Alberto Fucarino

The odyssey of Hsp60 from tumor cells to other destinations includes plasma membrane-associated stages and Golgi and exosomal protein-trafficking modalities.

Background In a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here. Principal Findings We found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle. Conclusions/Significance We propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.

Hsp60, amateur chaperone in amyloid-beta fibrillogenesis.

BACKGROUND Molecular chaperones are a very special class of proteins that play essential roles in many cellular processes like folding, targeting and transport of proteins. Moreover, recent evidence indicates that chaperones can act as potentially strong suppressor agents in Alzheimers disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aβ peptide and in vivo studies reveal that treatment with specific chaperones or their overexpression can ameliorate some distinct pathological signs characterizing AD. METHODS Here we investigate using a biophysical approach (fluorescence, circular dichroism (CD), transmission electron (TEM) and atomic force (AFM) microscopy, size exclusion chromatography (SEC)) the effect of the human chaperonin Hsp60 on Aβ fibrillogenesis. RESULTS We found that Hsp60 powerfully inhibits Aβ amyloid aggregation, by closing molecular pathways leading to peptide fibrillogenesis. CONCLUSIONS We observe that Hsp60 inhibits Aβ aggregation through a more complex mechanism than a simple folding chaperone action. The action is specifically directed toward the early oligomeric species behaving as aggregation seeds for on-pathway amyloid fibrillogenesis. GENERAL SIGNIFICANCE Understanding the specificity of the molecular interactions of Hsp60 with amyloid Aβ peptide allowed us to emphasize the important aspects to be taken into consideration when considering the recent promising therapeutic strategies for neurodegeneration.

Medium-term Culture of Normal Human Oral Mucosa: A Novel Three-dimensional Model to Study the Effectiveness of Drugs Administration

Tissue-engineered oral mucosal equivalents have been developed for in vitro studies for a few years now. However, the usefulness of currently available models is still limited by many factors, mainly the lack of a physiological extracellular matrix (ECM) and the use of cell populations that do not reflect the properly differentiated cytotypes of the mucosa of the oral cavity. For this reason, we have developed a novel three-dimensional culture model reflecting the normal architecture of the human oral mucosa, with the main aim of creating a better in vitro model where to test cellular responses to drugs administration. This novel 3D cell culture model (3D outgrowth) was set up using an artificial extracellular matrix (Matrigel™ ), allowing the interactions required for proper differentiation of the various citotypes which form the mucosal layer. Biopsies of human oral mucosa, in fragments of about 0.5 mm3, were placed onto 6.5mm Transwells, covered with Matrigel™ and grown in a specific culture medium. A gradual formation of an architectural structure similar to that of the in vivo oral mucosa was observed. Transmission electron and confocal microscopy were employed to characterize the newly developed model: the cell components (keratinocytes and fibroblasts) differentiated properly within the outgrowth and reconstituted, in vitro, the physiological structure of the human oral mucosa, including a stratified non-keratinized squamous layer composed of four different layers, a proper basal membrane and a lamina propria where fibroblasts produce ECM. Moreover, keratinocytes expressed CK5, CK13, CK19 and E-cadherin, whereas fibroblasts expressed collagen type I and IV, laminin and fibronectin. 3D outgrowths could be considered a valid alternative to animal models, and provide useful information for researchers interested in studying the responses of the human oral mucosa to locally delivered drugs or other exogenous treatments.

Medium-term culture of primary oral squamous cell carcinoma in a three- dimensional model: effects on cell survival following topical 5-fluororacile delivery by drug-loaded matrix tablets

Since the activity of several conventional anticancer drugs is restricted by resistance mechanisms and dose-limiting side-effects, the design of formulations for local application on malignant lesions seems to be an efficient and promising drug delivery approach. In this study, the effect of locally applied 5-FU on cell death was evaluated both in a SCC4/HEK001 model and in a newly proposed 3D outgrowth model of oral squamous cell carcinoma (OSCC). Initially, the optimal drug dose was established by delivery of solutions containing different amounts of 5-FU. The solution containing 1% (w/v) of 5-FU resulted effective in inducing cell death with complete eradication of cell colonies. Buccal tablets were designed to deliver 5-FU locoregionally to the cancer lesions of the oral cavity. Tablets were prepared using a drug loaded matrix of acrylic/methacrylic acid copolymer containing 1% (w/w) of 5-FU and applied on 3D outgrowths. The drug release from tablets appeared to be sufficient to induce cell death as confirmed by transmission electron microscopy and enzymatic assay (TUNEL). After 120 h of treatment, when about 90% of the drug had been discharged from the tablets into the culture environment, 5-FU caused loss of cell-cell communications and apoptotic cell death. After 192 h, a complete disaggregation of the 3D oral outgrowths and the death of all the cells was observed. Buccal matrix tablets could be considered a promising new approach to the locoregional treatment of OSCC. Risks of systemic toxicity are avoided since very low drug doses are delivered.

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit

ABSTRACT Background/Aim: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. Materials and Methods: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). Results: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo. Conclusions: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.

Stem Cell Populations and Regenerative Potential in Chronic Inflammatory Lung Diseases

Several acute and chronic inflammatory pathologies of the lung are accompanied by structural modifications of airway mucosa that vary depending on the severity, duration and type of the disease. These morphological changes, that determine organ dysfunction, are not always reversible. Indeed, the cycle of injury and repair, influencing airway wall re- generation, may sometimes break off and an exacerbation of the pathology may occur. The mechanisms at the base of airway remodelling during inflammation have been widely studied and numerous evidences indicate that the molecular dialogue among the cells of the mucosa has an essential role in orchestrating cell differentiation and tissue repair. In this review, we revise old notions on pulmonary morphology at the light of some of the most recent discoveries concerning stem cell differentiation, tissue homeostasis and organ regeneration of the lung.

Survey on the demand of sicilian physicians for a specific training on human cadavers and animals

Currently, surgical training of physicians in Italy has limited possibilities.Surgical training can be performed on dissection of human bodies as well as in animal laboratories, but experience is very poor. We conducted a survey through an anonymous questionnaire in order to evaluate the opinions of post-graduate physicians on their need for experience training on both human and animal bodies during their medical studies. A total of 165 young Sicilian physicians responded to the survey. Only 14 of them (8.5%) declared they had specific training on a live animal, while 46 (27.9%) reported they already attended cadaver labs. Over 70% assigned the maximum score to the utility of such courses as integration of medical academic offer. Our results showed that the majority of the subjects interviewed expressed a need for training using these practices and that it might be necessary to investigate patterns to promote the opportunity for direct practice on human and animal bodies.

Biological evaluation of PLLA membranes, with different pore diameters, to stimulate cell adhesion and growth in vitro

Polymeric membranes prepared via DIPS (Diffusion Induced Phase Separation) are widely studied and utilized as scaffolds for the regeneration of tissue. In this work, poly (L)-lactide membrane are prepared through a DIPS protocol starting from a ternary solution made of polymer, dioxane (solvent) and water (non-solvent). A three-dimensional, porous and mechanically stable membrane is desirable for ingrowth of human bronchial epithelial cells.

An ex vivo model of the human airways to study the effects of long-term exposure to Space conditions

An innovative 3D model of the human bronchial mucosa, a result of a long- term collaboration with Prof. Davies from the University of Southampton, UK, will be used to study, on board the International Space Station (ISS), the chang- es occurring in lungs exposed to the adverse environmental conditions found in Space. Our research group has developed a tissue engineered human airway model, providing a viable alternative to the use of animal models. The bronchial outgrowth offers numerous advantages compared to traditional approaches that use human tissue, including: the morphological analogy with the bronchial mucosa; the possibility to study long-term exposure (several months) to environmental factors (cigarette smoke, respiratory viruses, etc.) being able to analyze eventual changes in the long term; the possibility to add cellular and humoral components of the immune system in order to evaluate their effects. Recently, our application to the ILSRA 2014 solicitation by NASA/ESA, in which we propose to study the effects of prolonged stay on board the ISS on the development and function of human airway cells in our model, was successful and our project was selected. This project will involve several international partners, including, in addition to the University of Palermo and Southampton, the Euro-Mediterranean Institute of Science and Technology (IEMEST, Palermo), the University of Leiden (NL), the University of Galveston (Texas, USA), the Wyle Laboratories (Texas, USA) and NASA itself. The results obtained will be particularly useful in view of exploratory missions of our solar system planned in the near future. To verify the resilience of the model to the extreme environmental conditions on board the vectors that transport the samples to the ISS, and therefore collect essential data prior to the flight experiments, differentiated outgrowths obtained from healthy patients were exposed for up to 1 week to the following stress conditions: withdrawal of growth medium, reduced culture temperature (10 and 20°C), absence of CO2, intense rolling and shaking motions. TER values were measured 1 week prior to 1 week after the exposures to evaluate the effects of these stimuli. The data obtained show that even though the stimuli determined a slight degree of tissue alteration (reduction of TER) during exposure, the outgrowths promptly recovered their origi- nal structure and function.

Effects of antioxidants on CSE-induced cell death in human asthmatic primary bronchial epithelial cells

The link between cigarette smoke (CS) and lung inflammation is quite strong, however relatively little is still known on the effects of CS on human bronchial epithelial cells survival during asthma. In this study we focused our attention on the apoptotic effects of CS on healthy (HC) and asthmatic (AS) primary bronchial epithelial cells (PBEC) and on the role of antioxidants to protect epithelial cells from CSE-induced apoptosis. Twenty subjects (10 HC and 10 AS) were recruited for this study and PBEC were obtained by bronchoscopy. PBEC were treated with oxidants (H2O), anti-oxidants (GSH and AA) and cigarette smoke extracts (CSE). Early apoptosis (EA) and necrosis were measured by flow cytometry using Annexin-V and propidium iodide. After treatment with CSE 20%, AS showed an increased susceptibility to the CSE treatment compared to HC (24.34+/-9.61 vs 48.45+/-11.91, p=0.003). Similarly, when EA was taken into consideration, there was a significant increase of EA cells in the AS group treated with CSE compared to HC (33.12+/-10.38 vs 16.73+/-6.92, p<0.05). AA failed to protect both HS and AS PBEC from CSE-induced cell death. GSH instead was able to protect significantly both HS and AS from CSE-induced cell death. In particular, the association between GSH and CSE 20% determined a significant (p=0.005 in HC and p=0.003 in AS) increase of viability when compared to CSE alone and at the same time EA levels dropped considerably (p<0.05 in HC and p=0.003 in AS) down in the presence of this antioxidant Moreover, GSH treatment determined a significantly bigger (p=0.002) overall increase in viability in the AS group when compared to the HC group. In view of this data it could be possible to hypothesise that the typical imbalance in oxidants-antioxidants levels of asthmatic bronchial epithelial cells might be responsible for their increased susceptibility to oxidative stress.