Strategies to optimize capsid protein expression and single-stranded DNA formation of adeno-associated virus in Saccharomyces cerevisiae.

AIMS: Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single-stranded DNA (ssDNA) formation in Saccharomyces cerevisiae. METHODS AND RESULTS: Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation. CONCLUSIONS: This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation. SIGNIFICANCE AND IMPACT OF THE STUDY: New manufacturing strategies for AAV-based gene therapy vectors (rAAV) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production.

Altered systemic serologic parameters in patients with silicone mammary implants.

BACKGROUND: The most common local complication in patients with silicone mammary implants (SMIs) is excessive peri-SMI connective tissue capsule formation and its subsequent contracture. However, considerable controversy remains as to whether these implants also cause systemic side effects. The present study was undertaken to identify possible alterations of serological markers in SMI patients that may herald systemic side effects. METHODS: We investigated several systemic serological parameters in 143 individuals, 93 of whom had received SMIs and 50 were controls. The patients were grouped according to the severity of capsular contracture (Baker scores I-IV) and the duration of SMI implants (less than 1 year, between 1 and 5 years, more than 5 years). We also included control groups (female blood donors, nurses with possible professional silicone exposure). Patients with breast cancer and subsequent SMI-reconstruction were excluded from the study since they are generally considered immunocompromised. The following parameters were determined: anti-neutrophil cytoplasmatic autoantibodies (cANCA), anti-nuclear autoantibodies (ANA), anti-cardiolipin antibodies (CL-Ab), rheumatoid factor (RF), complement components (C3, C4), circulating immune complexes (CIC), procollagen III (a marker of active fibrosis), anti-polymer antibodies (APA) and soluble intercellular adhesion molecule-1 (sICAM-1). RESULTS: The following parameters were increased in the sera of SMI patients: CIC, procollagen III, APA, sICAM-1. CONCLUSIONS: We found a set of parameters in serum that correlate with fibrosis development and the duration of the implants in otherwise healthy SMI carriers. Future studies will clarify whether these serological abnormalities will be useful in predicting clinical disease, and also further assess the sensitivity and specificity of these parameters. Our present recommendation as a result of this study is that SMI patients with persistent abnormal serological parameters should be monitored closely by a clinical team that includes rheumatologists.

Spontaneous unilateral autoinflation of a saline-filled mammary implant.

We present the case of a woman with a massive volume increase in her right breast 12 years after breast augmentation with saline-filled silicone mammary implants (SMI). Tenderness of and pressure pain in the enlarged right breast were noted on physical examination. Intraoperatively, the right implant was seen to be markedly enlarged, altered in colour and filled with a brownish fluid as compared to the other side. No macroscopic damage, including to the valve of the enlarged SMI, was noticed. The liquid in the inflated SMI was subjected to biochemical analysis. Although neither cells nor nucleic acids were detected, 4 mg/ml protein was found in the liquid of the autoinflated SMI. On SDS-PAGE separation, these proteins resolved in a pattern similar to that of serum proteins. This observation was corroborated by Western blots for several serum proteins. Surprisingly, proteins in the SMI liquid were significantly more glycosylated and oxidised than were serum proteins; this finding indicates a process of protein ageing. We hypothesise that the reason for this in vivo expansion was a defective valve and not colloid osmotic swelling, as previously suggested.

Evaluation of the stimulatory effect of Epimedium alpinum L. methanolic extract on the immune response in vivo.

The effect of the methanolic extract of the underground parts of Epimedium alpinum L. (MEEA) on the immune response to Keyhole Limpet Hemocyanine (KLH) or alloantigens in vivo was studied in AO rats. Immunization of experimental animals with KLH or allogeneic lymphocytes together with MEEA was followed by an increase in cellularity of draining lymph nodes (LN) and enhanced proliferation of LN lymphocytes after their restimulation with specific antigens in vitro, compared to control rats immunized without MEEA. These effects correlated with an increase in relative values of B, MHC class II+, CD25+ and CD71+ cells, whereas percentages of T cells and both subsets of T cells (CD4+ and CD8+) were not significantly altered. As a consequence of higher LN cellularity, total numbers of all cell subsets in the MEEA-treated group of rats were significantly increased, compared to the corresponding control. The addition of MEEA together with KLH in vitro to LN lymphocytes of rats immunized with KLH or KLH and MEEA in vivo was manifested by significant increase (0.1 microg/ml of MEEA) and decrease (50 microg/ml and 100 microg/ml of MEEA) of cell proliferation, respectively. However, when LN lymphocytes from rats, immunized in vivo with KLH and MEEA, were stimulated in vitro with MEEA together with an anti-alphabeta T cell receptor monoclonal antibody (R73), their proliferation was siginificantly inhibited. Taken together, obtained results suggest that MEEA possesses immunostimulatory activity in vivo, but some components from the extract exert immunosuppressive effects in vitro on previously in vivo activated T cells.