In vivo electroporation restores the low effectiveness of DNA vaccination against HER-2/neu in aging.

Experimental evidence has been provided that cancer vaccines are less effective at older age than in young adults. In this study, we evaluated the possibility to recover the low effectiveness of DNA immunization against HER-2/neu increasing plasmid uptake by cells from old mice through electroporation with the aim to enhance the activation of specific immune responses. Young and old Balb/c mice received two immunizations with a pCMV-ECDTM DNA plasmid using plasmid intramuscular injection followed by electroporation (IM + E) or plasmid intramuscular injection alone (IM), and successively, they were challenged with syngeneic HER-2/neu overexpressing TUBO cells. Young mice were completely protected whereas less than 60% protection was observed in old mice after IM immunization. IM + E immunization completely protected old mice against a TUBO cell challenge. The protection was associated with increased transgene expression in the site of immunization and with the induction of both humoral and cell-mediated immunity in old mice. We conclude that the effectiveness of anticancer DNA vaccination in old ages may be improved increasing plasmid uptake and transgene expression through electroporation, suggesting the relevant role of the first steps of the immunization process in the success of cancer vaccines at older age.

In vivo effect of alpha-bisabolol, a nontoxic sesquiterpene alcohol, on the induction of spontaneous mammary tumors in HER-2/neu transgenic mice.

Breast cancer represents the most commonly diagnosed invasive malignancy in pre- and postmenopausal women in both developed and underdeveloped countries. Taking into account that treatment options, including surgery, have not been able to deal with the growing incidence of breast malignancy, it is required to develop mechanism-based novel agents for its prevention. Wide interest in some natural compounds as antiinflammatory agents and as alternative to the traditional medicines is increasing because they do not provoke any adverse effects and are effective in multiple organs, alpha-Bisabolol (BISA), a small oily sesquiterpene alcohol, was reported as chemopreventive agent in induced rat mammary carcinogenesis. The aim of the present study is to investigate the role played by two doses of BISA (via intramammary infusion) on the induction and development of mammary tumor in HER-2/neu transgenic mice as well as the BISA effect after tumor surgical resection. The main data show that (a) optimal dosage of BISA is 10 mg/mouse rather than 3.6 mg/mouse with no adverse effects (e.g., alopecia); (b) the number of the palpable tumor masses decreases in mice treated with 10 mg/mouse of BISA; (c) mice after surgical resection of the primary tumor and treatment with BISA (10 mg) are free from tumor for more weeks, after the surgical treatment; (d) using array analysis, some genes implicated in carcinogenesis mechanisms (NF-kappaBia, Map2k, Mapkl4, and HER2/ neu), angiogenesis process (Fgf), and inhibition of apoptosis (Birc5) are differently regulated after BISA treatment, with a downregulation of the HER2/neu as well as of Fgf and Birc5 genes; (e) the NK cell cytotoxicity increases in tumor-treated mice, especially after the removal of the first tumor mass. Such effectiveness could be important to achieve goals for a possible combination of BISA to conventional therapies in breast cancer and to tumor surgical removal (adjuvant therapy), as suggested for other sesquiterpene analogs.

Diet (zinc)-gene interaction related to inflammatory/immune response in ageing: possible link with frailty syndrome?

The pivotal role played by zinc-gene interaction in affecting the inflammatory response mediated by IL-6 in ageing, successful ageing (nonagenarians) and the most common age-related diseases is now recognized. Contradictory data emerging from association studies of IL-6 polymorphisms with longevity and chronic age-related diseases seem to arise from the interaction of this inflammatory pathway with dietary habits. Similar conclusions are expected to arise from association studies with the frailty syndrome. Some polymorphisms of genes related to vitamin B12 availability have been already found to be associated with frailty suggesting a possible link among diet-gene interaction and frailty in old age. Other studies in this field are urgently required because of their high potential for suggesting strategies in the care and prevention of frailty in ageing through dietary interventions, in which nutrient zinc may play a pivotal role taking into account that the high copper to zinc ratio is a significant index of the mortality in older people.

Accumulation of cells with short telomeres is associated with impaired zinc homeostasis and inflammation in old hypertensive participants.

Critical shortening of telomeres, likely associated with a considerable increase of senescent cells, can be observed in PBMC of individuals aged 80 and older. We investigated the relationship between critical telomere shortening and zinc status in healthy or hypertensive participants with or without cardiovascular disease in old and very old participants. Telomere shortening and accumulation of cells with short telomeres (percent of cells with short telomeres) in advancing age was evident in patients and healthy controls, but exacerbated in those patients aged 80 and older. Moreover, in very old patients, the accumulation of % CST may impair intracellular zinc homeostasis and metallothioneins expression, which itself is linked to an increased number of inflammatory agents, thereby suggesting the existence of a possible causal relationship between % CST and zinc homeostasis. The determination of % CST could be a more reliable means than the simple measure of telomere length as fundamental parameter in ageing to determine whether individuals are still able to respond to stress.

HER2/neu DNA vaccination for breast tumors.

Several studies of DNA vaccination against HER2/neu showed the effectiveness of immunization protocols in models of transplantable or spontaneous tumors. The DNA delivery system plays a crucial role in the success of DNA vaccination. In particular, our studies of DNA vaccination against HER2/neu tumor antigen showed that intramuscular injection of the vaccine followed by electroporation elicits an optimal protection against the development of spontaneous HER2/neu- tumors occurring in transgenic mice.

Zinc deficiency and IL-6 -174G/C polymorphism in old people from different European countries: effect of zinc supplementation. ZINCAGE study.

IL-6 SNP at position -174 is associated with age-related diseases characterized by an impaired Zn status. This polymorphism seems also relevant in regulating the expression of proteins, such as Metallothioneins (MT), involved in the modulation of Zn homeostasis. Since high IL-6 levels in elderly induce hypozinchemia, the IL-6-174 SNP may be useful to identify old subjects who are at risk for Zn deficiency. The objectives of this study are: (1) to choose old subjects who effectively need Zn supplementation and (2) to study the effect of Zn supplementation on Zn, immune and psychological status in genetically selected subjects. For this purpose, a baseline study comprising 895 healthy old subjects recruited in Central-Northern and Southern European Countries was carried out by evaluating their dietary intake, psychological and immune parameters as well as their Zn status. A Zn supplementation trial was performed in 110 old subjects selected on the basis of their plasma Zn levels and IL-6 SNP. After correcting for age and Zn intake, C- carriers displayed higher MT and lower levels of several parameters related to zinc status (plasma Zn, erythrocyte Zn and NO-induced release of Zn in PBMC) than C+ carriers. Better NK cell cytotoxicity and psychological functions (PSS, MMSE) were also found in C+ than C- carriers strictly related to the zinc status. However, independently by the polymorphism, all subjects with plasma zinc < or = 10.5microM showed the worst immune response and psychological functions. Supplementation was carried out in C+ and C- carriers with stable low plasma zinc levels ( < or =10.5microM at baseline and at 1 year follow-up) and in C- carriers with unstable plasma zinc (< or =10.5microM at baseline and >10.5microM at 1 year follow-up). C+ carriers with plasma zinc >10.5microM were not supplemented because showing the best immune and psychological conditions. After 48+/-2 days of supplementation with 10mg/day of Zn-aspartate, the NO-induced release of Zn, erythrocyte Zn and NK cell cytotoxicity increased in all groups selected for supplementation, including C- with unstable plasma zinc. In conclusion, the sole assessment of plasma Zn level is not reliable to exclude C- carriers from Zn supplementation. A possible explanation for the conflicting data on the identification of IL-6-174G as a "risk allele" based on different dietary intake in the studied population is also suggested.

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.

Evaluation of different plasmid DNA delivery systems for immunization against HER2/neu in a transgenic murine model of mammary carcinoma.

Studies of DNA vaccination against HER2/neu showed the effectiveness of immunization protocols in models of transplantable or spontaneous tumors; scarce information, however, has been provided to identify the procedure of DNA administration that more effectively contributes to the activation of immune system against spontaneously arising HER2/neu-positive tumors. We compared the effectiveness of different procedures of DNA vaccine delivery (intradermic injection (ID), gene gun (GG) delivery and intramuscular injection (IM) alone or with electroporation) in a murine transgenic model of mammary carcinoma overexpressing HER2/neu. We highlighted the role of DNA delivery system in the success of DNA vaccination showing that, among the analysed methods, intramuscular injection of the vaccine, particularly when associated to electroporation, elicits a better protection against HER2/neu spontaneous tumor development inducing antibody and cell-mediated immune responsiveness against HER2/neu and a Th1 polarization of the immune response.