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Background and aim of the study. COVID-19 is characterized by super spread events occurring in communities, e.g., hospitals. To limit virus diffusion among healthcare workers the use of personal protective equipment and screening tests are highly advised; also, isolation of virus positive professionals while moni-toring their health condition is recommended. This study aims to assess, in a cohort of COVID-19 positive quarantined healthcare workers, the perceived source of infection and exposure risk as well as the clinical evolution of the disease through a surveillance interview. Methods. A retrospective observational study ac-counting 896 observations on 93 healthcare professionals tested positive for COVID-19. Data were collected from the Nursing and Technical Directorate of Romagna, Ravenna, Local Health Company, Italy. Results. 99.5% of the positive workers accepted phone interviews with management staff. 2.6% of workers were positive with increasing records in the specialist medical area. Nurses and social health professionals were mostly affected. Patient exposure at a distance 2 hours was the first cause of positivity. In COVID-19 and territorial emergency departments, the first cause was the contact with colleagues. At the time of the infection, most of the staff wore a surgical mask. Cough, asthenia, fever, anosmia, dysgeusia, and rhinitis were common symptoms. Asymptomatic percentage was about 10%. The self-perceived physical condition was high (>7) and improved during the observation period. Conclusions. The diffusion rate of COVID-19 among healthcare workers is relatively low, probably due to the use of personal protective equip-ment. The distancing, also among colleagues, is a fundamental measure to reduce the possibility of infection. Symptoms are mild and can be controlled by surveillance measures. Constant contact with the organization is an essential strategy for promoting recovering of workers and reducing the spread of the virus within the healthcare organization. (www.actabiomedica.it).

BACKGROUND The persisting organ shortage in the field of transplantation recommends the use of marginal kidneys which poorly tolerate ischemic damage. Adenosine triphosphate (ATP) depletion during cold ischemia time (CIT) is considered crucial for graft function. We tested different strategies of kidney perfusion before transplantation in the attempt to improve the technique. MATERIAL AND METHODS Twenty human discarded kidneys from donors after brain death and with at least 20 hours of CIT were randomized to the following experimental groups (treatment time three-hours at 4°C): a) static cold storage (CS); b) static cold hyperbaric oxygenation (Hyp); c) hypothermic perfusion (PE); d) hypothermic perfusion in hyperbaric oxygenation (PE-Hyp); and e) hypothermic oxygenated perfusion (PE-O2). RESULTS Histological results showed that perfusion with or without oxygen did not produce any endothelial damage. A depletion of ATP content following the preservation procedure was observed in CS, PE, and Hyp, while PE-Hyp and PE-O2 were associated with a net increase of ATP content with respect to baseline level. In addition, PE-Hyp was associated with a significant downregulation of endothelial isoform of nitric oxide synthase (eNOS) gene expression and of hypoxia inducible factor-1α (HIF-1α). CONCLUSIONS Hyperbaric or normobaric oxygenation with perfusion improves organ metabolic preservation compared to other methods. This approach may prevent the onset of delayed graft function, but clinical trials are needed to confirm this.

Introduction. Intimal hyperplasia (IH) is characterized by concentric lumen constriction due to marked vascular cell proliferation and is associated with arteriovenous fistula (AVF) early failure in haemodialysis patients. Aim. The present study aimed at establishing ex-vivo cell cultures from patients enrolled for AVF procedure, in order to identify the main cellular and molecular mediators of IH. Methods. Tissues and cells were collected from i) normal veins taken when the first AVF was established, and ii) AVF with IH. Formalin-fixed paraffin-embedded tissue sections were stained with anti CD34, CD44 and MMP-9 monoclonal antibodies. Isolated vascular cells were investigated for bromo-deoxy-uridine incorporation, and migration ability; immunofluorescence (CD34, CD44, ASMA, SOX9) was used to characterize the cell phenotype. Results. Tissue detection of CD34+, CD44+ cells within the IH lesions suggested the involvement of endothelial cells in AVF failure. The expression of MMP-9 protein in the same area supported the activation of migration/matrix remodeling pathways, typical of mesenchymal cells. This same immunophenotype was observed in the cells harvested from IH lesions, together with the detection of ASMA. We observed significant growth differences between cells isolated from normal veins and AVF with IH. Indeed, cells from IH grew faster than cells from normal veins; moreover, IH cells expressed higher levels of SOX9, a transcription factor associated with cell proliferation, and exhibited a marked migration property. Conclusion. Our data evidence that, when compared with cells harvested from not diseased veins, cells derived from AVF with IH have increased proliferation, migration and remodeling abilities. The immunophenotype documented in tissue and cell studies suggests that the endothelial-mesenchymal transition is involved in IH formation and progression. This preliminary study will be expanded with further investigations on IH induction mechanisms and on possible therapeutic targets.

Introduction. The vascular calcification, recruiting osteo-progenitors under inflammatory stimulation, recapitulates the normal bone-biogenesis process. We recently identified a population of Mesenchymal Stem Cells (MSCs) within abdominal aortic aneurysm (AAA) tissues, hypothesizing their contribution to the atherosclerotic plaque calcification. Aim. The present study aimed at exploring the impact of inflammation, a feature of atherosclerosis, on osteogenic differentiation of vascular MSCs. Methods. The extent of calcification within AAA tissues was detected by Alizarin Red stain. MSCs were enzymatically isolated from human healthy cryopreserved thoracic aortas (hMSCs) and from AAA (AAA-MSCs) biopsies. hMSCs and AAA-MSCs were characterized by flow cytometry and compared in terms of osteogenic potential. In order to investigate the effect of the inflammatory milieu on plaque calcification, hMSCs were primed with a cytokine cocktail (TNF-a/IL-1b) for 24 h before osteogenic induction. The osteogenic differentiation was induced by a specific growth media for 21 days, followed by Alizarin Red stain of calcium deposits and osteogenic factors analysis. Results. Alizarin Red confirmed the presence of large calcified areas within the AAA wall. Healthy- and AAA-MSCs both expressed the typical MSC markers CD90, CD73, CD105 and CD44. AAA-MSCs had an higher degree of mineralization activity in comparison to the healthy MSC population, confirmed by the up-regulation of the osteogenic factors BMP-2 and OPN transcriptional levels (1.5 and 15 folds increase when compared to hMSCs, respectively). The inflammatory priming induced the expression of osteogenic factors BMP-2 and OPN genes and proteins in hMSCs. Accordingly, hMSCs pre-cultured under inflammatory conditions also had increased mineralization activity. Conclusion. Vascular MSCs actively regulates the atherosclerosis progression, becoming a crucial source of osteoprogenitors. This process is sharpened by the local inflammatory milieu, supporting a tight association among inflammation, osteogenic differentiation and plaque calcification.

Vascular Calcification, i.e., the ectopic accumulation of calcium phosphate salts in the vessel wall, is associated with atherosclerosis, aging, diabetes, and chronic kidney disease, which thusly contributes to increased cardiovascular morbidity and mortality. Previously considered to be a passive process, vascular calcification is now recognized as an active process similar to bone formation. However, the pathogenetic mechanisms involved in vascular calcification are particularly complex and remain inadequately understood, even though they impact significantly on patient prognosis as well as on the stenting placement and outcome of revascularization. Cellular, subcellular, genetic and molecular mechanisms have been proposed. Of particular importance is the role of the progenitor cells either resident in the vessel wall or circulating that are committed to an osteogenic program as well as transdifferentiation of vascular smooth-muscle cells. In addition, numerous evidences show that metastatic calcification is not as directly related to atherosclerosis as dystrophic is, indicating another mechanism of generation, involving additional metabolic pathways. A better understanding of the pathogenetic mechanisms corroborated with ultrastructural clues, as shown in the present review, will be essential for the development of new therapeutic strategies, in order to prevent and treat vascular calcification.

Le discrasie plasmacellulari sono patologie eterogenee causate da una espansione clonale di plasmacellule o linfociti B con produzione abnorme di immunoglobuline monoclonali intatte o frammentate in catene leggere e/o catene pesanti che si depositano causando danni in diversi organi e tessuti dell’organismo. Il rene è l’organo maggiormente coinvolto e il danno da accumulo di catene leggere che ne deriva può interessare tutte le componenti renali (glomerulo, tubulo e interstizio). Le principali entità patologiche associate al danno renale si manifestano con quadri clinici e alterazioni istologiche/morfologiche molto diversi e comprendono la nefropatia da cast, la tubulopatia prossimale, la nefrite tubulo-interstiziale acuta, la malattia da deposizione di catene leggere o pesanti e l’amiloidosi di tipo AL. Per la diagnosi di queste lesioni, la presenza di un prominente picco monoclonale identificato mediante dosaggio delle catene leggere presenti nel siero e/o nelle urine è affiancata all’esame istologico, immunologico e ultrastrutturale della biopsia renale per definire non solo l’isotipo dell’immunoglobulina coinvolta ma anche l’esatta localizzazione delle catene nefrotossiche nel parenchima renale. Riportiamo due casi di tubulopatia prossimale con cristalli in un paziente affetto da gammopatia monoclonale di significato non determinato (MGUS; caso n. 1) e in un paziente con mieloma multiplo (caso n. 2). Come riportato dalla letteratura, in questa condizione si osserva un accumulo di catene leggere monoclonali nei citoplasmi delle cellule tubulari prossimali che esita nella formazione di strutture cristalline provviste di forme geometriche differenti. In uno dei due casi, i cristalli risultavano presenti anche nel citoplasma dei podociti glomerulari (caso n. 2). L’indagine in immunofluorescenza ha dimostrato come gli inclusi fossero composti da catene leggere kappa. La microscopia elettronica riveste un ruolo determinante per documentare con certezza la presenza di inclusioni cristalline nella tubulopatia prossimale di pazienti affetti da discrasia plasmacellulare.

Mesenchymal Stem Cells (MSCs) possess important characteristics that could be exploited in therapeutic strategies for Type 1 Diabetes (T1D) and for certain complications of Type 2 Diabetes (T2D). MSCs can inhibit autoimmune, alloimmune and inflammatory processes. Moreover, they can promote the function of endogenous and transplanted pancreatic islets. Furthermore, they can stimulate angiogenesis. MSC functions are largely mediated by their secretome, which includes growth factors, exosomes, and other extracellular vesicles. MSCs have shown a good safety profile in clinical trials. MSC-derived exosomes are emerging as an alternative to the transplantation of live MSCs. MSCs harvested from different anatomical locations (e.g. bone marrow, umbilical cord, placenta, adipose tissue, and pancreas) have shown differences in gene expression profiles and function. Data from clinical trials suggest that umbilical cord-derived MSCs could be superior to bone marrow-derived MSCs for the treatment of T1D. Autologous MSCs from diabetic patients may present abnormal functions. BM-MSCs from T1D patients exhibit gene expression differences that may impact in vivo function. BM-MSCs from T2D patients seem to be significantly impaired due to the T2D diabetic milieu. In this review, we highlight how the harvesting site and donor derivation can affect the efficacy of MSC-based treatments for T1D and T2D.

This chapter is devoted to comparing two numerical approaches for modelling the dynamic behaviour of masonry structures. The constitutive equation of masonry-like materials with bounded compressive strength and the dynamic problem for both three-dimensional bodies and one-dimensional structures are presented. The numerical methods implemented in the codes NOSA and MADY, respectively for three- and one-dimensional structures, are outlined. With the aim of comparing the two numerical procedures, two examples, a masonry tower with rectangular cross-section and a masonry arch, are addressed. The structures are subjected to their own weight and to three accelerograms recorded during real earthquakes. The comparison with the numerical results obtained assuming a linear elastic behaviour for the materials constituting the tower and the arch has stressed the importance of accurate constitutive models for masonry.

In recent years exosome research pubblications are rapidly expanding (1). These small vesicles (30‐100 nm) of endocytic origin are thought to participate in cell—cell communication and protein and RNA delivery. A wide range of cells have been shown to release exosomes, but they have also been detected in several biological fluids, including plasma, urine, saliva and breast milk. In particular urinary exosomes have been proposed as starting material to detect protein biomarkers of renal dysfunction and structural injury or overall to shed much insight on the health status of the kidney (2, 3, 4). We aimed to validate methods for exosome isolation from urine in a large animal model, the pig one. The swine shares with the man anatomical and physiological characteristics that make it preferred species as a pre‐clinical model, in particular for kidney functions, surgical approaches and in the view to obtain a significant amount of biological specimens (urine and blood samples, tissue for renal biopsy) compared to other animals. Exosomes were purified by differential ultracentrifugation, identified by electron microscopy and described in morphology, shape, size and distribution using atomic force microscopy (AFM). Validation methods include Western blot with pan‐exosome markers and urinary specific exosome antibodies. Exosome protein content was analyzed through nanospray liquid chromatography‐tandem mass spectrometry (LM‐MS/MS). The total exosomal RNA purificated was evaluated using gel electrophoresis and a Bioanalyzer. We found that the vesicles displayed a typical exosome‐like size and morphology as analyzed by electron microscopy and AFM. Western blot and mass spectrometry further confirmed the presence of several exosome‐associated molecules. Exosome RNA profile detected is typical of these vesicles, lacking rRNA subunits which are prominent in analysis of cellular RNA. Characterization of isolated exosomes indicates that the described isolation methods are suitable for the subsequent RNA and protein profiling.(1) Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell‐to‐cell communication. Kidney Int. 2010 Nov;78(9):838‐48. (2) Fernández‐Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA. Tamm‐Horsfall protein and urinary exosome isolation. Kidney Int. 2010 Apr;77(8):736‐42. (3) Pisitkun T, Shen RF, Knepper MA. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A. 2004 Sep 7;101(36):13368‐73. (4) Lässer C, Eldh M, Lötvall J. Isolation and characterization of RNA‐containing exosomes. J Vis Exp. 2012 Jan 9;(59):e3037.

This paper describes the numerical modelling of the structural behaviour of the church of San Francesco in Lucca and the Rognosa Tower in San Gimignano. The constitutive equation of no-tension materials views masonries as nonlinear elastic materials with zero tensile strength and infinite or bounded compressive strength. As such equation has proven to be able to realistically model the most significant aspects of masonry's behaviour, it has been implemented in the finite element code NOSA-ITACA for solving static and dynamic problems of solids not withstanding tension. The numerical analyses of the church and the tower have been conducted via the NOSA-ITACA code. These case studies highlight the important role of numerical tools in assessing the mechanical behaviour of historical masonry buildings subjected to earthquakes, as well as in planning strengthening operations on such structures.

Introduction. Neoangiogenesis is crucial for the progression and vulnerability of atheromasic lesions. Since adult vasa vasorum, which represent the neoangiogenetic burden of healthy arteries, constitutively express Nestin and Wilms Tumor (WT1), the aims of the present study are: i) to describe and quantify Nestin and WT1 in plaque neovessels; ii) to investigate the relationship between neovessel phenotype and plaque instability. Methods. We prospectively evaluated 49 consecutive carotid endarterectomy specimens. Histopathological characteristics were separately collected, particularly the intraplaque histological complications. Immunohistochemistry was carried out for CD34, Nestin and WT1; the density of positivity was evaluated for each marker. RT-PCR was performed to assess Nestin and WT1 mRNA levels on the first 10 plaques and on 10 control arteries. Results. Six (12.2%) plaques showed no neoangiogenesis. In the others, the mean immunohistochemical densities of CD34, Nestin, and WT1-positive structures were 41.88, 28.84 and 17.68/mm2. Among the CD34+ neovessels, 68% and 42% expressed Nestin and WT1 respectively, i.e., nearly 36% of the neovessels resulted to be Nestin+/WT1-. Furthermore, complicated plaques (n=30) showed significantly more CD34 and Nestin-positive vessels than uncomplicated plaques (n=13; P=0.045 and P=0.009), while WT1 was not increased (P=0.139). RT-PCR confirmed that WT1 gene expression was 3-fold lower than Nestin gene in plaques (p=0.001). Conclusions. Plaque neoangiogenesis shows both a Nestin+/WT1- and a Nestin+/WT1+ phenotype. The Nestin+/WT1- neovessels are significantly more abundant in complicated (vulnerable) plaques. The identification of new transcription factors in plaque neoangiogenesis, and their possible regulation, can open new perspectives in the therapy of vulnerable plaques.

The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.