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.

The Exposure to 2.45 GHz Electromagnetic Radiation Induced Different Cell Responses in Neuron-like Cells and Peripheral Blood Mononuclear Cells.

Electromagnetic radiation emitted by commonly used devices became an issue for public health because of their harmful effects. Notably, 2.45 GHz electromagnetic radiation exposure has been associated with DNA damage and alterations in the central nervous system. We here investigated the effects of 2.45 GHz electromagnetic radiation on cell redox status by using human SH-SY5Y neuroblastoma cells, which were differentiated to neuronal-like cells, and peripheral blood mononuclear cells (PBMCs), which were exposed to an antenna emitting 2.45 GHz electromagnetic radiation for 2, 24, and 48 h. We evaluated cell viability and mitochondrial activity alterations by measuring reactive oxygen species (ROS), mitochondrial transmembrane potential (ΔΨm), NAD+/NADH ratio, mitochondrial transcription factor A (mtTFA), and superoxide dismutase 1 (SOD1) gene transcript levels. We also investigated apoptosis and autophagy, evaluating B-cell lymphoma 2 (BCL2), BCL2-associated X protein (BAX), and microtubule-associated protein 1A/1B-light chain 3 (LC3) gene transcript levels. Cell viability was significantly reduced after 24-48 h of exposure to radiation. ROS levels significantly increased in radiation-exposed cells, compared with controls at all exposure times. ΔΨm values decreased after 2 and 24 h in exposed SH-SY5Y cells, while in PBMCs, values decreased soon after 2 h of exposure. Alterations were also found in the NAD+/NADH ratio, mtTFA, SOD1, LC3 gene expression, and BAX/BCL2 ratio. Our results showed that neuron-like cells are more prone to developing oxidative stress than PBMCs after 2.45 GHz electromagnetic radiation exposure, activating an early antioxidant defense response.

Immunohistological Analysis of Lichen Sclerosus of the Foreskin in Pediatric Age: Could It Be Considered a Premalignant Lesion?

BACKGROUND: A major worry of juvenile penile LS is potential malignant degeneration to spinocellular carcinoma (SCC) in adulthood. LS is characterized by increased CD8+ and CD57+ cells, dermal sclerosis, epidermal atrophy, and hyperkeratosis. p53 and Ki67 are reliable premalignant markers. Our aim was to define the LS immunohistochemical profile of foreskin in children, focusing on tissue immune response and cell proliferation. METHODS: Thirty specimens of foreskins removed from pediatric patients during circumcision were included: six from ritual operation (A), twelve from phimosis (B), and twelve from phimosis with LS (C). Formalin-fixed paraffin-embedded sections were stained for histomorphology and immunohistochemistry. A quantitative evaluation for CD8, CD57, p53, and Ki-67 and a statistical analysis were performed. RESULTS: As compared to groups A and B, the samples from group C patients showed an acanthotic epidermis, a dermal band of lymphoid infiltrate with a significant enhancement of CD8+ CD57+ lymphocytes, and a keratinocytic hyperplasia with an overexpression of Ki67+ and p53+ cells. CONCLUSIONS: Immunohistological findings confirmed an immune reaction and proliferative behavior in juvenile LS of foreskin. We believe that radical circumcision should be the first treatment of choice in pediatric patients with clinical suspicious of LS for the potential risk of transformation to SCC in adulthood.

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.

Methods to investigate advanced glycation end-product and their application in clinical practice.

Advanced glycation end-products (AGEs) are heterogeneous compounds of irreversible adducts principally derived from nonenzymatic glycation and glycoxidation of proteins. An increase in AGEs may be involved in the pathogenesis of metabolic and cardiovascular diseases, chronic degenerative diseases, neurological diseases and cancer, and it has been suggested as a biomarker of oxidative stress. AGEs have been evaluated in different biological fluids, as well as in tissues. The most utilized techniques for AGE measurement can be divided into immunochemical methods, such as ELISA, and bioanalytical methods, including fluorescence spectroscopy, high-performance liquid chromatography, liquid chromatography-mass spectroscopy, gas chromatography-mass spectroscopy. However, the lack of reference values, well-established standard molecules, and standardized methods to measure these compounds, could limit the application of AGE evaluation for clinical purpose. Aim of this review is to provide an overview on the state of the art of the most employed techniques for detection and measurement of AGEs and their application in clinical practice.

How Much Does HIV Positivity Affect the Presence of Oral HPV? A Molecular Epidemiology Survey.

HIV-positive people showed a high oral prevalence of HPV-DNA and have a greater incidence of head and neck carcinomas compared to general population. We performed a molecular survey evaluating the presence of HPV-DNA in saliva of HIV-positive and HIV-negative subjects in order to quantify the risk represented by HIV-positivity. The sample was made up by 102 subjects: 40 HIV-positive, 32 HIV-negative with sexual risk behaviors (SRB) and 30 HIV-negative without risk factors. DNA was extracted from cellular pellets and HPV detection and genotyping were performed by PCR assays. In the HIV-positive group (of which 58.3% declared SRB) 33.33% of the sample were HPV-positive (33.33% to high-risk genotypes, 25.0% to low-risk genotypes and 41.66% to other genotypes). In the HIV-negative SRB group, HPV-positive subjects were 37.04% (60.0% to high risk genotypes, 20.0% to low risk genotypes, and 20.0% to other genotypes). Finally, in the control group, the HPV-positive subjects were 7.14% (50% to high-risk genotypes and 50% to low-risk genotypes). In the HIV group, concerning the HPV positivity, there was no significant difference between subjects with and without SRBs. In summary, we found a high oral HPV-DNA detection in HIV+ group, showing a strong relationship between HIV and HPV.

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.

Is Melatonin the Cornucopia of the 21st Century?

Melatonin, an indoleamine hormone produced and secreted at night by pinealocytes and extra-pineal cells, plays an important role in timing circadian rhythms (24-h internal clock) and regulating the sleep/wake cycle in humans. However, in recent years melatonin has gained much attention mainly because of its demonstrated powerful lipophilic antioxidant and free radical scavenging action. Melatonin has been proven to be twice as active as vitamin E, believed to be the most effective lipophilic antioxidant. Melatonin-induced signal transduction through melatonin receptors promotes the expression of antioxidant enzymes as well as inflammation-related genes. Melatonin also exerts an immunomodulatory action through the stimulation of high-affinity receptors expressed in immunocompetent cells. Here, we reviewed the efficacy, safety and side effects of melatonin supplementation in treating oxidative stress- and/or inflammation-related disorders, such as obesity, cardiovascular diseases, immune disorders, infectious diseases, cancer, neurodegenerative diseases, as well as osteoporosis and infertility.

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.

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.

Vitamin D status influences transcriptional levels of RANKL and inflammatory biomarkers which are associated with activation of PBMC.

Vitamin D status is involved in the risk of many chronic diseases including cancer, inflammatory and autoimmune disease. The RANK/RANKL/OPG system is also implicated in the orchestration of immune functions. We aimed to investigate the expression of RANKL, OPG and markers of inflammation and immune activation in peripheral blood mononuclear cells (PBMC) from healthy subjects with different 25(OH)D3 plasma levels. The 25(OH)D3 plasma concentrations were assessed by HPLC. The gene expression was evaluated by qRT-PCR. The expression of CYP27B1 was lower in subjects with 25(OH)D3 levels below 50 nmol/L (deficiency) than subjects with both insufficient and sufficient levels of 25(OH)D3. In subjects with deficiency, we observed the up-regulation of RANKL, TNF-α, IFN-γ, ICAM and LFA-1, and a reduction of the anti-inflammatory cytokines IL-13 and IL-4 in comparison to other subjects. Finally, we found a negative correlation between RANKL mRNA levels and 25(OH)D3 and between 25(OH)D3 and ICAM mRNA levels. A positive correlation between ICAM and RANKL was observed. Our results give evidence of the modulatory effects of circulating 25(OH)D3 levels on gene expression of biomarkers of immune activation in PBMC, suggesting the possible use of PBMC as ex-vivo model to characterize molecular mechanisms of immune/inflammatory response in hypovitaminosis conditions.

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.

The inverse relation between mitochondrial transmembrane potential and proteins α-helix in neuronal-like cells under static magnetic field and the role of VDAC.

In this study, a correlation between cell channel α-helices displacement and the mitochondrial transmembrane potential after exposure of 3, 7, 15 and 24 h of neuronal-like cells to a uniform magnetic field at the intensity of 2 mT was shown. Fourier Transform Infrared (FTIR) Spectroscopy and fluorescence techniques were used to analyze the secondary structure of protein content and mitochondrial transmembrane potential, respectively. The main result of this study was represented by a significant inverse relation between the mitochondrial transmembrane potential and the intensity of the Amide I band that can be associated with time exposure. Given that mitochondrial transmembrane potential should be related to the gating state of voltage-dependent anion channel (VDAC) in mitochondrial membrane, this result could have a relevant role in medicine. Indeed, VDAC's irregular behavior can be associated with several varieties of mitochondria-associated pathologies and various forms of cancer and neurodegeneration.

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.

CO2 Pneumoperitoneum Effects on Molecular Markers of Tumor Invasiveness in SH-SY5Y Neuroblastoma Cells.

INTRODUCTION: CO2 pneumoperitoneum can influence the biological behavior of neuroblastoma (NB). Angiogenesis and genetic features are responsible for malignant phenotype of this tumor. We examined the CO2 effects on N-Myc, vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) expression as critical biomarkers of tumor invasiveness, in NB cells without N-Myc amplification. MATERIALS AND METHODS: SH-SY5Y cells were exposed to CO2 (100%) at 15 mm Hg pressure for 4 hours and then moved to normal condition for 24 hours. Control cells were incubated with 5% CO2 for the same time. In control and CO2-exposed cells, the messenger ribonucleic acid (mRNA) levels of hypoxia-inducible factor (HIF)-1α, HIF-2α, VEGF-A, and MMP-2 were quantified by real-time polymerase chain reaction. N-Myc expression was evaluated by Western blot analysis. RESULTS: The exposure to 15 mm Hg CO2 (100%) for 4 hours induced an increase in HIF-1α, but not in HIF-2α, mRNA levels. No differences were observed in N-Myc expression between exposed and control cells at each incubation time. Similarly, no significant differences were found for VEGF-A and MMP-2 transcript levels. In CO2 exposed cells, we observed only a slight increase in both VEGF-A and MMP-2 mRNA levels after 4 and 24 hours in comparison to controls. CONCLUSION: In our study, the hypoxic environment induced by CO2 exposure does not affect the expression of critical biomarkers of NB aggressiveness, such as N-Myc, VEGF, and MMP-2, in human SH-SY5Y NB cells without N-Myc amplification. These data suggest that CO2 pneumoperitoneum might not adversely impact NB cell invasiveness; however, it is necessary to evaluate these effects in others in vitro and in vivo models.

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.

Transglutaminase 2 Up-Regulation Is Associated with Inflammatory Response in PBMC from Healthy Subjects with Hypovitaminosis D.

Recent evidence indicated that transglutaminase 2 (TG2) is involved in the adaptive immune response. Peripheral blood mononuclear cells (PBMC) have largely been used to characterize molecular mechanisms occurring in the activation of immune response. Given that the maintenance of immune system functions requires an optimal vitamin D status, we aimed to assess the involvement of TG2/NF-κB signaling in cytokine production in PBMC isolated from adult subjects with different vitamin D status. We observed TG2 up-regulation and a significant positive correlation between TG2 expression and tumor necrosis factor (TNF)-α mRNA levels in PBMC of recruited patients. The mRNA levels of TG2 and TNF-α were higher in PBMC of subjects having hypovitaminosis D, namely plasma 25(OH)vitamin D3 levels lower than 50 nmol/L, than in those with normal vitamin D levels. Moreover, NF-κB up-regulation and nuclear translocation were detected, concomitantly with TG2 as well as TNF-α increased expression, in PBMC of vitamin D-deficient subjects. The present findings confirm that an increase in TG2 expression exacerbates the activation of NF-κB and the production of pro-inflammatory cytokines, and suggest a link between vitamin D deficiency, TG2 up-regulation, and inflammation.

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®.