Maria Michela Muscettola

The lymphatic route. 1) Albumin and hyaluronidase modify the normal distribution of interferon in lymph and plasma.

When human recombinant interferon-α2 diluted in saline was injected s.c. into rabbits, the total amount recovered in thoracic lymph was less than 0.4%. Recoveries increased from 2- to 8-fold if interferon was injected in 4% albumin or with hyaluronidase, respectively. Albumin added to interferon acts as an interstitial fluid expander, thus favoring interferon absorption through lymphatics rather than blood capillaries. This strategy may increase the therapeutic index of interferon.

Phosphorylation-independent mTORC1 inhibition by the autophagy inducer Rottlerin

We recently found that Rottlerin not only inhibits proliferation but also causes Bcl-2- and Beclin 1-independent autophagic death in apoptosis-resistant breast adenocarcinoma MCF-7 cells. Having excluded a role for canonical signaling pathways, the current study was aimed to investigate the contribution of the AMPK/mTOR axis in autophagy induction and to search for the upstream signaling molecules potentially targeted by Rottlerin. Using several enzyme inhibitors, Western blotting analysis, mTOR siRNA and pull down assay, we demonstrate that the Rottlerin-triggered autophagy is mediated by inhibition of mTORC1 activity through a novel AMPK and mTORC1 phosphorylation-independent mechanism, likely mediated by the direct interaction between Rottlerin and mTOR.

High Doses of Ascorbate Kill Y79 Retinoblastoma Cells In vitro

Objectives: To tests the sensitivity of Y79 retinoblastoma cell lines to high doses of ascorbate, in vitro, and compare its effects with those of some chemotherapeutic agents routinely employed in the treatment of retinoblastoma.Methods: Y79 retinoblastoma cells have been exposed to increasing doses of either sodium ascorbate (SA) or Melphalan (MEL), to define a dose-response curve around the peak plasma concentrations reached by both chemicals when administered according to the existing therapeutic procedures and protocols. The assessment of cell number and viability was performed, before and after exposure, with both the manual (Trypan Blue Exclusion Test) and automated (flow cytometry) methods. Fluorescence microscopy and direct observation of cells in culture, with inverted microscope, were also performed.Results: Y79 cells are highly sensitive to the cytotoxic effect of SA, with cell viability reduced of over 90% in some experiments. As reported in the literature, this effect is directly cytotoxic and most probably mediated by acute oxidative stress on different cellular components. The same does not apply to Melphalan which, at the doses commonly used for therapeutic purposes, did not show any significant effect on cell viability, in vitro.Conclusion: To our knowledge, this is the first report showing that high doses of SA can actively kill retinoblastoma cells in vitro. While it is not surprising for SA, to show direct cytotoxic effect on tumor cells, the data reported herein represent the first evidence in favor of the possible clinical use of high doses of intravenous SA, to treat children affected by retinoblastoma. Given the many advantages of SA over the chemotherapeutic agents commonly employed to treat cancer (including its almost total absence of toxic or side effects, and its exclusive specificity for cancer cells), it is reasonable to assume, from the data reported herein, that the high doses of intravenous ascorbate, have the potential to represent a real revolution in the treatment of retinoblastoma.

Effect of 1,25-dihydroxyvitamin D3 on interferon gamma production in vitro

In recent years, receptors for calcitriol (the active form of vitamin D3) have been identified in monocytes and activated, but not resting, human B and T lymphocytes suggesting that it may be involved in immune regulation. Because lymphokines are central in the regulation and modulation of immune or inflammatory responses and since the calcium translocation is involved in the mitogen-induced activation of lymphocytes, we thought it interesting to study the role of calcitriol on interferon gamma (IFN-gamma) production in vitro. In this study, we report that calcitriol inhibits the IFN-gamma production by staphylococcal enterotoxin A-stimulated peripheral blood mononuclear cells (PBMC) in a dose-dependent fashion. The inhibitory effect was less potent in calcium ionophore A23187-stimulated PBMC and was absent in resting PBMC.

Expression of RXFP1 in skin of scleroderma patients and control subjects.

Objectives: Relaxin (RLX) is involved in extracellular matrix and collagen remodelling. The therapeutic role of the circulating isoform RLX-2 as an anti-fibrotic factor in systemic sclerosis (SSc) has been investigated. Several RLX family peptide receptors (RXFPs) are recognized in humans: RLX-2 is a ligand for RXFP1/LGR7 and RXFP2/LGR8. The aim of this study was to define the pattern of expression of LGR7 in different types of human skin cells and to compare normal skin with lesional and unaffected skin from patients with limited SSc (lSSc). Method: We analysed RXFP1 immunolocalization on skin biopsies and cultured fibroblasts from lSSc patients and control subjects. Western blot analysis was carried out on fibroblast lysates. Results: RXFP1 showed cytoplasmic localization on skin cells from control subjects and non-lesional skin from lSSc patients: keratinocytes, gland epithelial cells, endothelium, smooth muscle cells, and fibroblasts. Immunogold electron microscopy confirmed a diffuse epithelial cytoplasmic localization of RXFP1. A substantially lower RXFP1 expression was observed in scleroderma skin, with a lack of staining in most cells. Occasional weak reactivity was observed in cultured scleroderma fibroblasts, while control fibroblasts showed a diffuse cytoplasmic immunoreactivity of RXFP1, confirmed by Western blot analysis. Conclusions: The decreased cellular expression of RLX-2 receptor RXFP1 in scleroderma skin might represent a pro-fibrotic factor and contribute to the substantial inefficacy of RLX treatment in SSc, as reported in the literature. The pathophysiology of the decrease in RXFP1 may be linked to high RLX-2 serum levels previously detected in SSc, but it has yet to be elucidated.

Inhibitions of mTORC1 and 4EBP-1 are key events orchestrated by Rottlerin in SK-Mel-28 cell killing

Earlier studies demonstrated that Rottlerin exerts a time- and dose-dependent antiproliferative effect on SK-Mel-28 melanoma cells during 24u2009h of treatment, but cytotoxicity due to cell death began only after a 48u2009h exposure. In the current study, in order to identify the type of cell death in this cell line, which is notoriously refractory to most anticancer therapies, and to clarify the underlying mechanisms of this delayed outcome, we searched for apoptotic, necrotic/necroptotic and autophagic traits in Rottlerin-exposed cells. Although SK-Mel-28 cells are both apoptosis and autophagy competent, Western blotting analysis, caspase activity assay, nuclear imaging and the effects of autophagy, apoptosis and necroptosis inhibitors, indicated that Rottlerin cytotoxicity was due to none of the aforementioned death mechanisms. Nevertheless, in growth arrested cells, the death did occur after a prolonged treatment and most likely ensued from the observed blockage of protein synthesis that reached levels expected to be incompatible with cell survival. From a mechanistic point of view, we ascribed this effect to the documented inhibition of mTORC1 activity; mTORC1 inhibition on the one hand led to a not deadly, rather protective autophagic response but, on the other hand caused a near complete arrest of protein synthesis. Interestingly, no cytotoxicity was found towards normal skin fibroblasts, which only resulted mildly growth arrested by the drug.

Immune and endocrine aspects of physical and social environmental variations in groups of male rabbits in seminatural conditions

Endocrine and immune parameters, namely glucocorticoid receptors (GcR) and IFN-γ production in peripheral blood mononuclear cells (PBMC), and corticosterone in plasma, were studied in groups of four male rabbits observed in seminatural conditions in relation to agonistic behaviour and to seasonal variations. These parameters were selected on the basis of the findings that the presence of GcR in PBMC gives an indication of the in vivo biological effects of glucocorticoids and that PBMC cytokine production and plasma corticosterone are related to social behaviour. The frequency of active and passive behaviours was used to rank the animals. Seasonal variations were present for agonistic behaviours (Attack, Follow, Chase) and for plasma corticosterone, which were significantly higher in winter. IFN-γ production in PBMC was increased after social interactions in both seasons, while plasma corticosterone was increased only in winter. GcR capacity in PBMC was decreased after social interactions. The results indicate that social and physical environmental factors are correlated with immune-endocrine functions.

Restorative Effect of Bacillus subtilis Spores on Interferon Production in Aged Micea

Cytokines are autocrine, paracrine, and endocrine regulatory (g1yco)proteins that interact with specific cell receptors and have pleiotropic effects. They affect the activation state, proliferation, differentiation, maturation, and function of other cell types in the immune system as well as those in the major organ systems. It is well known that immune function declines with age in humans and experimental animals. One important component of this decline is reduced cytokine production, primarily murine interleukin-1 (IL-l),z4 1L-2:~~ and increased IL-3J and IL-4 secretion. Information about age-related production of another group of cytokines, the interferons (IFNs), is contradictory. The amount of IFN in the serum induced by Newcastle disease virus decreased with aging of the animal?,10 Another report shows an age-related decline in IFN-y production in PHAand ConA-stimulated mouse spleen cells,I1 and two reports demonstrate an increase of this cytokine in ConAstimulated murine ~plenocytes .~~J~ It is well established that Bacillus subtilis spores have immunomodulatory and immunostimulatory activities in experimental animals and man.14121 Intravenous injection of B. subtilis in mice is reported to induce plasma IFN-y productionzz and oral administration of B. subtilis spores in rabbits to induce high levels of plasma IFN-like activity.23 Because peritoneal and spleen cells from mice fed with B. subtilis spores produce significantly higher IFN-P and -y levels than those of c0ntrols,2~ we evaluated the influence of age on IFN production and the effect of oral administration of B. subtilis spores in restoring IFN production in aged mice.