Plasma PCSK9 levels and sepsis severity: an early assessment in the emergency department

Clinical and Experimental Medicine - The aim of the study is to evaluate the prognostic value of early PCSK9 levels in non-intubated septic patients admitted to the emergency department. This...     

Concomitant effect of topical ubiquinone Q10 and vitamin E to prevent keratocyte apoptosis after excimer laser photoablation in rabbits

PURPOSE: To investigate in vivo whether ubiquinone Q10 together with vitamin E protects rabbit corneas from keratocyte apoptosis after excimer laser irradiation. METHODS: Photorefractive keratectomy (PRK) was performed in both eyes of three New Zealand white rabbits. During 3 days before surgery, each right eye received four-times-daily instillation of an eye-drop solution containing ubiquinone Q10 0.20% and vitamin E 0.04%; each left eye was treated with a solution that did not contain ubiquinone or vitamin E. The central cornea was analyzed after surgery using the in situ end labelling (ISEL) technique of nicked DNA to detect DNA fragmentation. To determine the number of ISEL positive nuclei, an average of 70 random microscopic fields (five for each de-epithelialized tissue section) of 138,000 mu2 were examined in the right and left cornea samples at 250X by two different observers. RESULTS: Light microscopic examination of the sections from corneas treated before PRK showed that cells committed to apoptosis by PRK were about 50% compared to those of untreated controls. CONCLUSION: Treatment of rabbit eyes before PRK with ubiquinone Q10 lowered the number of apoptotic events.     

Antisense oligonucleotide drug design

Maneuvering single gene expression is not only an optimal way to study gene function but also an ambitious goal, which will lead to the treatment of a variety of human diseases whose main pathogenetic event is a genetic alteration. The recent efforts focusing on the genome project have led to array based, high throughput, gene expression analysis techniques that allow the study of complex molecular networks. Combining these powerful new technologies with modulation of gene expressions is making it possible to unravel complex molecular networks or, vice versa, to find new gene products responsible for pathological conditions on which exogenous modulation can be productive. Efficient and specific modulation of gene expression can be obtained either by producing transgenic or gene knockout organisms or cells (gene targeting), or by treating organisms or cells with short synthetic nucleic acid segments in antisense orientation with respect to the targeted mRNAs (mRNA targeting by antisense strategy). While genome manipulation is a time consuming and expensive approach, requiring invasive intervention, the "antisense strategy" is characterized by high flexibility resulting from safeness, specificity, reversibility, modulability, and low cost. The rationale of the antisense strategy is that, once one gene sequence is known, its expression can be silenced by application of synthetic single-strand nucleic acid segments (oligonucleotides) whose sequence is in antisense orientation compared to the targeted mRNA. Recently, this "informational" strategy has been boosted by the discovery of the RNA interference: a natural mechanism by which cells are thought to fight detrimental exogenous viruses and endogenous transposons. Despite promising futures, antisense-based therapeutics are far from being an established reality. This review analyses the recent improvements in antisense-based gene expression modulation, focuses on the treatment of diseases in the light of the past, and provides our personal findings on this topic.     

Development of new drugs in angiogenesis

Angiogenesis, the growth of new capillaries from pre-existing vessels, contributes to the development and progression of a variety of physio-pathological conditions. There is growing evidence that anti-angiogenic drugs will improve future therapies of diseases like cancer, rheumatoid arthritis and ocular neovascularisation. Conversely, therapeutic angiogenesis is an important homeostatic response contributing to limit the damage to ischemic tissues. Molecular processes involved in angiogenesis include stimulation of endothelial growth by cytokine production (i.e. vascular endothelial growth factor, VEGF; fibroblast growth factor-2, FGF-2), degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs), and migration of endothelial cells mediated by integrins (cell membrane adhesion molecules). Drugs targeting pathologic angiogenesis have been designed to interfere with any of these steps and are currently undergoing evaluation in early clinical studies. Important therapeutic strategies are: suppression of activity and signaling pathways activated by the major angiogenic regulators like VEGF and FGF-2; inhibition of function of alphav-integrins and MMPs; exploitation of endogenous anti-angiogenic molecules like angiostatin and endostatin. The strategy to "silence" endothelium with antiangiogenic drugs to starve tumors, provides a novel approach for cancer treatment. The unique targets of these drugs (endothelium) make them distinct from traditional cytotoxic chemotherapeutic agents. Conversely, gene transfer of angiogenesis inducers is the new approach for therapeutic neovascularization, which is under investigation using a variety of growth factors and a wide array of potential delivery systems, including the application of the gene as naked DNA or by viral vector. The status of pro- and anti-angiogenic therapies is here presented and discussed.     

Low effectiveness of DNA vaccination against HER-2/neu in ageing

We evaluated the effectiveness of vaccination with a HER-2/neu DNA plasmid to induce protective immunity against HER-2/neu overexpressing syngeneic TUBO tumour cells in old ages. Young and old Balb/c mice received three immunizations with a pCMVneuNT DNA plasmid and, successively, were challenged with TUBO cells. Young mice were completely protected whereas less than 60% protection was observed in old mice. Anti-p185(neu) antibodies were found in the sera from both young and old immunized mice, even if antibody production was significantly higher in young in comparison with old mice. Similarly, higher anti-p185(neu) lymphocyte proliferation was induced in young than in old mice. No anti-p185(neu) cytotoxicity was found in lymphocytes from old animals. We conclude that anticancer DNA vaccination has a lower effectiveness in old than in young ages.     

Downregulation of bcl-2 expression in lymphoma cells by bcl-2 ARE-targeted modified,synthetic ribozyme

Synthetic ribozymes are catalytic RNA molecules designed to inhibit gene expression by cleaving specific mRNA sequences. We investigated the potential of synthetic ribozymes to inhibit bcl-2 expression in apoptosis defective bcl-2 overexpressing tumors. A chemically stabilized hammerhead ribozyme has been targeted to the A+U-rich regulative element of bcl-2 mRNA that is involved in bcl-2 gene switch-off during apoptosis. The design of the ribozyme was based on the results of probing accessibility of the RNA target in cellular extracts with antisense DNA. The ribozyme was lipotransfected to a bcl-2 overexpressing human lymphoma cell line (Raji). The cellular uptake of this ribozyme resulted in a marked reduction of both bcl-2 mRNA and BCL-2 protein levels and dramatically increased cellular death by apoptosis. Our results suggest a potential therapeutic application of such ribozyme for the treatment of bcl-2 overexpressing tumors.     

Genetic regulation of alpha-1-antitrypsin levels in the neonatal period: The influence of the PiS allele and the PiM subtypes

Five groups of full-term newborn infants divided on the basis of the subtypes of M and one group of full-term newborn infants belonging to the phenotype M1S have been studied.A decrease in alpha-1-antitrypsin level in the M1S newborn infants was observed (P<0.001). A similar decrease was observed in a group of M1S preterm newborn infants in comparison with M1M1 and M2M2 newborn infants of the same gestational age.Moreover, full-term infants belonging to the subtype M1M2 also showed a decrease in the level of this inhibitor in comparison to the full-term infants belonging to other subtypes of M (P<0.01). Preterm newborn infants belonging to subtypes M1M2 and M1M1 showed identical circulating alpha-1-antitrypsin levels.When the phenotype is taken into account, data obtained from newborn infants free from perinatal pathology show that alpha-1-antitrypsin levels—but not alpha-2-macroglobulin levels—increase with fetal age.Moreover the specific compensatory response to the deficiency, consisting in an increase in alpha-2-macroglobulin levels, is absent in M1S and M1M2 neonates.The wide distribution of the M1M2 subtypes and, in some regions of the MS phenotype, suggests a need for genetic typing of the alpha-1-antitrypsin molecule in investigating the behaviour of this inhibitor in the perinatal period.Supported in part by a grant from Consiglio Nazionale delle Ricerche     

Effect of the silybin-phosphatidylcholine complex (IdB 1016) on the development of mammary tumors in HER-2/neu transgenic mice

Silybin, a main component of the milk thistle of Silybum marianum, has been reported to possess anticancer activity. We investigated the effects of IdB 1016, a complex of silybin with phosphatidylcholine, on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice. The mechanisms involved in the antitumor effect of IdB 1016 were evaluated by studying the apoptosis, senescent-like growth arrest, intratumoral leukocyte infiltrate, and the expression of HER-2/neu and p53 in tumoral mammary glands from transgenic mice and in human breast SKBR3 tumor cells. The administration of IdB 1016 delayed the development of spontaneous mammary tumors, reduced the number and size of mammary tumor masses, and diminished lung metastasization in HER-2/neu transgenic mice. In tumoral mammary glands from IdB 1016-treated mice, a down-regulation of HER-2/neu gene expression was associated with an increased senescent-like growth arrest of tumor cells, and an increased infiltrate of neutrophils, CD4, and CD8 T cells. Both senescent-like growth arrest and apoptosis were significantly increased and were associated with a reduced p185(HER-2/neu) protein and an increased p53 mRNA in SKBR3 in vitro treated with IdB 1016 in comparison with control cells. The results show the antitumor effect of IdB 1016 in the development of spontaneous mammary tumors in HER-2/neu transgenic mice. The effect of IdB 1016 might be related to the down-regulation of HER-2/neu expression and the induction of senescent-like growth arrest and apoptosis through a p53-mediated pathway in tumor cells.