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Chronic hepatitis C virus (HCV) infection is a major public health problem at a global level, causing an enormous burden of hepatic and extra-hepatic morbidity and mortality. Treatment of chronic HCV (CHC) has been revolutionized in the last few years by the introduction of highly effective and well tolerated direct acting antiviral agents (DAAs) able to achieve >90% rates of sustained virological response (SVR) in many groups of patients, including those previously excluded from interferon-based regimens. For such reason interferon-free regimens are now the treatments of choice for all patients. Successful anti-HCV treatment can stop liver disease progression and can solve the HCV-related extra hepatic manifestations, eventually reducing both liver-related and overall mortality. Together with the rapidly accumulating data about the evolution of treatment landscape, different guidelines from national and international Liver Scientific Societies have been published until today. However, these recommendations may not be applied worldwide as, due to high treatment costs, most of them identify as priority groups only patients with advanced liver disease. Moreover some types of patients pose clinical management problems for which even the guidelines do not always provide useful answers. With the aim of treatment optimization by filling some of the gaps of the current guidelines and addressing the remaining unmet needs in practice, a group of Italian experts, experienced on treatment of HCV infection, met in Stresa in February 2016. The summary of all the considerations arising from this two-day meeting and the final statements are reported in this position paper.

Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, several affinity reagents against fluorescent proteins have also been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfil their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or in living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.

L’artrosi e le tendinopatie sono patologie che presentano simili sintomi, quesiti clinici ed eziologia, entrambe caratterizzate da infiammazione e degenerazione tissutale. I trattamenti conservativi attualmente disponibili hanno dimostrato scarsa efficacia, portando interesse nello sviluppo di strategie di medicina rigenerativa, mirate a sostituire o rigenerare le cellule e i tessuti danneggiati. Le cellule staminali mesenchimali (MSC) rappresentano lo strumento più promettente per questi approcci, grazie alla loro capacità di proliferare e differenziarsi verso specifiche linee cellulari, oltre alle loro proprietà anti-infiammatorie e trofiche. Le principali fonti di MSC sono il midollo osseo e il tessuto adiposo, sebbene esse siano presenti in quasi tutti i distretti corporei, con localizzazione perivascolare. Infatti sono state recentemente descritte come periciti, cellule deputate a rispondere al danno tissutale ripristinando l'omeostasi. L'interesse verso fonti alternative di MSC è nato a causa della loro limitata capacità di differenziare in vivo, soprattutto verso la linea tenogenica e condrogenica. In questo contesto sono state identificate alcune sottopopolazioni di cellule tendinee e cartilaginee con abilità clonogeniche e differenziative, in grado di partecipare direttamente alla rigenerazione tissutale. Tuttavia, l'attività paracrina di questi progenitori, così come la loro reale natura di mesenchimali, è ancora dibattuta. Il primo obiettivo di questa ricerca è stato quindi quello di identificare i criteri minimi tipici delle MSC in queste sottopopolazioni in termini di capacità differenziative, proliferative e di immunofenotipo. Le cellule staminali/progenitrici del tendine (TSPC) sono risultate in possesso di questi criteri, mostrando anche una scarsa immunogenicità e una capacità immumodulatoria tipica delle ASC. Al contrario, le cellule cartilaginee possiedono una maggior capacità di differenziazione verso la cartilagine ialina, ma hanno, Osteoarthritis and tendinopathy are common orthopaedic conditions with similar etiology, symptoms and clinical issues. They both are characterized by chronic inflammation and tissue degeneration, and the current conservative treatments do not effectively contrast the pathologic progression. The clinical need for innovative treatment has led to the development of regenerative medicine strategies, aimed to replace or regenerate cells and tissues. In this setting, mesenchymal stem cells (MSCs) represents the most promising tool, thanks to their ability to proliferate and differentiate towards specific cell lineages, as well as their paracrine action on the immune system and on the tissue resident cells. Although the most used sources of MSCs are bone marrow and adipose tissue, they are present in almost all body compartments, with a perivascular localization. Indeed, MSCs have been recently described as pericytes, deputed to respond to damage and restore tissue homeostasis. The interest in new sources of MSCs resides in the discovery of their limited in vivo differentiation ability, especially towards specific lineages such as tenocytes and chondrocytes. To face this, subpopulations of tendon and cartilage derived cells were found to possess clonogenic and differentiation abilities, and they were proposed as possible candidates for specific tissue regeneration. Contrary to their well-known ability to participate directly to tissue regeneration, the paracrine activity of these specific progenitors is still unclear, as well as their real nature as MSCs. Then, the first goal of this research was to identify the MSCs minimal criteria in these subpopulations, in terms of differentiation ability, immunophenotype and proliferation. We found that tendon stem/progenitor cells (TSPCs) possess these minimal criteria, and share the low immunogenicity and the immumodulatory ability with adipose derived stem cells (ASCs). On the contrary, while cartilage cells are the most effective

Background: Tendon resident cells (TCs) are a mixed population made of terminally differentiated tenocytes and tendon stem/progenitor cells (TSPCs). Since the enrichment of progenitors proportion could enhance the effectiveness of treatments based on these cell populations, the interest on the effect of culture conditions on the TSPCs is growing. In this study the clonal selection and the culture in presence or absence of basic fibroblast growth factor (bFGF) were used to assess their influences on the stemness properties and phenotype specific features of tendon cells. Methods: Cells cultured with the different methods were analyzed in terms of clonogenic and differentiation abilities, stem and tendon specific genes expression and immunophenotype at passage 2 and passage 4. Results: The clonal selection allowed to isolate cells with a higher multi-differentiation potential, but at the same time a lower proliferation rate in comparison to the whole population. Moreover, the clones express a higher amounts of stemness marker OCT4 and tendon specific transcription factor Scleraxis (SCX) mRNA, but a lower level of decorin (DCN). On the other hand, the number of cells obtained by clonal selection was extremely low and most of the clones were unable to reach a high number of passages in cultures. The presence of bFGF influences TCs morphology, enhance their proliferation rate and reduce their clonogenic ability. Interestingly, the expression of CD54, a known mesenchymal stem cell marker, is reduced in presence of bFGF at early passages. Nevertheless, bFGF does not affect the chondrogenic and osteogenic potential of TCs and the expression of tendon specific markers, while it was able to downregulate the OCT4 expression. Conclusion: This study showed that clonal selection enhance progenitors content in TCs populations, but the extremely low number of cells produced with this method could represent an insurmountable obstacle to its application in clinical approaches. We obs

Methods: A bicentre, randomized, prospective open-label study aimed at defining a DNAaemia versus antigenaemia cutoff for guiding preemptive therapy of human cytomegalovirus (HCMV) infections in solid organ transplant recipients (SOTR) was completed. Overall, 99 patients were enrolled in the DNAaemia arm and 101 patients in the antigenaemia arm. Patients were randomized to be monitored for HCMV infection in the blood by either assay. Antiviral treatment was started in both seropositive and seronegative patients when levels greater than 300,000 DNA copies/ml blood or 100 pp65-positive leukocytes in the relevant arm were reached. Results: HCMV infection was detected in 81/99 (81.8%) patients in the DNAaemia arm and in 87/101 (86.1%) patients in the antigenaemia arm (P=ns). Antiviral treatment was given to 23/99 (23.0%) patients in the DNAaemia arm and 42/101 (41.0%) patients in the antigenaemia arm (P=0.01). In the DNAaemia arm, antiviral therapy was significantly delayed and duration of the first course of treatment was significantly greater than in the antigenaemia arm. However, total duration of treatment was comparable in the two arms. No case of HCMV disease occurred in patients treated after reaching the relevant cutoff. However, four patients (three in the antigenaemia arm, and one in the DNAaemia arm) suffered from HCMV disease prior to reaching the relevant cutoff. Conclusions: Compared with antigenaemia, a single DNAaemia cutoff: (i) significantly reduces the number of patients requiring treatment; (ii) may be safely adopted to guide preemptive therapy of both primary and reactivated HCMV infections in SOTR; and (iii) does not significantly modify the overall duration of treatment.

Background: Musculoskeletal disorders are regarded as a major cause of worldwide morbidity and disability, and they result in huge costs for national health care systems. Traditional therapies frequently turned out to be poorly effective in treating bone, cartilage, and tendon disorders or joint degeneration. As a consequence, the development of novel biological therapies that can treat more effectively these conditions should be the highest priority in regenerative medicine. Main body of the abstract: Mesenchymal stem cells (MSCs) represent one of the most promising tools in musculoskeletal tissue regenerative medicine, thanks to their proliferation and differentiation potential and their immunomodulatory and trophic ability. Indeed, MSC-based approaches have been proposed for the treatment of almost all orthopedic conditions, starting from different cell sources, alone or in combination with scaffolds and growth factors, and in one-step or two-step procedures. While all these approaches would require cell harvesting and transplantation, the possibility to stimulate the endogenous MSCs to enhance their tissue homeostasis activity represents a less-invasive and cost-effective therapeutic strategy. Nowadays, the role of tissue-specific resident stem cells as possible therapeutic target in degenerative pathologies is underinvestigated. Biophysical stimulations, and in particular extracorporeal shock waves treatment and pulsed electromagnetic fields, are able to induce proliferation and support differentiation of MSCs from different origins and affect their paracrine production of growth factors and cytokines. Short conclusions: The present review reports the attempts to exploit the resident stem cell potential in musculoskeletal pathologies, highlighting the role of MSCs as therapeutic target of currently applied biophysical treatments.

Purpose: The purpose of this study was to translate the Oxford Ankle Foot Questionnaire (OAFQ) into Italian, to perform a cross-cultural adaptation and to evaluate its psychometric properties. Methods: The Italian OAFQ was developed according to the recommended forward/backward translation protocol and evaluated in pediatric patients treated for symptomatic flatfoot deformity. Feasibility, reliability, internal consistency, construct validity [comparing OAFQ domains with Child Health Questionnaire (CHQ) domains] and responsiveness to surgical treatment were assessed. Results: A total of 61 children and their parents were enrolled in the study. Results showed satisfactory levels of internal consistency for both children and parent forms. The test–retest reliability was confirmed by high ICC values for both child and parents subscales. Good construct validity was showed by patterns of relationships consistent with theoretically related domains of the CHQ. After surgery, the mean OAFQ scores improved in all the domains after treatment with the subtalar arthroereisis, for both children and parent scales (p < 0.01). Effect size ranged from small to moderate for almost all domains. Conclusions: The Italian version of the OAFQ might be a reliable and valid instrument in order to evaluate interventions used to treat children’s foot or ankle problem, but needs further study on different clinical settings.

Tendinopathy is a big burden in clinics and it represents 45% of musculoskeletal lesions. Despite the relevant social impact, both pathogenesis and development of the tendinopathy are still under-investigated, thus limiting the therapeutic advancement in this field. The purpose of this study was to evaluate the dose-dependent and time-related tissue-level changes occurring in a collagenase-induced tendinopathy in rat Achilles tendons, in order to establish a standardized model for future pre-clinical studies. With this purpose, 40 Sprague Dawley rats were randomly divided into two groups, treated by injecting collagenase type I within the Achilles tendon at 1 mg/mL (low dose) or 3 mg/mL (high dose). Tendon explants were histologically evaluated at 3, 7, 15, 30 and 45 days. Our results revealed that both the collagenase doses induced a disorganization of collagen fibers and increased the number of rounded resident cells. In particular, the high dose treatment determined a greater neovascularization and fatty degeneration with respect to the lower dose. These changes were found to be time-dependent and to resemble the features of human tendinopathy. Indeed, in our series, the acute phase occurred from day 3 to day 15, and then progressed towards the proliferative phase from day 30 to day 45 displaying a degenerative appearance associated with a very precocious and mild remodeling process. The model represents a good balance between similarity with histological features of human tendinopathy and feasibility, in terms of tendon size to create lesions and costs when compared to other animal models. Moreover, this model could contribute to improve the knowledge in this field, and it could be useful to properly design further pre-clinical studies to test innovative treatments for tendinopathy.