Sensory block in Day Surgery.

BACKGROUND: The aim of our research is to evaluate the effectiveness of the spinal anesthesia versus Local Anesthesia within the context of Day Surgery. MATERIALS AND METHODS: This study is a clinical trial. 140 patients were enrolled (60 female, 80 male). Some parameters have been evaluated with scales ASA, Bromage Scale, Hollmen Scale, Numerical Rate Scale, and Patient Satisfaction that are now internationally recognized as valid to assess both the degree of anesthesia and the patient benefit. RESULTS: Data is mostly matching between the two groups, even though there are some differences. Every patient from the group SUB underwent a single sensory block due to the fact that everyone had a Bromage Score under 2 and Hollmen score between 2 and 3 CONCLUSIONS: Results showed the versatility of the SUB blockage in a Acute postoperative pain is associated to the surgical treatment and is still today unavoidable. We always tried to keep in check or even avoid the pain, thinking it is useless and ethically inacceptable. It is considered dangerous since it starts neurovegetative and neuroendocrinal cascade which lead to delayed functional and psychophysical recoverywider population, compared to the LAs, the better surgical planning, but mostly a better analgesia over the following 24 hours and a better satisfaction. Despite all these findings the patients continue to prefer the local anesthesia. KEY WORDS: Analgesia, Epidural, Post-operative, Ultrasound.

The genetic stability of a conditional live HIV-1 variant can be improved by mutations in the Tet-On regulatory system that restrain evolution.

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because more quickly replicating pathogenic virus variants may evolve after vaccination. As an alternative vaccine approach, we have previously presented a doxycycline (dox)-dependent HIV-1 variant that was constructed by incorporating the tetracycline-inducible gene expression system (Tet-On system) into the viral genome. Replication of this HIV-rtTA variant is driven by the dox-inducible transcriptional activator rtTA and can be switched on and off at will. A large scale evolution study was performed to test the genetic stability of this conditional live vaccine candidate. In several long term cultures, we selected for HIV-rtTA variants that no longer required dox for replication. These evolved variants acquired a typical amino acid substitution either at position 19 or 37 in the rtTA protein. Both mutations caused rtTA activity and viral replication in the absence of dox. We designed a novel rtTA variant with a higher genetic barrier toward these undesired evolutionary routes. The corresponding HIV-rtTA variant did not lose dox control in long term cultures, demonstrating its improved genetic stability.

Quantification of residual host cell DNA in adenoviral vectors produced on PER.C6 cells.

Recombinant adenoviral vectors for gene therapy and vaccination are routinely prepared on cultures of immortalized cells, allowing the production of vector batches of high titer and consistent quality. Quantification of residual DNA from the producing cell line is part of the purity tests for clinical lots. Stringent guidelines stipulate the maximum acceptable level of DNA per dose of vector, and this quantification is therefore a crucial piece of information for researchers and manufacturers alike. In this paper we describe an optimized assay based on real-time polymerase chain reaction (PCR) for the quantification of residual PER.C6 DNA in recombinant adenoviral vectors. In order to reduce the risk of introducing contaminations and to increase the throughput, the assay was designed to require minimum sample handling. Furthermore, DNA extraction from the samples is not necessary, thereby eliminating the need to account for possible sample losses. We also report the results of the assay qualification, demonstrating that the assay is accurate, precise, and sensitive. Finally, we applied the assay successfully to determine the level of host cell DNA in an adenovirus vector produced on PER.C6 cells throughout a standard purification process. Because of its specifications, we anticipate that the assay can have broad applicability to biologics other than adenoviral vectors produced on PER.C6 cells.

Common structure of rare replication-deficient E1-positive particles in adenoviral vector batches.

The use of the PER.C6 adenovirus packaging cell line in combination with a designated vector plasmid system, whereby the cell line and vector with E1 deleted have no sequence overlap, eliminates the generation of replication-competent adenovirus during vector production. However, we have found cytopathic effect (CPE)-inducing particles in 2 out of more than 40 large-scale manufacturing lots produced in PER.C6 cells. The CPE inducer was detected at a frequency of 1 event in 7.5 x 10(12) vector particles. Despite amplification, it was not readily purified, indicating that the agent itself is replication deficient and requires the parental recombinant adenovirus serotype 5 (rAd5) vector for replication and packaging. Therefore, we designated the agent as a helper-dependent E1-positive region containing viral particle (HDEP). Here, we report the molecular structure of the HDEP genome, revealing an Ad comprised of E1 sequences derived from PER.C6 cells flanked by inverted terminal repeat, packaging signal, and transgene sequences. These sequences form a palindromic structure devoid of E2, E3, E4, and late genes. Since only 5 bp were shared between E1 sequences in the PER.C6 genome and viral vector sequences, the data strongly suggested that insertion of genomic DNA into an adenoviral genome had occurred essentially via nonhomologous recombination. HDEPs have been found in unrelated virus batches and appear to share a common structure that may explain their mechanism of generation. This finding allowed development of an HDEP assay to screen batches of rAd5 produced on the PER.C6 cell line and resulted in detection of seven HDEP agents from four different transgene-virus vector constructs in separate batches of Ad.

Viral evolution as a tool to improve the tetracycline-regulated gene expression system.

We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.

Conditional virus replication as an approach to a safe live attenuated human immunodeficiency virus vaccine.

Despite intensive efforts, no safe and effective vaccine has been developed for the prophylaxis of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Studies with the simian immunodeficiency virus (SIV)/macaque model demonstrated that live attenuated viruses are the most effective vaccines tested thus far. However, due to ongoing low-level replication of the attenuated virus and the error-prone replication machinery, the attenuated virus may regain replication capacity and become pathogenic. We therefore designed a novel vaccine strategy with an HIV-1 virus that replicates exclusively in the presence of the nontoxic effector doxycycline (dox). This was achieved by replacement of the viral TAR-Tat system for transcriptional activation by the Escherichia coli-derived Tet system for inducible gene expression. This designer HIV-rtTA virus replicates in a strictly dox-dependent manner and may represent an improved vaccine strain because its replication can be turned on and off at will. Spontaneous virus evolution resulted in optimization of the components of the Tet system for their new function to support virus replication in human cells. The optimised Tet system may be of particular use in other applications such as inducible expression of gene therapy vectors in the brain.

Efficient human immunodeficiency virus replication requires a fine-tuned level of transcription.

Transcription represents a crucial step in the life cycle of human immunodeficiency virus (HIV) and is highly regulated. Here we show that the strength of the viral long terminal repeat (LTR) promoter is optimized for efficient replication. Artificially increasing the rate of LTR-driven transcription was strongly detrimental for viral fitness, and HIV was able to regain replication capacity by selecting for variants with a weaker LTR. Strikingly, the strength of the evolved promoter was equivalent to that of the wild-type LTR.