Preclinical immunotherapy with Cytokine-Induced Killer lymphocytes against epithelial ovarian cancer.

Despite improvements in surgery and medical treatments, epithelial ovarian cancer (EOC) remains the most lethal gynaecological malignancy. Aim of this study is to investigate the preclinical immunotherapy activity of cytokine-induced killer lymphocytes (CIK) against epithelial ovarian cancers, focusing on platinum-resistant settings. We generated CIK ex vivo starting from human peripheral blood samples (PBMCs) collected from EOC patients. Their antitumor activity was tested in vitro and in vivo against platinum-resistant patient-derived ovarian cancer cells (pdOVCs) and a Patient Derived Xenograft (PDX), respectively. CIK were efficiently generated (48 fold median ex vivo expansion) from EOC patients; pdOVCs lines (n = 9) were successfully generated from metastatic ascites; the expression of CIK target molecules by pdOVC confirmed pre and post treatment in vitro with carboplatin. The results indicate that patient-derived CIK effectively killed autologous pdOVCs in vitro. Such intense activity was maintained against a subset of pdOVC that survived in vitro treatment with carboplatin. Moreover, CIK antitumor activity and tumor homing was confirmed in vivo within an EOC PDX model. Our preliminary data suggest that CIK are active in platinum resistant ovarian cancer models and should be therefore further investigated as a new therapeutic option in this extremely challenging setting.

TOP2A gene copy gain predicts response of epithelial ovarian cancers to pegylated liposomal doxorubicin: TOP2A as marker of response to PLD in ovarian cancer.

OBJECTIVE: The treatment of platinum resistant/refractory epithelial ovarian cancer (EOC) is a challenge for oncologists. One of the most utilized drugs in these patients is pegylated liposomal doxorubicin (PLD). As PLD is active only in a small subset of patients and causes side effects, selection of responsive patients is an unmet need and might be guided by the status of the DNA topoisomerase II alpha (TOP2A) that is poisoned by the drug. METHODS: From 176 ovarian cancers treated in three institutions, we selected 38 patients treated with PLD monotherapy as second/third line of treatment. TOP2A gene copies were measured using Fluorescent In Situ Hybridization (FISH) and expression evaluated using immunohistochemistry. Patients' derived xenografts (PDXs) of ovarian cancers were used to assess the correlation between TOP2A protein expression and response to PLD. RESULTS: Clinical data showed that TOP2A gene gain that is paralleled by increased expression of the protein, was associated with a higher probability of clinical benefit from PLD. Treatment of PDXs demonstrated that only xenografts showing a high percentage of TOP2A expressing cells underwent tumor shrinkage when treated with PLD. CONCLUSIONS: These data show that TOP2A gene gain and protein over-expression might predict activity of PLD in platinum resistant/refractory EOC.

Hydrophilic/hydrophobic features of TiO2 nanoparticles as a function of crystal phase, surface area and coating, in relation to their potential toxicity in peripheral nervous system.

The hydrophilic/hydrophobic properties of a variety of commercial TiO(2) nanoparticles (NP), to be employed as inorganic filters in sunscreen lotions, were investigated both as such (dry powders) and dispersed in aqueous media. Water uptake and the related interaction energy have been determined by means of adsorption microcalorimetry of H(2)O vapor, whereas dispersion features in aqueous solutions were investigated by dynamic light scattering and electrokinetic measurements (zeta potential). The optimized dispersions in cell culture medium were employed to assess the possible in vitro neuro-toxicological effect on dorsal root ganglion (DRG) cells upon exposure to TiO(2)-NP, as a function of crystal phase, surface area and coating. All investigated materials, with the only exception of the uncoated rutile, were found to induce apoptosis on DRG cells; the inorganic/organic surface coating was found not to protect against the TiO(2)-induced apoptosis. The risk profile for DRG cells, which varies for the uncoated samples in the same sequence as the photo-catalytic activity of the different polymorphs: anatase-rutile>anatase>>rutile, was found not to be correlated with the surface hydrophilicity of the uncoated/coated specimens. Aggregates/agglomerates hydrodynamic diameter was comprised in the ~200-400 nm range, compatible with the internalization within DRG cells.

Calcium signals: analysis in time and frequency domains.

Cytosolic calcium signals play important roles in processes such as cell growth and motility, synaptic communication and formation of neural circuitry. These signals have complex time courses and their quantitative analysis is not easily accomplished; in particular it may be difficult to evidence subtle differences in their temporal patterns. In this paper, we use wavelet analysis to extract information on the structure of [Formula: see text] oscillations. To this aim we have derived a set of indices by which different [Formula: see text] oscillatory patterns and their change in time can be extracted and quantitatively evaluated. This approach has been validated with examples of experimental recordings showing changes in oscillatory behavior in cells stimulated with a calcium-releasing agonist.

Calcineurin primes immature gonadotropin-releasing hormone-secreting neuroendocrine cells for migration.

During development, many neurons display calcium-dependent migration, but the role of this messenger in regulating gene expression leading to this event has not yet been elucidated. Among the decoders of calcium signals is calcineurin, a Ca(2+)/calmodulin serine/threonine phosphatase that has been involved in both short-term and long-term cellular changes. By using immortalized GnRH-secreting neurons, we now show that, in vitro, Ca(2+)-dependent gene expression, proceeding via calcineurin and the transcription factor nuclear factor of activated T cells, is a key player controlling the chemomigratory potential of developing GnRH-secreting neurons. Furthermore, our data highlight the switch nature of this phosphatase, whose activation or inactivation guides cells to proceed from one genetic program to the next.

A transport mechanism for NAADP in a rat basophilic cell line.

NAADP is a second messenger that releases Ca2+ from intracellular stores. Surprisingly, it has been recently shown that extracellular application of NAADP is capable of inducing intracellular Ca2+ release. This is particularly important since the only mammalian enzymes known to catalyze the synthesis of this second messenger are located extracellularly. In the present manuscript, we have investigated whether mammalian cells possess a transport system capable of transporting this highly charged molecule into cells. Indeed, in RBL-2H3 cells, a rat basophilic cell line, and in SK-N-BE cells, a neuroblastoma cell line, [32P]NAADP is efficiently transported inside cells. NAADP transport is Na+ and Ca2+ dependent, is partially blocked by dipyridamole, but is unaffected by nitrobenzylthioinosine. RBL-2H3 cells also transport [32P]cADPR, but the differences in the pharmacological profile suggest that NAADP transport proceeds by a novel mechanism. Lastly, extracellular application of NAADP, but not NADP, induced a raise in intracellular Ca2+. This is the first demonstration that NAADP is transported into cells and highlights the possibility that, alongside a second messenger, NAADP might also act as an autocrine/paracrine signal.