Alice Gualerzi

Inhibition of Class I Histone Deacetylases Unveils a Mitochondrial Signature and Enhances Oxidative Metabolism in Skeletal Muscle and Adipose Tissue

Chromatin modifications are sensitive to environmental and nutritional stimuli. Abnormalities in epigenetic regulation are associated with metabolic disorders such as obesity and diabetes that are often linked with defects in oxidative metabolism. Here, we evaluated the potential of class-specific synthetic inhibitors of histone deacetylases (HDACs), central chromatin-remodeling enzymes, to ameliorate metabolic dysfunction. Cultured myotubes and primary brown adipocytes treated with a class I–specific HDAC inhibitor showed higher expression of Pgc-1α, increased mitochondrial biogenesis, and augmented oxygen consumption. Treatment of obese diabetic mice with a class I– but not a class II–selective HDAC inhibitor enhanced oxidative metabolism in skeletal muscle and adipose tissue and promoted energy expenditure, thus reducing body weight and glucose and insulin levels. These effects can be ascribed to increased Pgc-1α action in skeletal muscle and enhanced PPARγ/PGC-1α signaling in adipose tissue. In vivo ChIP experiments indicated that inhibition of HDAC3 may account for the beneficial effect of the class I–selective HDAC inhibitor. These results suggest that class I HDAC inhibitors may provide a pharmacologic approach to treating type 2 diabetes.

An innovative three-dimensional model of normal human skin to study the proinflammatory psoriatic effects of tumor necrosis factor-alpha and interleukin-17

BACKGROUND Among all cytokines involved in the pathogenesis and in the progression of psoriasis, Tumor Necrosis Factor (TNF)-alpha and interleukin (IL)-17 play a pivotal role. OBJECTIVE The aim of the present study was to mimic a psoriatic microenvironment and to investigate the early effects of TNF-alpha and IL-17 in a three-dimensional model of organotypic normal human skin. METHODS Human skin explants were obtained from plastic aesthetic surgery of healthy young women 20-40years old (n=7). The study was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Bioptic fragments were cultured at the air-liquid interface overnight in a Transwell system and further divided before adding either 50ng/ml IL-17 or 100ng/ml TNF-alpha or a combination of both cytokines. For each subject, a control sample was cultured without any cytokine. Samples were harvested 24 or 48h after cytokine incubation. At both time points and for all cytokine treatments a bioptic fragment obtained from each patient was processed. Epidermal proliferation, expressions of terminal differentiation (keratin 10, K10, and 14, K14) and of intercellular adhesion (occludin for tight junctions and E-cadherin for adherens junctions) biomarkers were investigated by indirect immunofluorescence. RESULTS IL-17 and TNF-alpha induced an early and statistically significant inhibition of keratinocyte proliferation (more than 80% compared with their respective controls). At 24h, the combination of both cytokines did not further reduce cell proliferation. Starting from 24h of incubation, a non-continuous occludin expression in the granular layer was observed after both IL-17 and TNF-alpha exposure. Immunolabelling for E-cadherin in adherens junctions, for K10 in the suprabasal layers, and for K14 in the basal layer was similar in all experimental groups and unaffected after cytokine treatment. CONCLUSIONS These results suggest that in this experimental model IL-17 and TNF-alpha induced an early alteration of the homeostasis of the inner proliferative layer and of the upper granular layer, as shown by cell proliferation inhibition and occludin expression.

Desmoglein 3 and keratin 10 expressions are reduced by chronic exposure to cigarette smoke in human keratinised oral mucosa explants

OBJECTIVE Oral mucosa is a physiological barrier against several exogenous stimuli, among which cigarette smoke represents a source of reactive oxidizing compounds. No morphological evidences exist on the smoke effects induced in the human oral epithelium. In this study we performed a preliminary light and transmission electron microscopy morphological evaluation focussing in particular on keratinocyte intercellular adhesion and terminal differentiation in chronic smokers. DESIGN Human biopsies were obtained from healthy young chronic smoker women (n=5) compared with a parallel group of non-smoker healthy volunteers (n=5), as the smoking habit among women is ever more spreading. Samples were processed for light and transmission electron microscopy. On paraffin sections Massons and Dane and Hermans histochemical staining were performed. Biomarker expressions of intercellular adhesion (desmoglein 3, Dsg3), terminal differentiation (keratin 10, K10 and keratin 14, K14), and basal membrane preservation (laminin) were investigated by immunofluorescence. RESULTS In both groups the epithelial structural integrity, homeostasis, and the basal membrane were comparable. Dsg3 and K10 expressions were affected in smokers with the former significantly reduced (p<0.05). Ultrastructural analysis showed hypertrophic keratinocytes in the upper spinous layer and morphologically preserved desmosomes throughout the epithelial compartment. CONCLUSIONS The reduction of Dsg3 and K10 expressions indicates that the overall process of keratinocyte terminal differentiation was altered. These preliminary results strongly suggest that Dsg3 and K10 can represent valuable immunomarkers to evaluate the tissue attempt to respond to an exogenous stress such as chronic cigarette smoke, but further samples need to be analysed.

Etanercept restores a differentiated keratinocyte phenotype in psoriatic human skin: a morphological study.

Tumor Necrosis Factor‐α (TNF‐α) plays a pivotal role in psoriasis, an immuno‐mediated and genetic skin disease. Anti‐TNF‐α inhibitors, such as etanercept, are widely used in clinical practice. By immunofluorescence, we investigated the expression of junctional transmembrane proteins in desmosomes (desmocollin‐1, Dsc1; desmoglein‐1, Dsg1), adherens junctions (E‐cadherin), tight junctions (occludin), biomarkers of keratinocyte differentiation (keratin‐10, K10; keratin‐14, K14; keratin‐16, K16; involucrin), epithelial proliferation and apoptosis in psoriatic skin before/after etanercept treatment (n = 5) and in control skin samples (n = 5). Occludin, K14, K16 and involucrin expressions were altered in psoriatic epidermis, while Dsc1, Dsg1, E‐cadherin and K10 localisations were comparable to controls. Etanercept promoted the restoration of the physiological condition as suggested by a more differentiated keratinocyte phenotype and a reduced epidermal proliferation rate.

Acute effects of cigarette smoke on three-dimensional cultures of normal human oral mucosa

Context: Human oral mucosa is the combustion chamber of cigarette, but scanty evidence is available about the early smoke effects. Objective: The present work aimed at evaluating from a morphological point of view whole smoke early effects on epithelial intercellular adhesion and keratinocyte terminal differentiation in a three-dimensional model of human oral mucosa. Materials and methods: Biopsies of keratinized oral mucosa of healthy nonsmoking women (n = 5) were collected. After culturing in a Transwell system, one fragment of each biopsy was exposed to the smoke of one single cigarette; the remnant represented the internal control. The distribution of epithelial differentiation markers (keratin-10, K10, and keratin-14, K14, for suprabasal and basal cells respectively), desmosomes (desmoglein-1, desmoglein-3), tight junctions (occludin), adherens junctions (E-cadherin, β-catenin), and apoptotic cells (p53, caspase 3) were evaluated by immunofluorescence. Results: Quantitative analysis of K14 immunolabeling revealed an overexpression in the suprabasal layers as early as 3 h after smoke exposure, without impairment of the epithelial junctional apparatus and apoptosis induction. Discussion and conclusion: These results suggested that the first significant response to cigarette smoke came from the basal and suprabasal layers of the human oral epithelium. The considered model maintained the three-dimensional arrangement of the human mucosa in the oral cavity and mimicked the inhalation/exhalation cycle during the exposure to cigarette smoke, offering a good possibility to extrapolate the reported observations to humans.

An in vitro model of human oral explants to study early effects of radiation mucositis

Mucositis is a frequent problem after irradiation of the oral mucosa. To study the early effects of irradiation on the desmosomal adhesion complex, explants of keratinized oral mucosa were exposed to a single dose of 2 Gy gamma irradiation. Biopsies were obtained from the upper dental arch of nine young healthy non-smoking women undergoing minor oral surgery. The biopsies were incubated in a Transwell culture system and, after irradiation, fixed in formalin 24 h later. Morphometric measurements of epithelial thickness revealed that it was unaffected by exposure to ionizing rays. Immunofluorescence analysis of desmosomal cadherin expression (desmoglein 1/desmoglein 3) demonstrated that the distribution of desmoglein 1 was not affected, whereas the expression of desmoglein 3 decreased in the suprabasal layers of irradiated samples. It is suggested that this has consequences for the mechanical integrity of the mucosa and promotes the development of radiation mucositis.

Morphological evaluation of tongue mucosa in burning mouth syndrome

OBJECTIVE The aim of the present study was to perform a morphological evaluation by immunofluorescence of biomarkers of keratinocyte intercellular adhesion, and of differentiation in the tongue mucosa of burning mouth syndrome patients (BMS), compared with a control group. DESIGN A prospective blinded evaluation of tongue mucosal specimens processed for light microscopy was performed. Intercellular adhesion was evaluated by investigating the expression of desmoglein 1, desmoglein 3, and of occludin. Keratin 10 and keratin 14 (markers of epithelial differentiation) were also evaluated, as keratin 16 (marker for activated keratinocytes after epithelial injury). Apoptotic cascade was investigated by p53 and activated caspase-3 expression. The basal membrane integrity was analysed through laminin immunoreactivity. RESULTS In both groups, a preserved three-dimensional architecture of the tongue was observed. Desmoglein 1 and desmoglein 3 epithelial distributions were similar in the desmosomes of patients and control subjects. Again, keratin 10 immunoreactivity and distribution pattern of keratin 14 in the epithelial compartment was similar in both groups. In control samples, keratin 16 immunoreactivity was scant throughout the epithelium with a punctuate and scattered cytoplasmic labelling. In contrast, in all BMS patients keratinocyte cytoplasm was homogeneously labelled for keratin 16, with a more intense staining than controls. Furthermore, keratin 16 staining progressively decreased proceeding towards the most superficial epithelial layers. CONCLUSIONS The results of this study are consistent with and support the clinically normal features of oral mucosa in BMS, and suggest that keratin 16 may be involved in the cell mechanisms underlying the syndrome occurrence.

Raman spectroscopy uncovers biochemical tissue-related features of extracellular vesicles from mesenchymal stromal cells

Extracellular vesicles (EVs) from mesenchymal stromal cells (MSC) are emerging as valuable therapeutic agents for tissue regeneration and immunomodulation, but their clinical applications have so far been limited by the technical restraints of current isolation and characterisation procedures. This study shows for the first time the successful application of Raman spectroscopy as label-free, sensitive and reproducible means of carrying out the routine bulk characterisation of MSC-derived vesicles before their use in vitro or in vivo, thus promoting the translation of EV research to clinical practice. The Raman spectra of the EVs of bone marrow and adipose tissue-derived MSCs were compared with human dermal fibroblast EVs in order to demonstrate the ability of the method to distinguish the vesicles of the three cytotypes automatically with an accuracy of 93.7%. Our data attribute a Raman fingerprint to EVs from undifferentiated and differentiated cells of diverse tissue origin, and provide insights into the biochemical characteristics of EVs from different sources and into the differential contribution of sphingomyelin, gangliosides and phosphatidilcholine to the Raman spectra themselves.

Detection and Characterization of Different Brain-Derived Subpopulations of Plasma Exosomes by Surface Plasmon Resonance Imaging

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases.

Simultaneous detection of multiple biomarkers by means of SERS on polymer nanopillar gold arrays

The detection of biomarkers by means of Surface Enhanced Raman Spectroscopy (SERS) is foreseen to became a very important tool in the clinical practice because of its excellent sensitivity and potential for the simultaneous detection of multiple biomarkers. In the present paper we describe how it was possible to build a sensor for the detection of genetic biomarkers involved in acute myeloid leukemia. The assay is based on the use of a specifically designed SERS substrate made of a 2D crystal structure of polymeric pillars embedded in a gold layer. This substrate is characterized by good enhancing properties coupled with an excellent homogeneity. The SERS substrate was conjugated with DNA probes complementary to a target sequence and used in a sandwich assay with gold nanoparticles labeled with a second DNA probe and a Raman reporter. The so developed assay allowed the detection of a leukemia biomarker (WT1 gene) and an housekeeping gene with low picomolar sensitivity. At last, we optimized the assay in order to tackle one of the main limitations of SERS based assay: the loss of signal that is observed when the Raman spectra are collected in liquid. Combining a preferential functionalization on the polymeric pillars with a different height of the polymer pillars from the gold layer the assay demonstrated its effectiveness even when measured in buffer.