Adenovirus (Ad) and adeno-associated virus (AAV) have attractive and complementary properties that can be exploited for gene transfer purposes. Ad vectors are probably the most efficient vehicles to deliver foreign genes both in vitro and in vivo . AAV exhibits the unique ability to establish latency by efficiently integrating at a specific locus of human chromosome 19 (AAVS1). Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs). In this study, we report the development of two helper-dependent adenoviral (HD) vectors, one carrying the Rep78 gene, the other an AAV-ITR-flanked transgene. Although Rep proteins have been demonstrated to interfere with Ad replication, HD Rep78 vector was successfully amplified on serial passages in 293CRE4 cells with a yield of 50–100 transducing units per cell. DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs. The high transduction efficiency, large cloning capacity, and high titer of the HD, combined with the site-specific integration machinery provided by AAV-derived components, make the Ad/AAV hybrid viruses a promising vehicle for gene therapy.

In experimental and clinical pain studies, the sex of subjects was rarely taken into account, even if nociceptive inputs appear to be processed and modulated by partially distinct neural mechanisms in each sex. In this study we analysed, in male and female mice, behavioural and neuronal responses in developing, maintaining, and recovering from neuropathic pain. Experiments were carried out in adult CD1 mice by using Chronic Constriction Injury (CCI) as neuropathic pain model. We investigated the temporal trend of mechanical nociceptive threshold together with functional recovery of the injured paw, and the immunofluorescence staining of proteins associated with nerve injury and repair and with spinal gliosis, 7 and 121days after CCI. A proteomic analysis on proteins extracted from sciatic nerves was also performed. Male mice showed a gradual decrease of CCI-induced allodynia, the complete recovery occurring 81days after the sciatic nerve ligation. On the contrary, in female mice, allodynia was still present 121days after CCI. Sex-dependent differences also resulted from immunofluorescence experiments: in sciatic nerve, the expression of P0 and Neu200 is greater in neuropathic males than in neuropathic females, suggesting faster nerve regeneration. Proteomic analysis confirmed sex-related differences of proteins associated with nerve regenerative processes. In addition, the reactive gliosis induced by CCI at day 7, as revealed by colocalization of glial fibrillary acidic protein (astrocytes) and CD11b (microglia) with phosphorylated p38, disappeared 121 days after CCI in male but not in female mice. These results may have important therapeutic implications for the treatment of neuropathic pain.

Axonal degeneration in peripheral nerves after injury is accompanied by myelin degradation initiated by Schwann cells (SCs). These cells activate autophagy, a ubiquitous cytoprotective process essential for degradation and recycling of cellular constituents. Concomitantly to nerve insult and axonal degeneration, neuropathic pain (NeP) arises. The role of SC autophagy in the mechanisms underlying NeP is still unknown. In this study, we examined the role of the autophagy during the early phase of Wallerian degeneration in NeP induction and chronification by using a murine model of peripheral nerve lesion (chronic constriction injury). We demonstrate that the autophagy inducer rapamycin, administered in the first week after nerve damage, induces long-lasting analgesic and antiinflammatory effects, facilitates nerve regeneration, and prevents pain chronification. Conversely, when autophagy is altered, by means of autophagic inhibitor 3-methyladenine administration or as occurs in activating molecule in Beclin-1-regulated autophagy transgenic mice (Ambra1(+/gt)), NeP is dramatically enhanced and prolonged. Immunohistochemical and ultrastructural evaluations show that rapamycin is able to increase autophagic flux in SCs, to accelerate myelin compaction, and to reduce inflammatory and immune reaction. Proteomic analysis combined with bioinformatic analysis suggests that a redox-sensitive mechanism could be responsible for SC autophagy activation. These data suggest that a deficiency of autophagic activity in SCs can be an early event in the origin of NeP chronification and that autophagy modulation may represent a powerful pharmacological approach to prevent the onset and chronification of NeP in the clinical setting.

Baculovirus vectors are efficient tools for gene transfer into mammalian cells in vitro. However, in vivo gene delivery by systemic administration is hindered by the vector inactivation mediated by the complement system. To characterize further the gene transfer efficacy of baculovirus we examined the vector transduction efficiency in skeletal muscle. Vectors expressing vesicular stomatitis virus glycoprotein (VSV-G) in the viral envelope were generated by inserting the VSV-G coding sequence downstream of the polyhedrin promoter. Two viruses were constructed to carry either the Escherichia coli beta-galactosidase (beta-Gal) gene or the mouse erythropoietin (EPO) cDNA cloned downstream of the cytomegalovirus immediate-early promoter and enhancer. The greater gene transduction efficiency of the Bac-G-betaGal vector was confirmed by comparing the beta-Gal expression level in a variety of human and mouse cell lines with that obtained on infection with Bac-betaGal, a vector that lacks VSV-G. Similarly, a 5- to 10-fold increase in beta-Gal expression between Bac-G-betaGal and Bac-betaGal was observed when mouse myoblasts and myotubes were infected. The same increase in beta-Gal expression was detected on injection of the Bac-G-betaGal vector in the quadriceps of BALB/c and C57BL/6 mice. In contrast, a 2-fold difference in transduction was observed between these two vectors in DBA/2J mouse strain. Last, expression of EPO cDNA was detected for at least 178 days in DBA/2J mice on Bac-G-EPO injection into the quadriceps whereas EPO expression declined to normal values by 35 days postinfection in BALB/c and C57BL/6 mice. Thus, these results indicate that baculovirus may be considered a useful vector for gene transfer in mouse skeletal muscle and that persistence of expression may depend on the mouse strain used.

Sex differences play a role in pain sensitivity, efficacy of analgesic drugs and prevalence of neuropathic pain, even if the underlying mechanisms are far from being understood. We demonstrate that male and female mice react differently to structural and functional changes induced by sciatic nerve ligature, used as model of neuropathic pain. Male mice show a gradual decrease of allodynia and a complete recovery while, in females, allodynia and gliosis are still present four months after neuropathy induction. Administration of 17β-estradiol is able to significantly attenuate this difference, reducing allodynia and inducing a complete recovery also in female mice. Parallel to pain attenuation, 17β-estradiol treated-mice show a functional improvement of the injured limb, a faster regenerative process of the peripheral nerve and a decreased neuropathy-induced gliosis. These results indicate beneficial effects of 17β-estradiol on neuropathic pain and neuronal regeneration and focuses on the importance of considering gonadal hormones also in clinical studies.