Editorial of Special Issue "The Role of Vitamin D in Human Health and Diseases 3.0".

After the successful collection of studies published in the past two Special Issues on the role of vitamin D in health and disease, this Special Issue, titled "The Role of Vitamin D in Human Health and Diseases 3 [...].

Iron Overload in Brain: Transport Mismatches, Microbleeding Events, and How Nanochelating Therapies May Counteract Their Effects.

Iron overload in many brain regions is a common feature of aging and most neurodegenerative diseases. In this review, the causes, mechanisms, mathematical models, and possible therapies are summarized. Indeed, physiological and pathological conditions can be investigated using compartmental models mimicking iron trafficking across the blood-brain barrier and the Cerebrospinal Fluid-Brain exchange membranes located in the choroid plexus. In silico models can investigate the alteration of iron homeostasis and simulate iron concentration in the brain environment, as well as the effects of intracerebral iron chelation, determining potential doses and timing to recover the physiological state. Novel formulations of non-toxic nanovectors with chelating capacity are already tested in organotypic brain models and could be available to move from in silico to in vivo experiments.

Vitamin D and Sulforaphane Decrease Inflammatory Oxidative Stress and Restore the Markers of Epithelial Integrity in an In Vitro Model of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is strictly linked to chronic oxidative stress, inflammation, loss of epithelial barrier integrity, and often with abnormal new blood vessel development. In this study, the retinal epithelial cell line ARPE-19 was treated with pro-inflammatory transforming growth factor-beta (TGF-ß) to investigate the activity of vitamin D (VD) and sulforaphane (SF) in abating the consequences of oxidative stress and inflammation. The administration of VD and SF lowered reactive oxygen species (ROS) levels, and abated the related expression of the pro-inflammatory cytokines interleukin-6 and interleukin-8 induced by TGF-ß. We evaluated mitochondrial respiration as a source of ROS production, and we discovered that the increased transcription of respiratory elements triggered by TGF-ß was prevented by VD and SF. In this model of inflamed epithelium, the treatment with VD and SF also reduced the secretion of VEGF, a key angiogenic factor, and restored the markers of epithelial integrity. Remarkably, all the observed biological effects were potentiated by the co-stimulation with the two compounds and were not mediated by VD receptor expression but rather by the ERK 1/2 pathway. Altogether, the results of this study reveal the powerful synergistic anti-inflammatory activity of SF and VD and lay the foundation for future clinical assessment of their efficacy in AMD.

Editorial of Special Issue "The Role of Vitamin D in Human Health and Diseases 2.0".

Vitamin D performs a differentiating, metabolic and anti-inflammatory function, through genomic, non-genomic and mitochondrial mechanisms of action [...].

DNA repair protein DNA-PK protects PC12 cells from oxidative stress-induced apoptosis involving AKT phosphorylation.

BACKGROUND: Emerging evidence suggest that DNA-PK complex plays a role in the cellular response to oxidative stress, in addition to its function of double strand break (DSB) repair. In this study we evaluated whether DNA-PK participates in oxidative stress response and whether this role is independent of its function in DNA repair. METHODS AND RESULTS: We used a model of H2O2-induced DNA damage in PC12 cells (rat pheochromocytoma), a well-known neuronal tumor cell line. We found that H2O2 treatment of PC12 cells induces an increase in DNA-PK protein complex levels, along with an elevation of DNA damage, measured both by the formation of γΗ2ΑX foci, detected by immunofluorescence, and γH2AX levels detected by western blot analysis. After 24 h of cell recovery, γΗ2ΑX foci are repaired both in the absence and presence of DNA-PK kinase inhibitor NU7026, while an increase of apoptotic cells is observed when DNA-PK activity is inhibited, as revealed by counting pycnotic nuclei and confirmed by FACS analysis. Our results suggest a role of DNA-PK as an anti-apoptotic factor in proliferating PC12 cells under oxidative stress conditions. The anti-apoptotic role of DNA-PK is associated with AKT phosphorylation in Ser473. On the contrary, in differentiated PC12 cells, were the main pathway to repair DSBs is DNA-PK-mediated, the inhibition of DNA-PK activity causes an accumulation of DNA damage. CONCLUSIONS: Taken together, our results show that DNA-PK can protect cells from oxidative stress induced-apoptosis independently from its function of DSB repair enzyme.

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of human malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with exposure to asbestos, with poor prognosis and no effective therapies. The strong inhibitory activities of growth hormone-releasing hormone (GHRH) antagonists have been demonstrated in different experimental human cancers, including lung cancer; however, their role in MPM remains unknown. We assessed the effects of the GHRH antagonists MIA-602 and MIA-690 in vitro in MPM cell lines and in primary MPM cells, and in vivo in MPM xenografts. GHRH, GHRH receptor, and its main splice variant SV1 were found in all the MPM cell types examined. In vitro, MIA-602 and MIA-690 reduced survival and proliferation in both MPM cell lines and primary cells and showed synergistic inhibitory activity with the chemotherapy drug pemetrexed. In MPM cells, GHRH antagonists also regulated activity and expression of apoptotic molecules, inhibited cell migration, and reduced the expression of matrix metalloproteinases. These effects were accompanied by impairment of mitochondrial activity and increased production of reactive oxygen species. In vivo, s.c. administration of MIA-602 and MIA-690 at the dose of 5 µg/d for 4 wk strongly inhibited the growth of MPM xenografts in mice, along with reduction of tumor insulin-like growth factor-I and vascular endothelial growth factor. Overall, these results suggest that treatment with GHRH antagonists, alone or in association with chemotherapy, may offer an approach for the treatment of MPM.

Editorial of Special Issue "The Role of Vitamin D in Human Health and Diseases".

Vitamin D has been described as a differentiative hormone, but this definition is reductive for a molecule targeting every tissue, produced in its active form by many kinds of cells and effective on the whole life of cells by different mechanisms, which lead to nuclear, non-genomic and mitochondrial effects [...].

The Role of Vitamin D in Supporting Health in the COVID-19 Era.

The genomic activity of vitamin D is associated with metabolic effects, and the hormone has a strong impact on several physiological functions and, therefore, on health. Among its renowned functions, vitamin D is an immunomodulator and a molecule with an anti-inflammatory effect, and, recently, it has been much studied in relation to its response against viral infections, especially against COVID-19. This review aims to take stock of the correlation studies between vitamin D deficiency and increased risks of severe COVID-19 disease and, similarly, between vitamin D deficiency and acute respiratory distress syndrome. Based on this evidence, supplementation with vitamin D has been tested in clinical trials, and the results are discussed. Finally, this study includes a biochemical analysis on the effects of vitamin D in the body's defense mechanisms against viral infection. In particular, the antioxidant and anti-inflammatory functions are considered in relation to energy metabolism, and the potential, beneficial effect of vitamin D in COVID-19 is described, with discussion of its influence on different biochemical pathways. The proposed, broader view of vitamin D activity could support a better-integrated approach in supplementation strategies against severe COVID-19, which could be valuable in a near future of living with an infection becoming endemic.

Thermomagnetic Resonance Effect of the Extremely Low Frequency Electromagnetic Field on Three-Dimensional Cancer Models.

In our recent studies, we have developed a thermodynamic biochemical model able to select the resonant frequency of an extremely low frequency electromagnetic field (ELF-EMF) specifically affecting different types of cancer, and we have demonstrated its effects in vitro. In this work, we investigate the cellular response to the ELF electromagnetic wave in three-dimensional (3D) culture models, which mimic the features of tumors in vivo. Cell membrane was modelled as a resistor-capacitor circuit and the specific thermal resonant frequency was calculated and tested on two-dimensional (2D) and three-dimensional (3D) cell cultures of human pancreatic cancer, glioblastoma and breast cancer. Cell proliferation and the transcription of respiratory chain and adenosine triphosphate synthase subunits, as well as uncoupling proteins, were assessed. For the first time, we demonstrate that an ELF-EMF hampers growth and potentiates both the coupled and uncoupled respiration of all analyzed models. Interestingly, the metabolic shift was evident even in the 3D aggregates, making this approach particularly valuable and promising for future application in vivo, in aggressive cancer tissues characterized by resistance to treatments.

Vitamin D and Beta-Glucans Synergically Stimulate Human Macrophage Activity.

Vitamin D and beta-glucans are both immunostimulants. Vitamin D exerts its beneficial effects on many components of the immune system. In macrophages, the hormone modulates both phagocytic activity and cytokine production; therefore, it plays an important role in mediating the innate immune response to infection. The immunomodulatory properties of beta-glucans are attributed to the ability of these fungal cell wall polysaccharides to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes and macrophages. The intracellular signaling pathways activated by beta-glucans lead to enhanced phagocytosis and cytokine response. In this study we investigated the possible potentiation of immunomodulatory properties of the combined treatment with vitamin D and beta-glucans. The effects of 100 nM 1,25-dihydroxyvitamin D3 or 100 µg/mL beta-glucans were evaluated in human macrophages in terms of cytokine production, intracellular vesicle acidification and changes in energy metabolism, three hallmarks of macrophage antimicrobial activation. We found that all the analyzed parameters were enhanced by the co-treatment compared to the response to single molecules. The results of this study support the validity of a novel therapeutic approach that could boost the immune response, taking advantage of the synergy between two natural compounds.

In vitro generation of primary cultures of human hyalocytes.

Purpose: A growing number of studies on animal models have demonstrated that some ocular diseases are the result of the interaction between hyalocytes and the ocular inflammatory setting. Endogenous and exogenous substances might alter the structure and behavior of hyalocytes that can contribute to the pathogenesis of some ocular diseases. Obtaining primary cultures of human hyalocytes could help understand the role of these cells in response to different treatments. Methods: Hyalocytes were isolated from eyes of 54 patient volunteers subjected to vitrectomy for different clinical reasons. By testing different matrices and growth media, we reproducibly generated primary cultures of hyalocytes that we characterized morphologically and biologically, basally and upon treatment with several agents (basic fibroblast growth factor (bFGF), transforming growth factor beta 1 (TGF-ß), platelet-derived growth factor subunit-BB (PDGF-BB), ascorbic acid, dexamethasone, and hydrogen peroxide). Results: We were able to generate primary cultures from vitreous human samples, growing the cells on collagen-coated plates in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum; primary cells expressed the hyalocyte markers. Specific cytoskeletal modifications were observed upon treatment with bFGF, TGF-ß, PDGF-BB, ascorbic acid, dexamethasone, and hydrogen peroxide. Only bFGF was able to promote cell growth and hyaluronic acid production. Conclusions: We describe for the first time the generation and the characterization of primary cultures of human hyalocytes from living donors. Although human hyalocytes share some characteristics with animal hyalocytes, human hyalocytes have their own features typical of the species, confirming how important human experimental models are for investigating human pathologies and their treatments.

Morphokinetic analysis of cleavage stage embryos and assessment of specific gene expression in cumulus cells independently predict human embryo development to expanded blastocyst: a preliminary study.

To assess whether morphokinetic features at the cleavage stage together with specific gene expression in cumulus cells (CCs) may be used to predict whether human embryos are able to achieve the expanded blastocyst stage on day 5. Eighty-one embryos were cultured using the Geri plus® time-lapse system. Twenty-seven embryos progressing to the expanded blastocyst stage (BL group) were compared with thirty-five embryos showing developmental arrest (AR group) and nineteen reaching the stage of early or not fully expanded blastocyst (nBL group). The analyzed morphokinetic variables were pronuclear appearance (tPNa), pronuclear fading (tPNf), and completion of cleavage to two, three, four, and eight cells (t2, t3, t4, and t8). CCs were analyzed by RT-qPCR for bone morphogenetic protein 15 (BMP15), cytochrome c oxidase subunit II (COXII), ATP synthase subunit 6 (MT-ATP6), connexin 43 (Cx43), and heme oxygenase-1 (HO-1). Embryos of BL group showed a significantly faster kinetic. BMP15, COXII, and MT-ATP6 mRNA expression was significantly higher in CCs of BL group embryos, whereas Cx43 and HO-1 mRNA levels were higher in AR group. Kinetic parameters and gene expression were not significantly different between either the BL and nBL groups or the AR and nBL groups. ROC curves showed that the most predictive cut-offs were t2 < 26.25 for morphokinetics and COXII > 0.3 for gene expression. Multivariable logistic regression analysis showed that morphokinetic variables and gene expression were both valuable, independent predictors of embryo development to expanded blastocyst. Our results suggest the possibility of developing integrated prediction models for early embryo selection at the cleavage stage.

Vitamin D Receptor Is Necessary for Mitochondrial Function and Cell Health.

Vitamin D receptor (VDR) mediates many genomic and non-genomic effects of vitamin D. Recently, the mitochondrial effects of vitamin D have been characterized in many cell types. In this article, we investigated the importance of VDR not only in mitochondrial activity and integrity but also in cell health. The silencing of the receptor in different healthy, non-transformed, and cancer cells initially decreased cell growth and modulated the cell cycle. We demonstrated that, in silenced cells, the increased respiratory activity was associated with elevated reactive oxygen species (ROS) production. In the long run, the absence of the receptor caused impairment of mitochondrial integrity and, finally, cell death. Our data reveal that VDR plays a central role in protecting cells from excessive respiration and production of ROS that leads to cell damage. Because we confirmed our observations in different models of both normal and cancer cells, we conclude that VDR is essential for the health of human tissues.

An engineering thermodynamic approach to select the electromagnetic wave effective on cell growth.

To date, the choice of the characteristics of the extremely low-frequency electromagnetic field beneficial in proliferative disorders is still empirical. In order to make the ELF interaction selective, we applied the thermodynamic and biochemical principles to the analysis of the thermo-chemical output generated by the cell in the environment. The theoretical approach applied an engineering bio-thermodynamic approach recently developed in order to obtain a physical-mathematical model that calculated the frequency of the field able to maximize the mean entropy changes as a function of cellular parameters. The combined biochemical approach envisioned the changes of entropy as a metabolic shift leading to a reduction of cell growth. The proliferation of six human cancer cell lines was evaluated as the output signal able to confirm the correctness of the mathematical model. By considering the cell as a reactive system able to respond to the unbalancing external stimuli, for the first time we could calculate and validate the frequencies of the field specifically effective on distinct cells.

Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes.

Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.

Endodontic Treatment of Chronic Apical Periodontitis Ameliorates Systemic Inflammation and Restores Impaired Cellular Responses to Insulin in an In Vitro Model.

INTRODUCTION: A growing body of research supports an association between periapical inflammation and an increased risk of developing systemic diseases. There is currently no scientific evidence to support a causal effect of inflammation on the onset of insulin resistance (IR) in patients with apical periodontitis (AP). The aim of this in vitro study was to evaluate any association between AP and levels of serum inflammatory factors potentially associated with the onset of IR, and to investigate the effect of root canal treatment (RCT) on these systemic inflammation markers and on the response in vitro to insulin. METHODS: A total of 27 control subjects and 27 patients with AP were enrolled. Patients with AP underwent RCT and were followed-up 6 and 12 months post-treatment. Enzyme-linked immunosorbent assays were used to evaluate serum levels of proinflammatory cytokines interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor (TNF)-α. The response in vitro to insulin was assessed by measuring glucose consumption in a human pancreatic epithelioid carcinoma cell line treated with sera from healthy and AP subjects. RESULTS: At baseline AP was associated with significant higher levels of IL-1, IL-6, and IL-8 in the serum of untreated (AP) patients vs controls (P < .001). Glucose consumption decreased in pancreatic cells incubated with baseline serum from patients with AP, in a manner proportional to total cytokines amount. Notably, endodontic treatment was associated with reduced levels of cytokines (P < .001) and improved response to insulin in AP group (P < .001). CONCLUSIONS: Our findings suggest that AP may promote inflammatory-driven IR in an in vitro model, and that RCT may ameliorate inflammatory mediators in vivo and the cellular response to insulin in vitro.

The exposure to extremely low frequency electromagnetic-fields inhibits the growth and potentiates the sensitivity to chemotherapy of bidimensional and tridimensional human osteosarcoma models.

We previously established a thermodynamical model to calculate the specific frequencies of extremely low frequency-electromagnetic field (ELF-EMF) able to arrest the growth of cancer cells. In the present study, for the first time, we investigated the efficacy of this technology on osteosarcoma, and we applied a precise frequency of the electromagnetic field on three human osteosarcoma cell lines, grown as adherent cells and spheroids. We evaluated the antitumour efficacy of irradiation in terms of response to chemotherapeutic treatments, which is usually poor in this type of cancer. Importantly, the results of this novel combinatorial approach revealed that the specific exposure can potentiate the efficacy of several chemotherapeutic drugs, both on bidimensional and tridimensional cancer models. The effectiveness of cisplatinum, methotrexate, ifosfamide and doxorubicin was greatly increased by the concomitant application of the specific ELF-EMF. Moreover, our experiments confirmed that ELF-EMF inhibited the proliferation and modulated the mitochondrial metabolism of all cancer models tested, whereas mesenchymal cells were not affected. The latter finding is extremely valuable, given the importance of preserving the cell reservoir necessary for tissue regeneration after chemotherapy. Altogether, this novel evidence opens new avenues to the clinical applications of ELF-EMF in oncology.

How the Competition for Cysteine May Promote Infection of SARS-CoV-2 by Triggering Oxidative Stress.

SARS-CoV-2 induces a broad range of clinical manifestations. Besides the main receptor, ACE2, other putative receptors and co-receptors have been described and could become genuinely relevant to explain the different tropism manifested by new variants. In this study, we propose a biochemical model envisaging the competition for cysteine as a key mechanism promoting the infection and the selection of host receptors. The SARS-CoV-2 infection produces ROS and triggers a massive biosynthesis of proteins rich in cysteine; if this amino acid becomes limiting, glutathione levels are depleted and cannot control oxidative stress. Hence, infection succeeds. A receptor should be recognized as a marker of suitable intracellular conditions, namely the full availability of amino acids except for low cysteine. First, we carried out a comparative investigation of SARS-CoV-2 proteins and human ACE2. Then, using hierarchical cluster protein analysis, we searched for similarities between all human proteins and spike produced by the latest variant, Omicron BA.1. We found 32 human proteins very close to spike in terms of amino acid content. Most of these potential SARS-CoV-2 receptors have less cysteine than spike. We suggest that these proteins could signal an intracellular shortage of cysteine, predicting a burst of oxidative stress when used as viral entry mediators.

Antioxidant and Anti-Inflammatory Activity of Combined Phycocyanin and Palmitoylethanolamide in Human Lung and Prostate Epithelial Cells.

Inflammation involving the innate and adaptive immune systems is a normal response to infection; however, when allowed to continue unchecked, inflammation may result in several pathologies. Natural molecules with antioxidant properties can target the key players of inflammation and exert beneficial health effects. In this study, human normal bronchial (Beas-2B) and prostate (HPrEpiC) epithelial cell lines were exposed to infectious stimulation and treated with phycocyanin (PC) and palmitoylethanolamide (PEA), with the aim of demonstrating the enhanced antioxidant and anti-inflammatory properties of the combination. The cotreatment protected from cytotoxicity and greatly abated both the production of radical oxygen species (ROS) and the transcription of several inflammatory cytokines. Oxidative stress and inflammation were curtailed by affecting three main pathways: (1) inhibition of cyclooxygenase-2 enzyme and consequent decrease of signaling generating ROS; (2) increased synthesis of glutathione and therefore strengthening of the natural antioxidant defenses of the cells; (3) decreased infection-driven mitochondrial respiratory burst which generates oxidative stress. Based on the mounting interest in using nutraceuticals as adjuvants in the clinical practice, the present study unveils new mechanisms of action and enhanced efficacy of PC and PEA, supporting the possible exploitation of this combination in human disorders.

Whey Derivatives and Galactooligosaccharides Stimulate the Wound Healing and the Function of Human Keratinocytes through the NF-kB and FOXO-1 Signaling Pathways.

Skin repair requires the activation of keratinocytes and is mediated by controlled inflammation and cell migration and proliferation, ending with the regeneration of well-differentiated cell layers. Whey derivatives contain galactooligosaccharides (GOS), which have potential beneficial effects on wound healing due to their activity as toll-like receptor ligands, although their direct nonprebiotic effects in the skin have not yet been described. In this study, we investigated the effects of different whey-derived products and purified GOS on a human keratinocyte cell line. We found that the inflammatory cytokine interleukin-8 (IL-8) was upregulated by nuclear factor kappa B (NF-kB) signaling triggered by whey derivatives and GOS and that wound healing was accelerated by promoting cell migration and the loss of E-cadherin in the absence of epithelial-mesenchymal transition. Interestingly, the treatments enhanced the mitochondrial function in association with the translocation of the Forkhead Box O1 (FOXO-1) transcription factor. Finally, we detected the increased expression of the differentiation markers induced by GOS and whey derivatives. All together, our results show that GOS-containing products can promote wound closure and skin health by direct activity on keratinocyte functions. Among the preparations tested, the fermented compound produced by autochthonous microorganisms was the most active in modulating keratinocyte activity, supporting the biological value of whey derivatives for health.