S3) dorsal horn region of ICR mice resulted in antinociceptive responses to systemic injection of opioid antagonist naloxone without altering the acute agonist morphine responses and no measurable tolerance or dependence development during subchronic naloxone treatment. To develop further such mutant MORs into a therapeutic agent in pain management, a less invasive method for virus delivery is needed. Thus, in current studies, the dsAAV2 was locally injected into the subarachnoid space of the spinal cord by intrathecal administration. Instead of using the MORS196A mutant, we constructed the dsAAV2 vector with the MORS196ACSTA mutant, a receptor mutant in which naloxone has been shown to exhibit full agonistic properties in vitro. After 2 weeks of virus injection, naloxone (10 mg/kg s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg s.c., b.i.d. for 6 days) and significant withdrawal symptoms. On the other hand, subchronic treatment with morphine (10 mg/kg s.c., b.i.d.) for 6 days induced significant tolerance (4.8-fold) and withdrawal symptoms. Furthermore, we found that morphine, but not naloxone, induced the rewarding effects (determined by conditioned place preference test). These data suggest that local expression of MORS196ACSTA in spinal cord and systemic administration of naloxone has the potential to be developed into a new strategy in the management of pain without addiction liability.
Assuntos
Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Manejo da Dor , Medição da Dor/efeitos dos fármacos , Receptores Opioides mu/efeitos dos fármacos , Receptores Opioides mu/genética , Animais , Condicionamento Operante/efeitos dos fármacos , Tolerância a Medicamentos , Técnicas de Transferência de Genes , Terapia Genética , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mutação , Transtornos Relacionados ao Uso de Opioides/psicologia , Recompensa , Espaço SubaracnóideoRESUMO
Uncontrolled growth and diffused invasion are major causes of mortality in patients with malignant gliomas. Nodal has been shown to have a central role in the tumorigenic signaling pathways of malignant melanoma. In this study, we show that grade IV human glioma cell lines expressed different levels of Nodal, paralleled to the potential for cell invasiveness. Treatment of glioma cell lines with recombinant Nodal (rNodal) increased matrix metalloproteinase 2 (MMP-2) secretion and cell invasiveness. The ectopic expression of Nodal in GBM glioma cells that expressed Nodal at low level resulted in increased MMP-2 secretion, enhanced cell invasiveness, raised cell proliferation rates in vitro, increased tumor growth in vivo, and was associated with poor survival in a mice xenograft model. In contrast, the knockdown of Nodal expression in U87MG glioma cells with high Nodal expression level had reduced MMP-2 secretion, less cell invasiveness, lower tumor growth in vivo and longer lifespan in mice with U87MG/shNodal cell xenografts. In addition, Nodal knockdown promoted the reversion of malignant glioma cells toward a differentiated astrocytic phenotype. Furthermore, our data support the notion that Nodal may regulate glioma progression through the induction of the leukemia inhibitory factor (LIF) and Cripto-1 through activated Smad.
Assuntos
Glioma/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Animais , Divisão Celular , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/enzimologia , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/patologia , Progressão da Doença , Fator de Crescimento Epidérmico/biossíntese , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Glioma/enzimologia , Glioma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Fator Inibidor de Leucemia/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Camundongos , Invasividade Neoplásica , Proteínas de Neoplasias/biossíntese , Proteínas Recombinantes/uso terapêutico , Proteínas Smad/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , TransfecçãoRESUMO
We previously reported that mutations in the mu-opioid receptor (MOR), S196L or S196A, rendered MOR responsive to the opioid antagonist naloxone without altering the agonist phenotype. Subsequently, a mouse strain carrying the S196A mutation exhibited in vivo naloxone antinociceptive activity without the development of tolerance. In this study we investigated the possibility of combining the in vivo site-directed delivery of MORS196A and systemic naloxone administration as a paradigm for pain management. Double-stranded adenoassociated virus type 2 (dsAAV2) was used to deliver MORS196A-EGFP by injecting the virus into the spinal cord (S2/S3) dorsal horn region of ICR mice. MORS196A-EGFP fluorescence colocalized with some calcitonin gene-related peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn 1 week after injection and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced similar antinociceptive responses and tolerance development or precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsAAV2-injected mice, naloxone produced antinociceptive effects at the spinal level but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsAAV2-MORS196A-EGFP did not induce tolerance, dependence, or reward responses. Thus, our current approach to activate a mutant receptor, but not the endogenous receptor, with an opioid antagonist represents an alternative to the use of traditional opioid agonists for pain management.
Assuntos
Dependovirus/genética , Genes Reporter/genética , Medição da Dor , Receptores Opioides mu/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Animais , Dependovirus/classificação , Tolerância a Medicamentos , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Masculino , Camundongos , Morfina/uso terapêutico , Mutação/genética , Naloxona/farmacologia , Dor/tratamento farmacológico , Dor/genética , Dor/metabolismo , Receptores Opioides mu/genética , Serina/genética , Serina/metabolismo , Fatores de TempoRESUMO
RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/terapia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular , Engenharia Genética , Vetores Genéticos/administração & dosagem , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Camundongos , Camundongos Transgênicos , RNA de Cadeia Dupla/administração & dosagem , Fatores de TempoRESUMO
Adeno-associated virus (AAV) is a promising gene vector based on a single-stranded (ss) DNA virus. Its transgene expression requires the conversion of ssDNA to double-stranded (ds) genome, a slow process responsible for the delayed transduction and occasional inefficiency. By mutating the inverted terminal repeat, we have made novel AAV vectors that predominantly package the self-complementary dsDNA genome. The dsAAV consistently demonstrated superior and accelerated transduction in vitro and in vivo. Dramatic increases in transgene expression were observed in most of the cell lines examined, including B16 melanoma and 3LL lung cancer that are difficult to be transduced by the conventional ssAAV vectors. Similar increases were also observed in vivo in a variety of tissues including muscle and liver. The dsAAV transduced a vast majority of the hepatocytes for more than 6 months, while the ssAAV transduced only a small fraction. In addition to circumventing the requirement for DNA synthesis, the dsAAV exhibited higher in vivo DNA stability and more effective circularization than the ssAAV, suggesting potential molecular mechanisms for the faster, stronger and prolonged transgene expression.
Assuntos
Dependovirus/genética , Vetores Genéticos/genética , Transdução Genética/métodos , Animais , Southern Blotting , Linhagem Celular Tumoral , Replicação do DNA/genética , DNA de Cadeia Simples/genética , DNA Viral/genética , Genoma Viral , Hepatócitos/virologia , Humanos , Mutação/genética , Transgenes/genéticaRESUMO
We have utilized a recombinant adeno-associated viral (AAV) vector carrying the angiostatin gene as an anti-angiogenesis strategy to treat the malignant brain tumor in a C6 glioma/Wistar rat model. Angiostatin, as a potent angiogenesis inhibitor, shows high promises as an anti-cancer drug through the inhibition of tumor neovessel formation. However, sustained in vivo protein delivery is required to achieve the therapeutic effects. The AAV vector has been proven to be able to deliver sustained and high-level gene expression in vivo, and therefore, is well suited to such a purpose. In this study, we implanted 5 x 10(5) C6 glioma cells into the rat brain 7 days before gene therapy. Intratumoral injection of a high-titer AAV-angiostatin vector has rendered efficacious tumor suppression and resulted in long-term survival in 40% of the treated rats, whereas the control AAV-GFP vector did not have any therapeutic benefits. In addition, we have investigated the combined gene therapy of an adenoviral vector carrying the suicidal thymidine kinase gene along with the AAV-angiostatin vector. The combined therapy offered the best tumor-suppressive effects and increased long-term survival to 55% in the treated rats. Our study has demonstrated the potential of using AAV as a safe and effective vector for anti-angiogenic gene therapy of brain tumors.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Glioma/terapia , Fragmentos de Peptídeos/uso terapêutico , Plasminogênio/uso terapêutico , Adenoviridae/genética , Inibidores da Angiogênese/genética , Angiostatinas , Animais , Expressão Gênica , Vetores Genéticos/administração & dosagem , Injeções Intralesionais , Masculino , Modelos Animais , Transplante de Neoplasias , Fragmentos de Peptídeos/genética , Plasminogênio/genética , Ratos , Ratos Wistar , Simplexvirus/enzimologia , Timidina Quinase/genética , Transfecção , Células Tumorais CultivadasRESUMO
To explore the potential of using the recombinant adeno-associated viral (rAAV) vector, expressing glial cell line-derived neurotrophic factor (GDNF) as the gene therapy for stroke, we injected rAAV vectors expressing GDNF (rAAV-GDNF) into the cortex of rats which had been experiencing transient bilateral common carotid artery ligation and right middle cerebral artery ligation for 90 min. GDNF levels in cortical tissues of rAAV-GDNF-injected animals were significantly higher than in the control animals injected with rAAV-expressing lacZ (rAAV-lacZ), indicating that rAAV can deliver and express the GDNF gene in cortical tissues. Triphenyltetrazolium chloride tissue stain analysis revealed that the rAAV-delivered GDNF gene could rescue the brain tissues from ischemia-induced injury. Cortical tissues which received rAAV-GDNF injections had both significantly smaller total volumes of infarction and smaller areas of infarction on each brain slice than those which were injected with rAAV-lacZ. An in situ labeling analysis demonstrated significantly less apoptotic cells in cortical tissues rescued by rAAV-GDNF, indicating prevention of apoptosis as the mechanism of cortical cell protection. Moreover, immunohistochemistry staining of Neu-N indicated that the rescued brain tissues contained the same number of Neu-N-positive neuronal cells as contralateral undamaged brain tissues. This provides strong evidence that cortical neuronal cells can be rescued by GDNF gene therapy. Indeed, these findings show that the rAAV is a potential delivery vector of GDNF gene for the therapy of stroke.
Assuntos
Adenoviridae/genética , Isquemia Encefálica/terapia , Terapia Genética/métodos , Vetores Genéticos , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores/metabolismo , Animais , Apoptose , Isquemia Encefálica/patologia , Células Cultivadas , Córtex Cerebral/patologia , DNA Complementar , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Marcação In Situ das Extremidades Cortadas , Rim/citologia , Óperon Lac , Masculino , Microinjeções , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/genética , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/terapiaRESUMO
We monitored the compositional change of host RNA polymerase in Xp 10 infected cells by one-step immunoprecipitation with core enzyme-specific antiserum. Results showed a rapid loss of sigma subunit from the RNA polymerase complex by 3 min after infection. In addition, some putative binding proteins were reduced to various extents. In contrast, increasing levels of several other polypeptides were detected. The de novo host protein synthesis was inhibited within 1 min after Xp 10 infection. On the other hand, a sequential expression of phage specific proteins was found and can be categorized as early, middle, and late stage. The alteration of host RNA polymerase and the shutdown of early class proteins took place in parallel.