TCTP is a critical survival factor that protects cancer cells from oxidative stress-induced cell-death.

The translationally controlled tumor protein (TCTP) displays growth-promoting and antiapoptotic properties. To gain information on the role of TCTP in cancer disease, we studied the modulation of TCTP and cell survival under stress conditions on tumor cell lines of different origins. When cancer cells were exposed to a mild oxidative stress, such low doses of Arsenic trioxide (ATO) or hydrogen peroxide (H(2)O(2)), up-regulation of TCTP was observed in cells survived to the treatment. Differently, a strong oxidative hit provided by ATO combined with glutathione (GSH) depletion or condition of glucose deprivation caused a down-modulation of TCTP followed by cell death. Clones with a forced expression of TCTP or with silenced TCTP were obtained from the breast cancer cell line MDA-MB-231. The sensitivity to oxidative stress was strongly enhanced in down-modulated TCTP cells while decreasing in cells with high levels of TCTP. Together these results indicate that TCTP is a survival factor that protects cancer cells from oxidative stress-induced cell-death. We propose TCTP as a "stress hallmark" that may be exploited as a therapeutic target to decrease the resistance of cancer cells to anticancer therapy

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice

© 2019 Elsevier Ltd Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl− and pH. This construct, called ClopHensor, consists of a H+- and Cl−-sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a red fluorescent protein (DsRedm). Stimulation of hippocampal Schaffer collaterals proved that the sensor is functionally active. To reveal the expression pattern of ClopHensor across the brain of Thy1::ClopHensor mice, we obtained transparent brain samples using the CLARITY method and imaged them with confocal and light-sheet microscopy. We then developed a semi-quantitative approach to identify brain structures with high intrinsic sensor fluorescence. This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl−/pH in normal and pathological conditions. This article is part of a Special Issue entitled: SI: Miledi´s contributions. Item Group:IG00516

CLARITY analysis of the Cl/pH sensor expression in the brain of transgenic mice

© 2019 Elsevier Ltd Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl− and pH. This construct, called ClopHensor, consists of a H+- and Cl−-sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a red fluorescent protein (DsRedm). Stimulation of hippocampal Schaffer collaterals proved that the sensor is functionally active. To reveal the expression pattern of ClopHensor across the brain of Thy1::ClopHensor mice, we obtained transparent brain samples using the CLARITY method and imaged them with confocal and light-sheet microscopy. We then developed a semi-quantitative approach to identify brain structures with high intrinsic sensor fluorescence. This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl−/pH in normal and pathological conditions. This article is part of a Special Issue entitled: SI: Miledi´s contributions. Item Group:IG00516

Thymosin α1 and cancer: action on immune effector and tumor target cells

Since it was first identified, thymosin alpha 1 (Tα1) has been characterized to have pleiotropic effects on several pathological conditions, in particular as a modulator of immune response and inflammation. Several properties exerted by Tα1 may be attributable to a direct action on lymphoid cells. Tα1 has been shown to exert an immune modulatory activity on both T cell and natural killer cell maturation and to have an effect on functions of mature lymphocytes, including stimulating cytokine production and cytotoxic T lymphocyte-mediated cytotoxic responses. In previous studies we have shown that Tα1 increases the expression of major histocompatibility complex class I surface molecules in murine and human tumor cell lines and in primary cultures of human macrophages. In the present paper, we describe preliminary data indicating that Tα1 is also capable of increasing the expression of tumor antigens in both experimental and human tumor cell lines. This effect, which is exerted at the level of the target tumor cells, represents an additional factor increasing the antitumor activity of Tα1

Evaluation of antigen specific recognition and cell mediated cytotoxicity by a modified lysispot assay in a rat colon carcinoma model

Antigen-specific CD8+ cytotoxic T lymphocytes represent potent effector cells of the adaptive immune response against viruses as well as tumours. Therefore assays capable at exploring the generation and function of cytotoxic T lymphocytes represent an important objective for both clinical and experimental settings

Thymosin α1 as a stimulatory agent of innate cell-mediated immune response

The innate immune response and its cellular effectors-peripheral blood mononuclear cells and differentiated macrophages-play a crucial role in detection and elimination of pathogenic microorganisms. Chemotherapy and some immunosuppressive drugs used after organ transplantation and for treatment of autoimmune diseases have, as main side effect, bone marrow suppression, which can lead to a reduced response of the innate immune system. Hence, many immune-depressed patients have a higher risk of developing bacterial and invasive fungal infections compared with immune-competent individuals. Thymosin α1 (Tα1) immunomodulatory activity on effector cells of the innate immunity has been extensively described, even if its mechanism of action is not completely understood. Here, we report some of the main knowledge on this topic, focusing on our in vitro and in vivo work in progress that reinforce the validity of Tα1 as a stimulatory agent for detection and elimination of pathogens by differentiated macrophages and for restoring immune parameters after chemotherapy-induced myelosuppression

Evidence for electro-chemical interactions between multi-walled carbon nanotubes and human macrophages

cited By 18International audienceCarbon multi-walled nanotubes (MWCNTs) may have several dangerous effects on different cell systems, but the mechanisms responsible for their cytotoxicity are not well known yet. At present, very little is known about the electrical interactions between nanomaterials and cells. We aimed to verify whether MWCNT electrical properties could affect the so called "charge-sensitive" cell parameters, interacting with cellular electrical activity. Human macrophages were challenged with two fully characterised MWCNT samples, one tested as-prepared (MWCNT), the other one purified (by annealing at 2400 °C) and better electro-conductive (a-MWCNT). Our findings show that a-MWCNTs are less cytotoxic but possess a higher inflammatory potential, as compared to MWCNTs. Moreover, only annealed and better conductive MWCNTs affect significantly the mitochondrial membrane polarity, the intracellular pH and the reorganisation of cytoskeleton actin filaments, cell functions strictly dependent on electro-chemical mechanisms. Based on our results, there is evidence for electro-chemical interactions taking place between cell membranes and electro-conductive MWCNTs. Such a specific behaviour could have wide-range applications in the biomedical field, not only concerning those cellular systems (neuronal and bone cells) sensitive to electrical stimuli, but also other cell systems

Evidence for electro-chemical interactions between multi-walled carbon nanotubes and human macrophages

cited By 18International audienceCarbon multi-walled nanotubes (MWCNTs) may have several dangerous effects on different cell systems, but the mechanisms responsible for their cytotoxicity are not well known yet. At present, very little is known about the electrical interactions between nanomaterials and cells. We aimed to verify whether MWCNT electrical properties could affect the so called "charge-sensitive" cell parameters, interacting with cellular electrical activity. Human macrophages were challenged with two fully characterised MWCNT samples, one tested as-prepared (MWCNT), the other one purified (by annealing at 2400 °C) and better electro-conductive (a-MWCNT). Our findings show that a-MWCNTs are less cytotoxic but possess a higher inflammatory potential, as compared to MWCNTs. Moreover, only annealed and better conductive MWCNTs affect significantly the mitochondrial membrane polarity, the intracellular pH and the reorganisation of cytoskeleton actin filaments, cell functions strictly dependent on electro-chemical mechanisms. Based on our results, there is evidence for electro-chemical interactions taking place between cell membranes and electro-conductive MWCNTs. Such a specific behaviour could have wide-range applications in the biomedical field, not only concerning those cellular systems (neuronal and bone cells) sensitive to electrical stimuli, but also other cell systems