Morphological analysis of the acute effects of cigarette smoke on normal human oral mucosa explants

Cigarette smoke is one of the five leading risk factors for mortality in the world and smoking related lung cancer is the main cause of deaths from cancer in both sexes in the United States. This habit is particularly popular in Western low- and middle- income countries, with about one billion males and 250 million females smoking in the world. Human oral mucosa is the combustion chamber of cigarette, but scanty evidence is available about the early smoke effects. 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. Biopsies of keratinized oral mucosa of healthy non smoking 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. Quantitative analysis of K14 immunolabeling revealed an over expression in the suprabasal layers as early as 3 hours after smoke exposure, without impairment of the epithelial junctional apparatus and apoptosis induction. The present study showed that the early response of the normal human oral mucosa to acute exposure to the smoke of one single cigarette induced K14 expression in suprabasal oral keratinocytes, without impairment of the epithelial junctional apparatus and apoptosis induction, suggesting that the first significant response to cigarette smoke arise from the basal and suprabasal layers of the human oral epithelium. The novelty of the present work consists in the combination of human oral explants with a non hermetic smoke exposure system. The main improvement offered by this setting is the ability of reproducing the cyclic condition by which cigarette smoke normally comes in contact with the oral mucosa. Furthermore, it allows administering simultaneously both gaseous phase and particulate matter of cigarette smoke

Chronic alendronate therapy impairs epithelial morphology and homeostasis in the human oral mucosa

Alendronate (ALN) is a nitrogen containing bisphosphonate (BP) widely used for the chronic treatment of osteoporotic patients, especially women over 60 years old. The diffusion of BPs in clinical practice has brought attention to one of their most serious side-effects, osteonecrosis of the jaw (ONJ) [1]. Several theories have been proposed to explain its pathogenesis, but the effect of BPs on the oral mucosa is still matter of debate despite its extensive involvement and injury in ONJ. This study aimed at evaluating from a morphological point of view the effects of ALN therapy on the oral epithelium of clinically healthy keratinized oral mucosa. Six women over 60 years old undergoing chronic therapy (2-7 years) with oral ALN after diagnosis of osteoporosis were recruited and compared to a gender and age matched group (n=6). Smoking habit, past history of head and neck cancer treatment, and concomitant assumption of steroidal and antiangiogenic drugs were excluding criteria. Proliferation, apoptosis, intercellular adhesion, and terminal differentiation were investigated by immunofluorescence and transmission electron microscopy (TEM). A significant decrease in keratinocyte proliferation was detected in the oral epithelium of patients undergoing ALN therapy compared to the control group (237.62 BrdU/mm2 ± 92.22 vs 104.16 BrdU/mm2 ± 66.20; p = 0.0002), without any sign of apoptosis induction by light microscopy and TEM. The presence of well established adherens and tight junctions was accompanied by profound alterations in desmosomal ultrastructure and molecular composition in the uppermost layers of the oral epithelium of the ALN group. Proceeding from the lower spinous to the granular layer, TEM analysis showed a progressive reduction in desmosomal thickness paralleled by a lower immunostaining for desmoglein 1 and desmoglein 3 in the suprabasal keratinocytes. In the upper epithelial layers, intermediate filaments gradually aggregated forming electron-dense bundles detached from the desmosomal plaque and a significant decrease in keratin 10 expression was observed. Taken together the reported results suggested a profound impairment in structure and function of the clinically healthy oral epithelium related to chronic ALN assumption. For the first time our results show that epithelial homeostasis in human oral mucosa is profoundly affected by nitrogen containing BPs, confirming previous in vitro studies [2-4] and strongly supporting the need of further investigation on the molecular mechanisms involved in ONJ pathogenesis

Skin morphological analysis upon treatment with anti-TNF-alpha agents in psoriatic patients

Psoriasis is the most common immune-mediated skin disease worldwide, with an estimated incidence of about 2%. It is characterized by erythematous scaly plaques and its pathogenesis is mostly due to an interplay among epidermal cells, immunocompetent cells, and pro-inflammatory cytokines. Previous studies on psoriatic skin demonstrated delayed terminal differentiation, keratinocyte hyperproliferation, and abnormal occludin expression in tight junctions. However, evidences about desmosomal cadherin distribution in psoriatic patients are not available so far, and only scattered studies have been carried out on adherens junctions. In psoriasis, Tumor Necrosis Factor- alpha (TNF-alpha) plays a central role, strongly supporting the treatment with anti-TNF-alpha agents, but the effects of these agents on epidermal intercellular adhesion, terminal differentiation, and proliferation have still to be elucidated. In this preliminary study, we investigated by immunofluorescence the expression of transmembrane proteins in tight junctions (occludin), adherens junctions (E-cadherin), and desmosomes (desmocollin-1 and desmoglein-1) in normal (N=5) and psoriatic skin before/after treatment with anti-TNF-alpha agents (N=5). Differentiation biomarkers (keratin-10, keratin-14, and involucrin) and epithelial proliferation were also evaluated. In psoriatic epidermis occludin, keratin-14, and involucrin were expressed also in the spinous layer, differently from controls. Desmoglein-1, desmocollin-1, E-cadherin, and keratin-10 localizations were comparable in psoriatic and healthy subjects. Moreover, in all considered patients the hyperproliferative condition was accompanied by an unusual suprabasal distribution of replicating keratinocytes. Interestingly, the distribution pattern of all considered biomarkers of intercellular adhesion and terminal differentiation was reverted to the physiological condition upon treatment with anti-TNF-alpha agents. What’s more, the proliferative rate registered in anti-TNF-alpha treated patients was reduced and similar to controls, even though scattered suprabasal dividing cells were still present. Our results highlight that anti-TNF-alpha biologicals, next to their proven inhibitory action on the cytokinic pathway, are effective in restoring an efficient junctional apparatus, a more differentiated phenotype, and the typical proliferation rate in the epidermis of psoriatic patients

Considerations towards a roadmap for collection, handling and storage of blood extracellular vesicles

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids (?liquid biopsies?). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.Non peer reviewe

Comparing the effects of augmented virtual reality treadmill training versus conventional treadmill training in patients with stage II-III Parkinson’s disease: the VIRTREAD-PD randomized controlled trial protocol

BackgroundIntensive treadmill training (TT) has been documented to improve gait parameters and functional independence in Parkinson’s Disease (PD), but the optimal intervention protocol and the criteria for tailoring the intervention to patients’ performances are lacking. TT may be integrated with augmented virtual reality (AVR), however, evidence of the effectiveness of this combined treatment is still limited. Moreover, prognostic biomarkers of rehabilitation, potentially useful to customize the treatment, are currently missing. The primary aim of this study is to compare the effects on gait performances of TT + AVR versus TT alone in II-III stage PD patients with gait disturbance. Secondary aims are to assess the effects on balance, gait parameters and other motor and non-motor symptoms, and patient’s satisfaction and adherence to the treatment. As an exploratory aim, the study attempts to identify biomarkers of neuroplasticity detecting changes in Neurofilament Light Chain concentration T0-T1 and to identify prognostic biomarkers associated to blood-derived Extracellular Vesicles.MethodsSingle-center, randomized controlled single-blind trial comparing TT + AVR vs. TT in II-III stage PD patients with gait disturbances. Assessment will be performed at baseline (T0), end of training (T1), 3 (T2) and 6 months (T3, phone interview) from T1. The primary outcome is difference in gait performance assessed with the Tinetti Performance-Oriented Mobility Assessment gait scale at T1. Secondary outcomes are differences in gait performance at T2, in balance and spatial–temporal gait parameters at T1 and T2, patients’ satisfaction and adherence. Changes in falls, functional mobility, functional autonomy, cognition, mood, and quality of life will be also assessed at different timepoints. The G*Power software was used to estimate a sample size of 20 subjects per group (power 0.95, α < 0.05), raised to 24 per group to compensate for potential drop-outs. Both interventions will be customized and progressive, based on the participant’s performance, according to a predefined protocol.ConclusionThis study will provide data on the possible superiority of AVR-associated TT over conventional TT in improving gait and other motor and non-motor symptoms in persons with PD and gait disturbances. Results of the exploratory analysis could add information in the field of biomarker research in PD rehabilitation

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Advances in the Field of Micro- and Nanotechnologies Applied to Extracellular Vesicle Research: Take-Home Message from ISEV2021

Extracellular Vesicles (EVs) are naturally secreted nanoparticles with a plethora of functions in the human body and remarkable potential as diagnostic and therapeutic tools. Starting from their discovery, EV nanoscale dimensions have hampered and slowed new discoveries in the field, sometimes generating confusion and controversies among experts. Microtechnological and especially nanotechnological advances have sped up biomedical research dealing with EVs, but efforts are needed to further clarify doubts and knowledge gaps. In the present review, we summarize some of the most interesting data presented in the Annual Meeting of the International Society for Extracellular Vesicles (ISEV), ISEV2021, to stimulate discussion and to share knowledge with experts from all fields of research. Indeed, EV research requires a multidisciplinary knowledge exchange and effort. EVs have demonstrated their importance and significant biological role; still, further technological achievements are crucial to avoid artifacts and misleading conclusions in order to enable outstanding discoveries

Characterization of the COPD Salivary Fingerprint through Surface Enhanced Raman Spectroscopy: A Pilot Study

Chronic Obstructive Pulmonary Disease (COPD) is a debilitating pathology characterized by reduced lung function, breathlessness and rapid and unrelenting decrease in quality of life. The severity rate and the therapy selection are strictly dependent on various parameters verifiable after years of clinical observations, missing a direct biomarker associated with COPD. In this work, we report the methodological application of Surface Enhanced Raman Spectroscopy combined with Multivariate statistics for the analysis of saliva samples collected from 15 patients affected by COPD and 15 related healthy subjects in a pilot study. The comparative Raman analysis allowed to determine a specific signature of the pathological saliva, highlighting differences in determined biological species, already studied and characterized in COPD onset, compared to the Raman signature of healthy samples. The unsupervised principal component analysis and hierarchical clustering revealed a sharp data dispersion between the two experimental groups. Using the linear discriminant analysis, we created a classification model able to discriminate the collected signals with accuracies, specificities, and sensitivities of more than 98%. The results of this preliminary study are promising for further applications of Raman spectroscopy in the COPD clinical field

Raman spectroscopy as a quick tool to assess purity of extracellular vesicle preparations and predict their functionality

Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application