Treatment of mouse limb ischemia with an integrative hypoxia-responsive vector expressing the vascular endothelial growth factor gene.

Constitutive vascular endothelial growth factor (VEGF) gene expression systems have been extensively used to treat peripheral arterial diseases, but most of the results have not been satisfactory. In this study, we designed a plasmid vector with a hypoxia-responsive element sequence incorporated into it with the phiC31 integrative system (pVHAVI) to allow long-term VEGF gene expression and to be activated under hypoxia. Repeated activations of VEGF gene expression under hypoxia were confirmed in HEK293 and C2C12 cells transfected with pVHAVI. In limb ischemic mice, the local administration of pVHAVI promoted gastrocnemius mass and force recovery and ameliorated limb necrosis much better than the group treated with hypoxia-insensitive vector, even this last group had produced more VEGF in muscle. Histological analyses carried out after four weeks of gene therapy showed increased capillary density and matured vessels, and reduced number of necrotic cells and fibrosis in pVHAVI treated group. By our study, we demonstrate that the presence of high concentration of VEGF in ischemic tissue is not beneficial or is less beneficial than maintaining a lower but sufficient and long-term concentration of VEGF locally.

Bupivacaine injection leads to muscle force reduction and histologic changes in a murine model.

OBJECTIVE: To evaluate the effect of bupivacaine on muscle force and histology. We hypothesize that bupivacaine will worsen the muscle's physiological activity. SETTING: Controlled laboratory experiment. METHODS: Bupivacaine (0.5 mL, 0.5%) was injected into the mid belly and distal portions of the right gastrocnemius in 32 Wistar male rats (the left gastrocnemius was used as a control). After 5, 14, 21, and 28 days, in groups of 4, muscle force was evaluated and the animals were euthanized by an overdose of anesthetic for histologic evaluation. One-way analysis of variance was used to analyze data from force and weight measurements. Only the values of P < .05 were considered to be statistically significant. RESULTS: Bupivacaine causes a process of degeneration-regeneration of the muscle fibers and it also causes a reduction in muscle force, which is significant at 2 and 3 weeks and does not normalize at 4 weeks. The muscle injury is obvious after 5 days, and the degenerative process is predominant at 2 and 3 weeks. We found an increase in muscle mass in the acute phase and a decrease in muscle force. CONCLUSION: Although our results do not allow a direct clinical application, we believe that caution should be warranted when intramuscular bupivacaine is used.

Therapeutic ultrasound promotes plasmid DNA uptake by clathrin-mediated endocytosis.

BACKGROUND: Ultrasound (US) has been widely used to improve the efficiency of nonviral vector transfection. The mechanism of plasmid uptake is usually attributed to sonoporation, although there is not clear evidence for this attribution. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, could be involved in this process. METHODS: NIH3T3 cells were transfected with plasmid vector pEGFP-N3 (4.7 kb) using a therapeutic US without microbubbles. Bioeffects such as calcium influx, reactive oxygen species (ROS) generation and membrane potential alterations were accessed with fluorescent dyes in real-time by confocal microscopy after US insonation. Localization of labeled plasmid DNA in cells was also monitored with endocytosis markers using an immunofluorescence assay. RESULTS: US at 2 W/cm(2) with a duty-cycle of 20% for 30 s resulted in approximately 40% transfection efficiency but, at 1 W/cm(2) , resulted in a very low level of transfection. Both the production of ROS and calcium influx were augmented during the insonation, although they were stopped soon after turning off US, with the exception of calcium influx with 1 W/cm(2) . US also changed the cell membrane potential to the hyperpolarization state, which returned to the normal state soon after insonation. Labeled plasmids DNA could be co-localized with clathrin-mediated endocytosis marker but not with caveolin-1. CONCLUSIONS: The present data indicate that plasmid DNA uptake promoted by US should occur via clathrin-mediated endocytosis.

Caracterizaçäo de anticorpos monoclonais dirigidos a lipofosfoglicano de formas promastigotas de Leishmania(Leishmania) amazonensis: papel protetor na infecçäo / Characterization of monoclonal antibodies against lipophosphoglycan of promastigote forms of leishmania(Leishmania) amazonensis: protector role on infection

Este estudo analisou o papel do lipofosfoglicano (LPG) de formas promastigotas de L. (L.) amazonensis, na infectividade do parasita, utilizando anticorpos monoclonais (MoAbs) VST-1 (IgG3) e VST-2 (IgM) dirigidos contra LPG. Neste trabalho foi também utilizado o MoAb ST-3 (IgG3), produzido contra glicoesfingolipídeos de formas amastigotas, que reconhece em promastigotas de L. (L.) amazonensis a fraçao de LPG. Por imunofluorescência o MoAb VST-1 apresentou marcaçao de superfície com formas promastigotas de L. (L.) amazonenses; o MoAb VST-2 reconheceu promastigotas de L. (L.) amazonenses e L. (L.) major, enquanto que o MoAb ST-3 reconheceu formas promastigotas de L. (L.) amazonensis e L. (V.) braziliensis. Estes resultados foram confirmados por radioimunoensaio (RIA) celular. Por PAGE-SDS, foi demonstrado que LPGs de L (L.) amazonensis e de L. (L.) major apresentam-se polidispersos com pesos moleculares aparentes entre 35 e 80 kDa e entre 8 e 45 kDa, respectivamente. Por Westem 81ot, a reatividade dos MoAbs VST-1 e ST-3 com LPG purificado de L. (L.) amazonenses foi intensa, por outro lado, o MoAb VST-2 reagiu preferencialmente com LPG de L. (L.) major. Os MoAbs VST-1, VST-2 mostraram-se específicos para LPG, nao reconhecendo outras fraçoes de glicolipídeos quando analisados por RIA. f A reatividade para os MoAbs VST-1, VST-2 e ST-3 por RIA foi favorecida quando o LPG purificado foi adsorvido em superfície positiva após tratamento da placa de poliestireno com poli-L-lisina, ou em superfície contendo componentes lipídicos de membrana, como fosfatidil colina e colesterol. Foi constatado um aumento da sensibilidade do método em cerca de uma ordem de grandeza em relaçao ao ensaio tradicional, na placa de poliestireno sem tratamento prévio. Nestas condiçoes se verificou que os MoAbs VST-1 e ST-3 detectam baixas concentraçoes de LPG como 100 ng/poço, e o MoAb VST-2, cerca de 1 ng do antígeno/poço. Estudos de caracterizaçao...(au)