Expression and Characterization of Human Vascular Endothelial Growth Factor Produced in SiHa Cells Transduced with Adenoviral Vector.

The vascular endothelial growth factor (VEGF) is an essential factor to pathologic angiogenesis. Disruption of VEGF/VEGF receptor interaction in cancer patients inhibits the development of new and pre-existing tumor blood vessels. Consequently, VEGF becomes an important therapeutic target for handling solid tumors. In this work, human VEGF was produced in the culture supernatant of SiHa cells transduced with a replication-defective adenoviral vector (pAdhVEGF121) encoding this molecule. The 35 kDa VEGF121 homodimer was obtained from clarified culture media as a glycosylated protein. VEGF121 expression levels were strictly dependent on the adenoviral viral load used. VEGF121 was produced with purity over 98% after a single step chromatography by immobilized metal affinity chromatography. Additionally, VEGF121 binds Bevacizumab antibody with a KD of 7 nM. Biological characterization by mitogenic assay in HUVEC and ECV-304 cells showed that VEGF121 stimulates cell proliferation in a dose-dependent manner in both cells. Finally, the neovascularization activity of VEGF121 was demonstrated by vascular permeability assays in matrigel plug-bearing mice, showing significantly increased vasculature leakage after treatment with VEGF121. Consequently, transduction of SiHa cells with adenovirus is a suitable alternative for manufacture heterologous proteins of therapeutic interest.

LOX-1 activation by oxLDL triggers an epithelial mesenchymal transition and promotes tumorigenic potential in prostate cancer cells.

Obesity is related to an increased risk of developing prostate cancer with high malignancy stages or metastasis. Recent results demonstrated that LOX-1, a receptor associated with obesity and atherosclerosis, is overexpressed in advanced and metastatic prostate cancer. Furthermore, high levels of oxLDL, the main ligand for LOX-1, have been found in patients with advanced prostate cancer. However, the role of LOX-1 in prostate cancer has not been unraveled completely yet. Here, we show that LOX-1 is overexpressed in prostate cancer cells and its activation by oxLDL promotes an epithelial to mesenchymal transition, through of lowered expression of epithelial markers (E-cadherin and plakoglobin) and an increased expression of mesenchymal markers (vimentin, N-cadherin, snail, slug, MMP-2 and MMP-9). Consequently, LOX-1 activation by oxLDL promotes actin cytoskeleton restructuration and MMP-2 and MMP-9 activity inducing prostate cancer cell invasion and migration. Additionally, LOX-1 increased the tumorigenic potential of prostate cancer cells and its expression was necessary for tumor growth in nude mice. In conclusion, our results suggest that oxLDL/LOX-1 could be ones of mechanisms that explain why obese patients with prostate cancer have an accelerated tumor progression and a greater probability of developing metastasis.

Analysis of hepatitis C virus core encoding sequences in chronically infected patients reveals mutability, predominance, genetic history and potential impact on therapy of Cuban genotype 1b isolates.

BACKGROUND AND OBJECTIVES: Hepatitis C virus (HCV) genotypes are relevant to epidemiological questions, vaccine development, and clinical management of chronic HCV infection. In the present work, we aimed at investigating HCV genotype, variability and genetic history of HCV isolates in Cuba from a sample of chronically infected patients. MATERIAL AND METHODS: A prospective study, involving 73 Cuban anti-HCV positive patients, was carried out. RT-PCR and phylogenetic analysis was employed to determine HCV genotypes. Divergence dates and demographic parameters in a Bayesian coalescent framework were estimated, as implemented in BEAST v1.4.8. RESULTS: HCV RNA was undetectable in 15 patients that received antiviral therapy. All HCV RNA positive patients, 58, were infected with genotype 1, three of them with subtype 1a and 55 with subtype 1b. The analysis of the DNA sequence coding for a core fragment, spanning nt positions 435-816 (relative to strain H77), revealed high percent (96.7% +/- 0.8%) nucleotide identity within Cuban HCV subtype 1b sequences. However, 56.7% and 20% of 30 analyzed individuals had changes in the core region in a six-month interval, at the nucleotide and amino acid level, respectively. Mutations involving aa changes were mainly found in the region encompassed between aa 70 and 106 of the core protein, with only one isolate showing a point mutation at position 43. Interestingly, some of the observed changes seem to be reversions and might in fact contribute to reducing the variability of this region. The estimated date for the most recent common ancestor of HCV genotype 1b Cuban isolates is 1969 (CI, 1953 to 1977). DISCUSSION: Analysis of HCV core encoding sequences from chronic patients reveals mutability of genotype 1b isolates in Cuba, which seem to be predominant and rapidly multiplied during the eighty decade of last century, and might limit the benefits obtained from current antiviral therapy.

Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial.

SUMMARY: Hepatitis C virus (HCV) is a worldwide health problem. No vaccine is available against this pathogen and therapeutic treatments currently in use are of limited efficacy. In the present study, the immunogenicity of the therapeutic vaccine candidate CIGB-230, based on the mixture of pIDKE2, a plasmid expressing HCV structural antigens, with a recombinant HCV core protein, Co.120, was evaluated. CIGB-230 was administered by intramuscular injection on weeks 0, 4, 8, 12, 16 and 20 to 15 HCV-chronically infected individuals, non-responders to previous treatment with interferon (IFN) plus ribavirin. Interestingly, following the final immunization, neutralizing antibody responses against heterologous viral pseudoparticles were modified in eight individuals, including six de novo responders. In addition, 73% of vaccinees exhibited specific T cell proliferative response and T cell IFN-gamma secretory response 24 weeks after primary immunization with CIGB-230. Furthermore, 33.3% of individuals developed de novo cellular immune response against HCV core and the number of patients (46.7% at the end of treatment) with cellular immune response against more than one HCV structural antigen increased during vaccination (P = 0.046). In addition, despite persistent detection of HCV RNA, more than 40% percent of vaccinated individuals improved or stabilized liver histology, particularly reducing fibrosis, which correlated with cellular immune response against more than one HCV antigen (P = 0.0053). In conclusion, CIGB-230 is a promising candidate for effective therapeutic interventions based on its ability for enhancing the immune response in HCV chronically infected individuals.