Sulforaphane Effects on Neuronal-like Cells and Peripheral Blood Mononuclear Cells Exposed to 2.45 GHz Electromagnetic Radiation.

Exposure to 2.45 GHz electromagnetic radiation (EMR) emitted from commonly used devices has been reported to induce oxidative stress in several experimental models. Our study aims to evaluate the efficacy of sulforaphane, a well-known natural product, in preventing radiation-induced toxic effects caused by a 24 h exposure of SH-SY5Y neuronal-like cells and peripheral blood mononuclear cells (PBMCs) to 2.45 GHz EMR. Cells were exposed to radiation for 24 h in the presence or absence of sulforaphane at different concentrations (5-10-25 µg/mL). Cell viability, mitochondrial activity alterations, the transcription and protein levels of redox markers, and apoptosis-related genes were investigated. Our data showed a reduction in cell viability of both neuronal-like cells and PBMCs caused by EMR exposure and a protective effect of 5 µg/mL sulforaphane. The lowest sulforaphane concentration decreased ROS production and increased the Mitochondrial Transmembrane Potential (Δψm) and the NAD+/NADH ratio, which were altered by radiation exposure. Sulforaphane at higher concentrations displayed harmful effects. The hormetic behavior of sulforaphane was also evident after evaluating the expression of genes coding for Nrf2, SOD2, and changes in apoptosis markers. Our study underlined the vulnerability of neuronal-like cells to mitochondrial dysfunction and oxidative stress and the possibility of mitigating these effects by supplementation with sulforaphane. To our knowledge, there are no previous studies about the effects of SFN on these cells when exposed to 2.45 GHz electromagnetic radiation.

Ozoile Reduces the LPS-Induced Inflammatory Response in Colonic Epithelial Cells and THP-1 Monocytes.

Inappropriate activation of immune functions in intestinal epithelial cells can lead to inflammation that is characterized also by infiltration into intestinal tissue of monocytes/macrophages. Current therapies for intestinal inflammation include anti-inflammatory, immunosuppressive and biological drugs. Ozoile (stable ozonides) has been reported to exert anti-inflammatory effects. However, ozonated oil has been used mainly for topical applications and no data are available about its effects on intestinal cells or immune cells. In this study, we evaluated Ozoile effects on human HT-29 colonic cells and THP-1 monocytic cells stimulated with LPS to induce inflammation. HT-29 and THP-1 cells were treated with LPS in the presence/absence of Ozoile for 4 h. Biomarkers of inflammation, some members of tight junctions and the adhesion molecule ICAM were assessed by qRT-PCR. Protein expression was analyzed by Western blotting. The release of TNF-α and IL-1ß was measured by ELISA. In HT-29, Ozoile inhibited LPS-induced expression of TNF-α, IL-1ß, ZO-1, CLDN1, NOS2 and MMP-2 and increased the expression of Nrf2 and SOD2 antioxidant proteins. In THP-1 cells, the LPS induction of TNF-α, IL-1ß and ICAM was counteracted by Ozoile treatment. Our in vitro results demonstrate the effectiveness of Ozoile in reducing the inflammatory response in intestinal and monocytic cells. Further in vivo studies are necessary to confirm its possible use for intestinal inflammatory conditions.

Rotenone-induced oxidative stress in THP-1 cells: biphasic effects of baicalin.

BACKGROUND: Several results demonstrated that microglia and peripheral monocytes/macrophages infiltrating the central nervous system (CNS) are involved in cell response against toxic compounds. It has been shown that rotenone induces neurodegeneration in various in vitro experimental models. Baicalin, a natural compound, is able to attenuate cell damage through anti-oxidant, anti-microbial, anti-inflammatory, and immunomodulatory action. Using THP-1 monocytes, we investigated rotenone effects on mitochondrial dysfunction and apoptosis, as well as baicalin ability to counteract rotenone toxicity. METHODS AND RESULTS: THP-1 cells were exposed to rotenone (250 nM), in the presence/absence of baicalin (10-500 µM) for 2-24 h. Reactive Oxygen Species production (ROS), mitochondrial activity and transmembrane potential (Δψm), DNA damage, and caspase-3 activity were assessed. Moreover, gene expression of mitochondrial transcription factor a (mtTFA), interleukin-1ß (IL-1ß), B-cell lymphoma 2 (Bcl2) and BCL2-associated X protein (Bax), together with apoptotic morphological changes, were evaluated. After 2 h of rotenone incubation, increased ROS production and altered Δψm were observed, hours later resulting in DNA oxidative damage and apoptosis. Baicalin treatment at 50 µM counteracted rotenone toxicity by modulating the expression levels of some proteins involved in mitochondrial biogenesis and apoptosis. Interestingly, at higher baicalin concentrations, rotenone-induced alterations persisted. CONCLUSIONS: These results give evidence that exposure to rotenone may promote the activation of THP-1 monocytes contributing to enhanced neurodegeneration. In this context, baicalin at low concentration exerts beneficial effects on mitochondrial function, and thus may prevent the onset of neurotoxic processes.

CO2 pneumoperitoneum effects on proliferation and apoptosis in two different neuroblastoma cell lines.

PURPOSE: The proto-oncogene MYCN is considered a transcription factor involved in the regulation of neuroblastoma (NB) cell biology. Since minimally invasive-surgery represents a debated treatment of NB, we investigated CO2 effects on proliferative activity and apoptotic pathway in two NB cell lines, SH-SY5Y (MYCN-non-amplified) and IMR-32 (MYCN-amplified). METHODS: SH-SY5Y and IMR-32 were exposed to CO2 (100%) at a pressure of 15 mmHg for 4 h and then moved to normal condition for 24 h. Cell proliferation, caspase 3 activity and transcript levels of BAX, BCL-2, cyclin B, cyclin D and MMP-2 were evaluated. RESULTS: CO2 exposure caused a decrease in cell proliferation associated to increases in BAX/BCL-2 ratio and caspase 3 activity in SH-SY5Y, while opposite effects have been found in IMR-32. CO2 exposure induced a decrease of cyclin B1 in SH-SY5Y, while an increase in cyclin B1 and D1 was observed in IMR-32. A slight up-regulation of MMP-2 expression in SH-SY5Y and a significant increase of 2.2 folds in IMR-32 was observed (p < 0.05). CONCLUSIONS: Our results suggest that CO2 exposure may cause different effects on various NB cell lines, likely due to MYCN amplification status. Further in vitro and in vivo studies are needed to highlight the role of laparoscopy on NB behaviour.

Changes in the Biomarkers of Oxidative/Nitrosative Stress and Endothelial Dysfunction Are Associated with Cardiovascular Risk in Periodontitis Patients.

Patients with cardiovascular disease (CVD) and periodontitis (PT) show shared risk factors as result of the altered molecular mechanisms associated with pathological conditions. The aim of our study was to evaluate if the plasma biomarkers associated with endothelial dysfunction may also be related to alterations in the inflammatory status in peripheral blood mononuclear cells (PBMC). Patients with PT, coronary heart disease (CHD), or both diseases as well as controls were enrolled. Plasma levels of coenzyme Q10 (CoQ10), 3-nitrotyrosine (NT), and asymmetric dimethylarginine (ADMA) were assessed using HPLC. mRNA levels of caspase-1 (CASP1), NLR family pyrin domain containing 3 (NLRP3), and tumor necrosis factor-α (TNF-α) in PBMC from the recruited subjects were quantified using real-time PCR. Patients with PT + CHD showed lower CoQ10 plasma levels and increased concentrations of NT in comparison to healthy subjects. ADMA levels were higher in CHD and PT + CHD patients compared to controls. Transcript levels of CASP1, NLRP3, and TNF-α were up-regulated in PBMC from all patient groups when compared to healthy subjects. Our results suggest a possible causal link between oxidative stress, high levels of NT and ADMA, and inflammasome activation, which may be involved in the endothelial inflammatory dysfunction leading to the pathogenesis and progression of CHD in PT patients.

Baicalin-Induced Autophagy Preserved LPS-Stimulated Intestinal Cells from Inflammation and Alterations of Paracellular Permeability.

Several studies have demonstrated a relevant role of intestinal epithelial cells in the immune response and in chronic inflammatory conditions, including ulcers, colitis, and Crohn's disease. Baicalin (BA), extracted from the root of Scutellaria baicalensis, has various beneficial healthy effects, including anti-inflammatory activity. However, few studies have evaluated BA effects on autophagic signaling in epithelial cell response to inflammatory stimuli. To explore possible beneficial effects of BA, HT-29 cells were exposed to lipopolysaccharide (LPS), in presence or absence of BA, for 4 h. We evaluated mRNA levels of autophagy-related genes and cytokines, triggering inflammatory response. Furthermore, the expression of claudin 1, involved in the regulation of paracellular permeability was analyzed. BA treatment repressed LPS-induced expression of TNF-α and IL-1ß. The down-regulation of autophagy-related genes induced by LPS was counteracted by cell pretreatment with BA. Under these conditions, BA reduced the NF-κB activation caused by LPS. Also, BA restored mRNA and protein levels of claudin 1, which were reduced by LPS. In conclusion, in intestinal epithelial cells BA regulates the NF-κB activation and modulates both autophagic and inflammatory processes, leading to an improvement of paracellular permeability. These results suggest that the anti-inflammatory effects of BA can be associated to the regulation of autophagic flux.

The Critical Importance of Molecular Biomarkers and Imaging in the Study of Electrohypersensitivity. A Scientific Consensus International Report.

Clinical research aiming at objectively identifying and characterizing diseases via clinical observations and biological and radiological findings is a critical initial research step when establishing objective diagnostic criteria and treatments. Failure to first define such diagnostic criteria may lead research on pathogenesis and etiology to serious confounding biases and erroneous medical interpretations. This is particularly the case for electrohypersensitivity (EHS) and more particularly for the so-called "provocation tests", which do not investigate the causal origin of EHS but rather the EHS-associated particular environmental intolerance state with hypersensitivity to man-made electromagnetic fields (EMF). However, because those tests depend on multiple EMF-associated physical and biological parameters and have been conducted in patients without having first defined EHS objectively and/or endpoints adequately, they cannot presently be considered to be valid pathogenesis research methodologies. Consequently, the negative results obtained by these tests do not preclude a role of EMF exposure as a symptomatic trigger in EHS patients. Moreover, there is no proof that EHS symptoms or EHS itself are caused by psychosomatic or nocebo effects. This international consensus report pleads for the acknowledgement of EHS as a distinct neuropathological disorder and for its inclusion in the WHO International Classification of Diseases.

Up-regulation of HIF-1α is associated with neuroprotective effects of agmatine against rotenone-induced toxicity in differentiated SH-SY5Y cells.

Agmatine, a metabolite generated by arginine decarboxylation, has been reported as neuromodulator and neuroactive substance. Several findings suggest that agmatine displays neuroprotective effects in several models of neurodegenerative disorders, such as Parkinson's disease (PD). It has been hypothesized that biogenic amines may be involved in neuroprotection by scavenging oxygen radicals, thus preventing the generation of oxidative stress. Mitochondrial dysfunction, that leads to a reduction of oxygen consumption, followed by activation of prolyl hydroxylase and decrease of hypoxia-inducible factor 1 alpha (HIF-1α) levels, has been demonstrated to play a role in PD pathogenesis. Using rotenone-treated differentiated SH-SY5Y cells as the in vitro PD model, we here investigated the molecular mechanisms underlying agmatine neuroprotective effects. Our results showed that the preliminary addition of agmatine induces HIF-1α activation, and prevents the rotenone-induced production of free radical species, and the activation of apoptotic pathways by inhibiting mitochondrial membrane potential decrease and caspase 3 as well as cytochrome c increase. Notably, these effects are mediated by HIF-1α, as indicated by experiments using a HIF-1α inhibitor. The present findings suggest that the treatment with agmatine is able to counteract the neuronal cell injury evoked by mitochondrial toxins.

Vitamin D Status Modulates Inflammatory Response in HIV+ Subjects: Evidence for Involvement of Autophagy and TG2 Expression in PBMC.

Conflicting results on the involvement of vitamin D deficiency in inflammatory and immune response in HIV+ subjects are reported. We aimed to characterize the possible influence of vitamin D status on changes in expression of tissue transglutaminase gene (TGM2) and other genes involved in inflammatory response and autophagy in peripheral blood mononuclear cells (PBMC) from HIV+ subjects. HIV+ subjects (n = 57) under antiretroviral therapy (ART) and healthy controls (n = 40) were enrolled. mRNA levels of 1-alpha-hydroxylase (CYP27B1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), TGM2, microtubule-associated protein 1A/1B-light chain 3 (LC3), autophagy-related 5 homolog (ATG5), and Beclin 1 (BECN1) were quantified by real-time PCR. In HIV+ subjects, 25(OH)D3 plasma levels were negatively correlated with time since HIV diagnosis. In PBMC from HIV+ subjects, increases in gene expression of TNF-α and IFN-γ in comparison to controls were observed. The highest increase in TNF-α transcripts was observed in HIV+ subjects with deficient 25(OH)D3 levels. Autophagy-related genes LC3, ATG5, and BECN1 were down-regulated in HIV+ subjects. Moreover, TGM2 transcripts were up-regulated in PBMC from HIV+ subjects with 25(OH)D3 deficiency. Changes observed in PBMC from HIV+ subjects appeared to be dependent on vitamin D status. The present results suggest that vitamin D deficiency is associated with changes in the expression of markers of inflammation and autophagy, resulting in immune cell dysfunction.

The SNP rs2298383 Reduces ADORA2A Gene Transcription and Positively Associates with Cytokine Production by Peripheral Blood Mononuclear Cells in Patients with Multiple Chemical Sensitivity.

Systemic inflammation and immune activation are striking features of multiple chemical sensitivity (MCS). The rs2298383 SNP of ADORA2A gene, coding for adenosine receptor type 2A (A2AR), has been involved in aberrant immune activation. Here we aimed to assess the prevalence of this SNP in 279 MCS patients and 238 healthy subjects, and its influence on ADORA2A, IFNG and IL4 transcript amounts in peripheral blood mononuclear cells of randomly selected patients (n = 70) and controls (n = 66) having different ADORA2A genotypes. The ADORA2A rs2298383 TT mutated genotype, significantly more frequent in MCS patients than in controls, was associated with a three-fold increased risk for MCS (O.R. = 2.86; C.I. 95% 1.99-4.12, p < 0.0001), while the CT genotype, highly prevalent among controls, resulted to be protective (O.R. = 0.33; C.I. 95% 0.224-0.475, p < 0.0001). Notably, ADORA2A mRNA levels were significantly lower, while IFNG, but not IL4, mRNA levels were significantly higher in TT MCS patients compared with controls. A significant negative correlation was found between ADORA2A and both IFNG and IL4, while a significant positive correlation was found between IFNG and IL4. These findings suggest that A2AR defective signaling may play a relevant role in PBMC shift towards a pro-inflammatory phenotype in MCS patients.

Hypoxia-Dependent Expression of TG2 Isoforms in Neuroblastoma Cells as Consequence of Different MYCN Amplification Status.

Transglutaminase 2 (TG2) is a multifunctional enzyme and two isoforms, TG2-L and TG2-S, exerting opposite effects in the regulation of cell death and survival, have been revealed in cancer tissues. Notably, in cancer cells a hypoxic environment may stimulate tumor growth, invasion and metastasis. Here we aimed to characterize the role of TG2 isoforms in neuroblastoma cell fate under hypoxic conditions. The mRNA levels of TG2 isoforms, hypoxia-inducible factor (HIF)-1α, p16, cyclin D1 and B1, as well as markers of cell proliferation/death, DNA damage, and cell cycle were examined in SH-SY5Y (non-MYCN-amplified) and IMR-32 (MYCN-amplified) neuroblastoma cells in hypoxia/reoxygenation conditions. The exposure to hypoxia induced the up-regulation of HIF-1α in both cell lines. Hypoxic conditions caused the up-regulation of TG2-S and the reduction of cell viability/proliferation associated with DNA damage in SH-SY5Y cells, while in IMR-32 did not produce DNA damage, and increased the levels of both TG2 isoforms and proliferation markers. Different cell response to hypoxia can be mediated by TG2 isoforms in function of MYCN amplification status. A better understanding of the role of TG2 isoforms in neuroblastoma may open new venues in a diagnostic and therapeutic perspective.

Intracellular Fate and Impact on Gene Expression of Doxorubicin/Cyclodextrin-Graphene Nanomaterials at Sub-Toxic Concentration.

The graphene road in nanomedicine still seems very long and winding because the current knowledge about graphene/cell interactions and the safety issues are not yet sufficiently clarified. Specifically, the impact of graphene exposure on gene expression is a largely unexplored concern. Herein, we investigated the intracellular fate of graphene (G) decorated with cyclodextrins (CD) and loaded with doxorubicin (DOX) and the modulation of genes involved in cancer-associated canonical pathways. Intracellular fate of GCD@DOX, tracked by FLIM, Raman mapping and fluorescence microscopy, evidenced the efficient cellular uptake of GCD@DOX and the presence of DOX in the nucleus, without graphene carrier. The NanoString nCounter™ platform provided evidence for 34 (out of 700) differentially expressed cancer-related genes in HEp-2 cells treated with GCD@DOX (25 µg/mL) compared with untreated cells. Cells treated with GCD alone (25 µg/mL) showed modification for 16 genes. Overall, 14 common genes were differentially expressed in both GCD and GCD@DOX treated cells and 4 of these genes with an opposite trend. The modification of cancer related genes also at sub-cytotoxic G concentration should be taken in consideration for the rational design of safe and effective G-based drug/gene delivery systems. The reliable advantages provided by NanoString® technology, such as sensibility and the direct RNA measurements, could be the cornerstone in this field.

Search for Novel Diagnostic Biomarkers of Prostate Inflammation-Related Disorders: Role of Transglutaminase Isoforms as Potential Candidates.

Investigations on prostate inflammation-related disorders, including acute and chronic prostatitis, chronic pelvic pain syndrome, benign prostate hyperplasia (BPH), and prostate cancer (PCa), are still ongoing to find new, accurate, and noninvasive biomarkers for a differential diagnosis of those pathological conditions sharing some common macroscopic features. Moreover, an ideal biomarker should be useful for risk assessment of prostate inflammation progression to more severe disorders, like BPH or PCa, as well as for monitoring of treatment response and prognosis establishment in carcinoma cases. Recent literature evidence highlighted that changes in the expression of transglutaminases, enzymes that catalyze transamidation reactions leading to posttranslational modifications of soluble proteins, occur in prostate inflammation-related disorders. This review focuses on the role specifically played by transglutaminases 4 (TG4) and 2 (TG2) and suggests that both isoenzymes hold a potential to be included in the list of candidates as novel diagnostic biomarkers for the above-cited prostate pathological conditions.

Stable Ozonides with Vitamin E Acetate versus Corticosteroid in the Treatment of Lichen Sclerosus in Foreskin: Evaluation of Effects on Inflammation.

BACKGROUND: Lichen sclerosus (LS) is a disease of the skin of unclear etiology that can occur in the foreskin. Topical therapy with corticosteroids is recommended, but they can have side effects. OBJECTIVES: We aimed to compare the effects of ozonides with vitamin E acetate (OZOILE) versus topical corticosteroid in children undergoing circumcision. METHOD: Twenty children undergoing circumcision were treated before surgery: 10 children with OZOILE cream and 10 with 0.1% mometasone furoate once a day for 7 days. Ten age-matched patients with LS of the foreskin without any treatment were recruited as controls. Transcript levels of proinflammatory and anti-inflammatory cytokines and e-cadherin were evaluated in removed foreskins by qRT-PCR. RESULTS: OZOILE and steroid topical treatment produced a similar reduction of TNF-α and IL-1ß mRNA levels in foreskins from patients with LS when compared to untreated patients (p < 0.001). OZOILE and steroid treatment caused an increase in the transcript levels of IL-13 and e-cadherin in the foreskin of patients affected by LS in comparison to untreated foreskin (p < 0.001). CONCLUSIONS: On the basis of our biochemical data, a randomized clinical trial might be useful to verify the actual clinical effect of OZOILE as alternative treatment to corticosteroids in children affected by LS of the foreskin.

Olfactory-Related Quality of Life in Multiple Chemical Sensitivity: A Genetic-Acquired Factors Model.

Genetic polymorphisms as well as environmental exposures to chemical compounds, iatrogenic, psychological, and physical trauma may play a pathophysiological role in multiple chemical sensitivity (MCS) olfactory complaints, given that xenobiotic metabolism is influenced by sequence variations in genes of metabolizing enzymes. Thus, the aim of the present study was to depict-by means of multiple regression analysis-how different genetic conditions, grouped according to their function as well as clinical background and environmental exposure may interfere with those olfactory complaints referred by MCS patients. Therefore, MCS patients after gene polymorphism sequencing, the olfactory-related quality of life score-calculated by means of the Questionnaire of Olfactory Disorder in forty-six MCS patients-have been found to significantly rely on the phase I and II enzymes score and exposure to previous compounds and surgical treatments. The present work-implementing for the first time a genetic-acquired factors model on a regression analysis-further reinforces those theories, positing MCS as a complex, multifactorial, disease in which the genetic risk related to phase I and II enzymes involved in xenobiotic detoxification, olfactory, and neurodegenerative diseases play a necessary, but probably not sufficient role, along the pathophysiological route of the disease.

Association of Higher Advanced Oxidation Protein Products (AOPPs) Levels in Patients with Diabetic and Hypertensive Nephropathy.

Background and Objectives: Diabetes mellitus (DM) and hypertension (HT) are characterized by cell damage caused by inflammatory and metabolic mechanisms induced by alteration in reduction-oxidative status. Serum advanced oxidation protein products (AOPP) are new markers of protein damage induced by oxidative stress. We evaluated serum levels of AOPP in a cohort of patients with DM and HT, with or without renal complications, compared with a control healthy population. Materials and Methods: The study group comprised of 62 patients with type 2 DM and 56 with HT. The 62 patients affected by DM were further distinguished in 24 subjects without renal impairment, 18 with diabetic nephropathy (DN), 20 with chronic kidney disease (CKD) stage 2-3 secondary to DN. The subgroup of 56 patients with primary HT comprised 26 subjects without renal complications and 30 with CKD (stage 2-3) secondary to HT. Thirty healthy controls, matched for age and sex, were recruited among blood donors. Results: Increased AOPP levels were found in DM patients compared with healthy subjects, although not significantly. This index was higher and more significant in patients with DN and CKD secondary to DN than in DM patients without nephropathy (p < 0.05) or controls (p < 0.0001). Patients with HT and with kidney impairment secondary to HT also had significantly higher AOPP serum levels than controls (p < 0.01 and p < 0.0001, respectively). There were no significant differences in mean AOPP levels among DM and HT patients. Conclusion: Our study showed that oxidative stress was higher in diabetic or hypertensive subjects than in healthy controls and, in particular, it appeared to be more severe in patients with renal complications. We suggest that the assessment of AOPP in diabetic and hypertensive patients may be important to predict the onset of renal failure and to open a new perspective on the adoption of antioxidant molecules to prevent CKD in those settings.

Role of Genetic Background in Cardiovascular Risk Markers Changes in Water Polo Players.

Methylene-tetrahydrofolate reductase (MTHFR) and paraoxonase 1 (PON1) gene polymorphisms have been associated with hyperhomocysteinemia and oxidative stress increase, that are established cardiovascular risk factors. Given that intense physical activity may increase the susceptibility to adverse cardiovascular outcomes, here we investigated the effects of MTHFR C677T and A1298C as well as PON1 Q192R gene polymorphisms on cardiovascular risk markers in twenty-eight male water polo elite players. The mean plasma levels of homocysteine (Hcy) and advanced oxidation protein products (AOPP) were above reference limits in resting conditions, and increased after competition. Moreover, a positive correlation was found between Hcy and AOPP concentrations, and also between their variations (ratio post-exercise/pre-exercise values) and the variations of lactic dehydrogenase (LDH) and creatine kinase (CK) activities, known as muscle damage markers. The highest Hcy and AOPP values were found in subjects having either MTHFR CT/AC or TT/AA, and PON1 QR192 genotype, respectively. After exercise, Hcy concentrations significantly increased in CT/AC or TT/AA subjects than in athletes having other MTHFR genotypes. A training-induced increase in plasma levels of LDH and CK activities, as well as myoglobin concentrations, was also observed, even if significant differences were found only for CK activity in athletes with MTHFR CT/AC or TT/AA athletes.

Health Risks of Hypovitaminosis D: A Review of New Molecular Insights.

Hypovitaminosis D has become a pandemic, being observed in all ethnicities and age groups worldwide. Environmental factors, such as increased air pollution and reduced ultraviolet B (UVB) irradiation, as well as lifestyle factors, i.e., decreased outdoor activities and/or poor intake of vitamin D-rich food, are likely involved in the etiology of a dramatic reduction of vitamin D circulating levels. The insufficiency/deficiency of vitamin D has long been known for its association with osteoporosis and rickets. However, in the last few decades it has become a serious public health concern since it has been shown to be independently associated with various chronic pathological conditions such as cancer, coronary heart disease, neurological diseases, type II diabetes, autoimmune diseases, depression, with various inflammatory disorders, and with increased risk for all-cause mortality in the general population. Prevention strategies for these disorders have recently involved supplementation with either vitamin D2 or vitamin D3 or their analogs at required daily doses and tolerable upper-limit levels. This review will focus on the emerging evidence about non-classical biological functions of vitamin D in various disorders.

Anti-Inflammatory and Tissue Regenerative Effects of Topical Treatment with Ozonated Olive Oil/Vitamin E Acetate in Balanitis Xerotica Obliterans.

Balanitis xerotica obliterans (BXO) is a chronic inflammatory skin disorder, considered the male genital variant of lichen sclerosus. Anti-inflammatory drugs are commonly used in BXO. We evaluated the effects of an innovative formulation of ozonated olive oil with vitamin E acetate (OZOILE®) on the inflammatory status and tissue remodeling in male children with BXO. The mRNA transcripts of proteins involved either in inflammation or in dynamics of tissue regeneration were analyzed by quantitative real-time PCR, in foreskins affected by BXO removed from patients untreated or treated with OZOILE® cream for 7 days before circumcision. We found a significant reduction in mRNA levels of IL-1ß, TNF-α, INF-γ, transglutaminase 2 and NOS2 in foreskins treated with OZOILE® in comparison to untreated ones (p < 0.001). No significant differences were observed in NF-κB activation in the specimens obtained from treated and untreated patients. Hence, OZOILE® treatment up-regulated hypoxia-inducible factor (HIF)-1alpha, vascular endothelial growth factor (VEGF) and E-cadherin gene expression (p < 0.001). The treatment with OZOILE® showed effective results in children affected by BXO by reducing the inflammatory process and stimulating mechanisms for tissue regeneration of the foreskin. A randomized clinical trial on a large number of children affected by BXO might be useful to verify the efficacy of topical treatment with OZOILE®.

Transglutaminase 2 is involved in amyloid-beta1-42-induced pro-inflammatory activation via AP1/JNK signalling pathways in THP-1 monocytes.

Deposition of amyloid-beta (Aß) peptides has been shown to induce the release of inflammatory factors by activated microglia and brain infiltrating monocytes/macrophages. Interestingly, the enzyme transglutaminase 2 (TG2) has been shown to play a key role in neuroinflammation and regulation of transcription factors involved in immunomodulation. In this study, we aimed to better elucidate the mechanisms underlying TG2 involvement in the pro-inflammatory signaling pathway activated by fibrillar Aß1-42 in THP-1 monocytes. Cell exposure for 24 h to 500 nM Aß1-42, induced the up-regulation of CD14, CD16, and TG2, suggesting THP-1 cell functional activation. Aß1-42 also increased the production of reactive oxygen species, that was reduced by the pre-incubation with genistein (25 µg/ml), a soy isoflavone with antioxidant properties. Moreover, IL-1ß and IL-6 mRNA transcript and protein levels were eightfold increased in Aß1-42-treated THP-1 monocytes. Interestingly, these effects were significantly reduced by R283 (~45%), a specific inhibitor of TG activity, and genistein (~40%). Aß1-42 induced the activation of p54/p46 JNK, as well as ERK 1/2 at a lower extent. The inactivation of ERK1/2 signalling pathway, but not JNK, by either genistein or U0126, a MEK1/2 inhibitor, was not able to blunt Aß1-42-induced TG2 up-regulation, that, instead, was significantly reduced by R283. Aß1-42 also induced AP-1 activation that was not significantly affected by genistein or U0126, while was strongly reduced by R283. Our preliminary findings first suggest that TG2 up-regulation is involved in the pro-inflammatory activation of THP-1 monocytes induced by Aß1-42 via AP1/JNK signalling pathways.