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1.
J Microbiol Immunol Infect ; 43(2): 85-92, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20457423

RESUMO

BACKGROUND/PURPOSE: The postweaning multisystemic wasting syndrome, caused by the porcine circovirus type 2 (PCV-2), is a major disease that poses a significant threat to the global swine industry. The purpose of this study was to establish a real-time polymerase chain reaction (PCR) method for the quantification of PCV-2 and to enable the rapid differentiation of porcine circoviruses type 1 and 2 (PCV-1 and PCV-2). Such a method would significantly speed up the process of clinical diagnosis, and could also be used to study the pathogenic mechanisms of diseases associated with PCV-2. METHODS: Multiplex real-time PCR, together with LightCycler PCR data analysis software, was used for the quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2. A 263-bp DNA fragment was amplified from the 3' end of the open reading frame-2 of PCV-2 by nested PCR, and its DNA sequence was verified as having 100% identity with a PCV-2 standard (NCBI accession number: AF055394). The 263-bp DNA fragment was cloned into the pGEM-T easy vector, and the recombinant plasmid was serially diluted and quantified using real-time PCR. A standard curve was then constructed for quantification of the PCV-2 levels in field samples. The differentiation of PCV-1 and PCV-2 was carried out by analyzing the melting temperatures of the genotype-specific PCR products. RESULTS: To quantify the PCV-2 levels in field samples, a standard curve (1 x 10(2) -1 x 10(9) copies/microL) was constructed. PCV-2 concentrations as low as 1 x 10(2) copies/microL could be detected in specimens taken from the lymph nodes or infected tissues in samples of PCV-2-infected pigs. The diagnosis of PCV-1 and PCV-2 infections and the quantification of the viral load in the field samples could be completed within 45 minutes after extracting the viral DNA using a commercial extraction kit. CONCLUSION: This study demonstrate that real-time PCR is a clinically feasible method for the accurate quantification of PCV-2, and for the rapid differentiation of PCV-1 and PCV-2.


Assuntos
Circovirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Síndrome Definhante Multissistêmico de Suínos Desmamados/diagnóstico , Carga Viral/métodos , Animais , Circovirus/classificação , Circovirus/genética , Primers do DNA/genética , DNA Viral/genética , Síndrome Definhante Multissistêmico de Suínos Desmamados/virologia , Suínos , Fatores de Tempo , Temperatura de Transição
2.
Brain Res Mol Brain Res ; 133(2): 198-207, 2005 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-15710236

RESUMO

Paraplegia is a catastrophic complication of thoracic aortic surgery. At present, there is no effective mean to prevent the ischemia-induced spinal cord trauma. Gene delivery of neurotrophic factors may hold promises for prevention of spinal injury. In the present study, we evaluated the effect of glial cell line-derived neurotrophic factor (GDNF) gene delivery on prevention of the pathological changes due to spinal ischemia. Recombinant adenovirus vectors encoding GDNF (Ad-GDNF) and green fluorescent protein (Ad-GFP) were used for gene transfer studies. Treatment with cobalt chloride induced dose-dependent bcl-2 and synaptophysin downregulation in spinal neuronal cells, which could be effectively reversed by GDNF gene transfer. Intrathecal injection of Ad-GDNF led to maximal GDNF expression in spinal cord within 2-7 days. Thus, after intrathecal administration of adenovirus vectors for 3 days, Sprague-Dawley rats received transient aortic occlusion to induce spinal ischemia and were monitored for behavior deficits. The Ad-GDNF-treated rats showed significantly lower paraplegia rate (40%) than that of Ad-GFP- or saline-treated groups (75-85%; P<0.01). In addition, the Ad-GDNF-treated rats exhibited significantly improved locomotor function comparing with rats of control groups (P<0.001). Histological analysis revealed that GDNF gene delivery profoundly attenuated the infiltration of leukocytes in spinal cord after ischemic insults. Furthermore, GDNF gene delivery prominently attenuated the ischemia-induced neuronal loss in dorsal horn lamina VI-VIII and reduction in synaptophysin expression in spinal cords. In conclusion, GDNF gene transfer confers protection to the neuronal cells and synapses networks, thereby alleviated the paraplegia due to spinal ischemia.


Assuntos
Terapia Genética , Fatores de Crescimento Neural/uso terapêutico , Paraplegia/terapia , Isquemia do Cordão Espinal/complicações , Adenoviridae/fisiologia , Animais , Comportamento Animal , Western Blotting/métodos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Cobalto/farmacologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/terapia , Técnicas de Transferência de Genes , Vetores Genéticos/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Imuno-Histoquímica/métodos , Inflamação/etiologia , Inflamação/prevenção & controle , Masculino , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/virologia , Paraplegia/etiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Sinaptofisina/metabolismo , Fatores de Tempo
3.
Pain ; 104(1-2): 159-67, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12855325

RESUMO

Opioids remain the most efficacious pharmacological agents for various clinical pain syndromes. Recently, various engineered cells capable of secreting opioidergic peptides have been applied to relieve pain in animal models. In vivo gene delivery by viruses encoding endogenous opioids has also been used with success. In this study, we attempted non-viral intrathecal in vivo gene delivery by electroporation to induce analgesia. Thirty Sprague-Dawley rats were used in this study, six in each of five groups. Rats were treated as follows: vehicle without electroporation (group A), vehicle with electroporation (group B), 100 microg of pCMV-hPOMC plasmid without electroporation (group C), or 100 microg of pCMV-hPOMC plasmid with electroporation (group D). Group E was treated with both pCMV-hPOMC plasmid and electroporation, and given naloxone (1mg/kg) 1h before the formalin test. The tail flick, paw withdrawal latency from radiant heat, and formalin test results for each groups were compared. Radioimmunoassay (RIA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the levels of expression of beta-endorphin in the spinal cord. beta-Endorphin expression was localized by immunohistochemistry. A significant decrease in the number of flinches in phase 2 of the formalin test was observed in the group treated with both plasmid and electroporation (group D), whereas the other measures of pain did not differ between groups. RIA and RT-PCR both showed increased expression of beta-endorphin in group D. The expression of beta-endorphin was highest in laminae I and II of the dorsal horn of the spinal cord. We conclude that electroporation successfully delivered intrathecally administered pCMV-hPOMC into the dorsal horn cells of the spinal cord, and induced analgesia in phase 2 of the formalin test in rats.


Assuntos
Analgesia/métodos , Modelos Animais de Doenças , Eletroporação/métodos , Medição da Dor/efeitos dos fármacos , Pró-Opiomelanocortina/administração & dosagem , Animais , DNA Complementar/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Humanos , Masculino , Dor/tratamento farmacológico , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Medula Espinal/química , Medula Espinal/efeitos dos fármacos , beta-Endorfina/análise
4.
Neurosci Lett ; 333(3): 155-8, 2002 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-12429371

RESUMO

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Abeta-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. Synaptophysin is a presynaptic vesicle protein, useful in the identification of synaptogenesis. Here we investigated whether synaptogenesis as measured by the expression of synaptophysin protein correlates with symptoms of neuropathic pain in rats with a chronic constriction injury (CCI) of the sciatic nerve. We used immunohistochemistry, Western immunoblotting and densitometry to study the distribution of synaptophysin and to quantify relative protein. Synaptophysin was increased in the ipsilateral dorsal horn with a peak level on day 14 and returned to baseline on day 21 post-CCI. Synaptophysin levels temporally correlated with thermal hyperalgesia but not with tactile allodynia. Our results indicate that thermal hyperalgesia in CCI significantly correlates with synaptogenesis within the superficial layers of the dorsal horn.


Assuntos
Nervo Isquiático/lesões , Medula Espinal/metabolismo , Sinaptofisina/metabolismo , Animais , Antígenos Nucleares/metabolismo , Western Blotting/métodos , Lateralidade Funcional/fisiologia , Imuno-Histoquímica/métodos , Ligadura , Masculino , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Sensação Térmica/fisiologia , Fatores de Tempo
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