Jacopo J.V. Branca

A Novel Role for a Major Component of the Vitamin D Axis: Vitamin D Binding Protein-Derived Macrophage Activating Factor Induces Human Breast Cancer Cell Apoptosis through Stimulation of Macrophages

The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This al1ows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

Effects of Vitamin D3 and Paricalcitol on Immature Cardiomyocytes: A Novel Role for Vitamin D Analogs in the Prevention of Cardiovascular Diseases

Cardiovascular diseases are more prevalent in patients with chronic kidney disease than in the general population and they are considered the leading cause of death in patients with end-stage renal disease. The discovery that vitamin D3 plays a considerable role in cardiovascular protection has led, in recent years, to an increase in the administration of therapies based on the use of this molecule; nevertheless, several studies warned that an excess of vitamin D3 may increase the risk of hypercalcemia and vascular calcifications. In this study we evaluated the effects of vitamin D3, and of its selective analog paricalcitol, on immature cardiomyocytes. Results show that vitamin D3 induces cAMP-mediated cell proliferation and significant intracellular calcification. Paricalcitol, however, induces cell differentiation, morphological modifications in cell shape and size, and no intracellular calcification. Furthermore, vitamin D3 and paricalcitol differently affect cardiomyoblasts responses to acetylcholine treatment. In conclusion, our results demonstrate that the effects of vitamin D3 and paricalcitol on cardiomyoblasts are different and, if these in vitro observations could be extrapolated in vivo, they suggest that paricalcitol has the potential for cardiovascular protection without the risk of inducing intracellular calcification.

Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia

The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species‐specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc‐protein‐derived macrophage‐activating factor (OA‐GcMAF) on two microglial cell lines, murine BV‐2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV‐2 and in C13NJ cells and that this effect was not reversed with OA‐GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA‐GcMAF in both cell lines. OA‐GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7‐2 protein, in BV‐2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA‐GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin‐induced cell damage and apoptosis were nearly completely reversed by OA‐GcMAF treatment in both BV‐2/SH‐SY5Y and C13NJ/SH‐SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV‐2 cell line may represent an excellent model for studying human microglia.

Gc-protein-derived macrophage activating factor counteracts the neuronal damage induced by oxaliplatin

Oxaliplatin-based regimens are effective in metastasized advanced cancers. However, a major limitation to their widespread use is represented by neurotoxicity that leads to peripheral neuropathy. In this study we evaluated the roles of a proven immunotherapeutic agent [Gc-protein-derived macrophage activating factor (GcMAF)] in preventing or decreasing oxaliplatin-induced neuronal damage and in modulating microglia activation following oxaliplatin-induced damage. The effects of oxaliplatin and of a commercially available formula of GcMAF [oleic acid-GcMAF (OA-GcMAF)] were studied in human neurons (SH-SY5Y cells) and in human microglial cells (C13NJ). Cell density, morphology and viability, as well as production of cAMP and expression of vascular endothelial growth factor (VEGF), markers of neuron regeneration [neuromodulin or growth associated protein-43 (Gap-43)] and markers of microglia activation [ionized calcium binding adaptor molecule 1 (Iba1) and B7-2], were determined. OA-GcMAF reverted the damage inflicted by oxaliplatin on human neurons and preserved their viability. The neuroprotective effect was accompanied by increased intracellular cAMP production, as well as by increased expression of VEGF and neuromodulin. OA-GcMAF did not revert the effects of oxaliplatin on microglial cell viability. However, it increased microglial activation following oxaliplatin-induced damage, resulting in an increased expression of the markers Iba1 and B7-2 without any concomitant increase in cell number. When neurons and microglial cells were co-cultured, the presence of OA-GcMAF significantly counteracted the toxic effects of oxaliplatin. Our results demonstrate that OA-GcMAF, already used in the immunotherapy of advanced cancers, may significantly contribute to neutralizing the neurotoxicity induced by oxaliplatin, at the same time possibly concurring to an integrated anticancer effect. The association between these two powerful anticancer molecules would probably produce the dual effect of reduction of oxaliplatin-induced neurotoxicity, together with possible synergism in the overall anticancer effect.

Selenium and zinc: Two key players against cadmium-induced neuronal toxicity

Cadmium (Cd), a worldwide occupational pollutant, is an extremely toxic heavy metal, capable of damaging several organs, including the brain. Its toxicity has been related to neurodegenerative diseases such as Alzheimers and Parkinsons diseases. The neurotoxic potential of Cd has been attributed to the changes induced in the brain enzyme network involved in counteracting oxidative stress. On the other hand, it is also known that trace elements, such as zinc (Zn) and selenium (Se), required for optimal brain functions, appears to have beneficial effects on the prevention of Cd intoxication. Based on this protective effect of Zn and Se, we aimed to investigate whether these elements could protect neuronal cells from Cd-induced excitotoxicity. The experiments, firstly carried out on SH-SY5Y catecholaminergic neuroblastoma cell line, demonstrated that the treatment with 10 μM cadmium chloride (CdCl2) for 24 h caused significant modifications both in terms of oxidative stress and neuronal sprouting, triggered by endoplasmic reticulum (ER) stress. The evaluation of the effectiveness of 50 μM of zinc chloride (ZnCl2) and 100 nM sodium selenite (Na2SeO3) treatments showed that both elements were able to attenuate the Cd-dependent neurotoxicity. However, considering that following induction with retinoic acid (RA), the neuroblastoma cell line undergoes differentiation into a cholinergic neurons, our second aim was to verify the zinc and selenium efficacy also in this neuronal phenotype. Our data clearly demonstrated that, while zinc played a crucial role on neuroprotection against Cd-induced neurotoxicity independently from the cellular phenotype, selenium is ineffective in differentiated cholinergic cells, supporting the notion that the molecular events occurring in differentiated SH-SY5Y cells are critical for the response to specific stimuli.

Morphological and Functional Characterization of IL-12Rβ2 Chain on Intestinal Epithelial Cells: Implications for Local and Systemic Immunoregulation

Interaction between intestinal epithelial cells (IECs) and the underlying immune systems is critical for maintaining intestinal immune homeostasis and mounting appropriate immune responses. We have previously showed that the T helper type 1 (TH1) cytokine IL-12 plays a key role in the delicate immunological balance in the gut and the lack of appropriate levels of IL-12 had important consequences for health and disease, particularly with regard to food allergy. Here, we sought to understand the role of IL-12 in the regulation of lymphoepithelial cross talk and how this interaction affects immune responses locally and systemically. Using a combination of microscopy and flow cytometry techniques we observed that freshly isolated IECs expressed an incomplete, yet functional IL-12 receptor (IL-12R) formed solely by the IL-12Rβ2 chain that albeit the lack of the complementary IL-12β1 chain responded to ex vivo challenge with IL-12. Furthermore, the expression of IL-12Rβ2 on IECs is strategically located at the interface between epithelial and immune cells of the lamina propria and using in vitro coculture models and primary intestinal organoids we showed that immune-derived signals were required for the expression of IL-12Rβ2 on IECs. The biological relevance of the IEC-associated IL-12Rβ2 was assessed in vivo in a mouse model of food allergy characterized by allergy-associated diminished intestinal levels of IL-12 and in chimeric mice that lack the IL-12Rβ2 chain on IECs. These experimental models enabled us to show that the antiallergic properties of orally delivered recombinant Lactococcus lactis secreting bioactive IL-12 (rLc-IL12) were reduced in mice lacking the IL-12β2 chain on IECs. Finally, we observed that the oral delivery of IL-12 was accompanied by the downregulation of the production of the IEC-derived proallergic cytokine thymic stromal lymphopoietin (TSLP). However, further analysis of intestinal levels of TSLP in IL-12Rβ2−/− mice suggested that this event was not directly linked to the IEC-associated IL-12Rβ2 chain. We interpreted these data as showing that IEC-associated IL12Rβ2 is a component of the cytokine network operating at the interface between the intestinal epithelium and immune system that plays a role in immune regulation.

Morphological analysis of neurons: Automatic identification of elongations.

Our study is focused on the development of a new method for the automatic analysis of cell images. We focused on neurons (cells line SH-SY5Y) treated/untreated with ultrasound and stained with Haematoxylin-Eosin. The aim of the algorithm is the automatic detection of the cell body as well as the determination of the number and the length of neuron elongations. Starting point of the algorithm was the convolution of an image with a bank of rotating Gaussian kernels and the construction of a module map. Then several strategies were implemented to detect cell bodies and to detect and extract data about cell elongations. We have also realized a graphical user interface allowing the loading, saving and processing of images. Results show that this method is able to properly and efficiently detect cell contours and elongations. The automated evaluation is in strong agreement with manual evaluation performed by an expert operator, with an average error of 11% with most parameter combinations. This tool constitutes an important support in biological research activities, where operators need to analyze a large number of images to investigate about cell morphology before and after a treatment.

Effect of ultrasounds on neurons and microglia: Cell viability and automatic analysis of cell morphology

Abstract Ultrasounds, besides their well-established medical imaging role, influence the homeostasis of complex anatomical systems including the physiology of neurons and glia and the permeability of the blood brain barrier. In this study, neurons and microglial cells were treated with ultrasounds (commonly used in diagnostics) and differences in cell proliferation and morphology were evaluated in comparison to control, untreated cells. Cell proliferation was evaluated by standard viability assessment, while the quantitative analysis of cell morphology, usually performed by edge and line detection algorithms, required the development of a new special algorithm. In fact, traditional software methodologies do not provide the appropriate tools for morphological analysis of neurons and microglial cells, typically characterized by a roughly triangular body and numerous elongations of different lengths resulting in a complex neuron–microglia network. This new method, based on a modified Hough Transform algorithm using a matching operator instead of the common gradient filter, enabled the automatic identification of cell elongations and branches, the extraction of related information, and the comparison of the data between control and treated neurons, as well as microglial cells. Results, based on the development of the new algorithm, showed that in ultrasound-treated cells, the number of elongations, as well as their maximum and mean lengths, increased significantly in comparison to control, untreated cells. These results were consistent with the standard microscopic evaluation. Furthermore, a significant correlation between cell morphology and proliferation suggested that ultrasounds induced cell differentiation affecting cell morphology, as well as the ability of neurons and microglial cells to form complex networks. Our results suggest the possibility of using ultrasounds, currently utilized in diagnostics, to reconstitute neuronal and microglial circuits that are often altered in neurodegenerative and neurodevelopmental disorders.

Vitamin D binding protein-derived macrophage activating factor stimulates proliferation and signalling in a human neuronal cell line.

Vitamin D (vitD), vitD binding protein-derived macrophage activating factor (DBP-MAF), and vitD receptor (VDR) are essential for adult neurogenesis [1], and this effect could be responsible for the recently reported effects of DBP-MAF on autism spectrum disorders (ASD) [2]. In order to test this hypothesis, we challenged a human neuronal cell line (SH-SY5Y, IZSLER) with DBP-MAF (Immuno Biotech), and we studied cAMP formation (cAMP EIA kit, Abnova), cell proliferation (MTT assay, Sigma Aldrich), apoptosis (human caspase 3 act, Invitrogen) and cell morphology. SH-SY5Y cells represent a validated in vitro model of human neurons in neurodegenerative diseases [3]. DBP-MAF induced rapid (15 min) formation of cAMP in a dose-dependent manner (0.4-40 ng/ml) as well as increase in cell proliferation at 24-48 and 72 h. Cell morphology was consistent with neurogenesis and an increase in the number of cells with high synthetic activity was observed. No apoptosis following DBP-MAF treatment was observed. Our results open the way to exploit these newly described effects to treat neurodegenerative disorders from Parkinson’s and Alzheimer’s diseases to Myalgic Encephalomyelitis and ASD.

Oxaliplatin-induced blood brain barrier loosening: a new point of view on chemotherapy-induced neurotoxicity

Oxaliplatin is a key drug in the treatment of advanced metastatic colorectal cancer. Despite its beneficial effects in tumor reduction, the most prevalent side-effect of oxaliplatin treatment is a chemotherapy-induced neuropathy that frequently forces to discontinue the therapy. Indeed, along with direct damage to peripheral nerves, the chemotherapy-related neurotoxicity involves also the central nervous system (CNS) as demonstrated by pain chronicity and cognitive impairment (also known as chemobrain), a newly described pharmacological side effect. The presence of the blood brain barrier (BBB) is instrumental in preventing the entry of the drug into the CNS; here we tested the hypothesis that oxaliplatin might enter the endothelial cells of the BBB vessels and trigger a signaling pathway that induce the disassembly of the tight junctions, the critical components of the BBB integrity. By using a rat brain endothelial cell line (RBE4) we investigated the signaling pathway that ensued the entry of oxaliplatin within the cell. We found that the administration of 10 μM oxaliplatin for 8 and 16 h induced alterations of the tight junction (TJs) proteins zonula occludens-1 (ZO-1) and of F-actin, thus highlighting BBB alteration. Furthermore, we reported that intracellular oxaliplatin rapidly induced increased levels of reactive oxygen species and endoplasmic reticulum stress, assessed by the evaluation of glucose-regulated protein GRP78 expression levels. These events were accompanied by activation of caspase-3 that led to extracellular ATP release. These findings suggested a possible novel mechanism of action for oxaliplatin toxicity that could explain, at least in part, the chemotherapy-related central effects.