Novel intracellular signalling regulators of cartilage progenitor cell populations

Cartilage is a viscoelastic tissue that absorbs shocks and facilitates low friction of the joint. Adult articular cartilage is limited in its ability to self-repair. Lesions or gradual wear-and-tear affect cartilage integrity and lead to damage that, when untreated, can ultimately develop to osteoarthritis. Osteoarthritis is a considerable health burden and is a leading cause of disability worldwide. In order to address this burdensome condition, several treatments have been developed. In particular, therapies that allow delivery of bone marrow and its constituent cell populations to the site of cartilage damage to form a regenerative clot have proved promising in repairing joint cartilage defects. In order to improve such regenerative technique, further research is required to provide a deeper understanding of the mechanisms underlying cartilage regeneration. One of the critical players are the cells that originally reside in the cartilage surrounding the damage. How these resident cells contribute to the activity of cells within the repair tissue at the site of cartilage damage is largely undescribed. In this thesis chondrocytes from three different cartilage areas were compared: chondrocytes from 1) the superficial (SZ) and 2) the middle-deep (MDZ) zone of non-weight bearing femoral condyles, and from 3) the osteoarthritic zone (OAZ) of patients undergoing knee replacement. More inflammatory factors and cytokines are present in MSCs co-culture with OA cartilage chips, and with MDZ cartilage chips. To assess how chondrocyte-MSC crosstalk would affect MSCs chondrogenesis, cartilage chips from the three different zones were co-cultured with BMSC pellets. Results indicated that the SZ induce chondrogenic differentiation of BMSCs, whereas MDZ and OAZ have a negative effect, compared to control conditions. The findings suggest that the presence of SZ, which has been reported to reduce with age, is important to direct BMSCs differentiation towards the chondrogenic fate. In order to better understand the molecular mechanism that differentiate SZ from MDZ and OA chondrocytes, DACTs (Dapper antagonist of catenin) were studied. DACT1 and DACT2 are known to be Wnt and TGFβ pathways regulators, but their role in chondrocytes and MSCs has not been described before. Both proteins are present in chondrocytes throughout the osteoarthritic human tissue, including in chondrocytes forming cell clusters. On the non-weight bearing and visually undamaged cartilage, DACT1 and DACT2 expression is localised to the articular surface. In mouse embryos (E.15.5), DACT2 is expressed at the interzone, site of developing synovial joint, indicating that DACT2 is expressed in cells that give rise to the articular joint. Subsequently the expression of DACT1 and DACT2 was analysed in MSCs: both are expressed in synovial and bone marrow-derived MSCs. Following this observation an RNAi knockdown experiment showed that DACT1 knockdown in both chondrocyte and MSCs causes the cells to undergo apoptosis within 24 hours. To understand the pathway regulated by DACT1, next generation sequencing gene expression analysis was performed on BMSCs where DACT1 had been reduced by RNAi. The study showed that loss of DACT1 influences the expression (p<0.05) of genes involved in both TGFβ and Wnt pathways and putative link to relevant cell regulatory pathways (Ingenuity® Pathway Analysis). SMURF2 and other genes involved in protein phosphorylation and degradation are downregulated following DACT1 knockdown. This suggests that DACT1 is an upstream regulator of SMAD protein phosphorylation affecting proliferation and survival of MSCs. The data presented in this thesis has indicated that different types of chondrocytes are present within human articular cartilage and that they cross talk with MSCs differently according to their regional origin. This information offers a new level of complexity to consider to improve regenerative techniques. In addition, this work describes for the first time, the presence and biological relevance of DACT1 and DACT2 in chondrocytes and MSCs. DACT1 is involved in MSCs survival and is downregulated in OA which suggest that this is an important regulatory protein. Further studies of DACT1 could help elucidate mechanisms involved in OA, but also uncover the relevance of cartilage progenitors loss in the development of cartilage degeneration

Danno d'organo subclinico in soggetti a basso rischio cardiovascolare

Titolo: Danno d’organo subclinico in soggetti a basso rischio cardiovascolare Scopo dello studio: Le attuali linee guida per la definizione del rischio cardiovascolare suggeriscono l’uso di un calcolo del rischio globale basato sui tradizionali fattori di rischio per tutti gli adulti asintomatici. Questi punteggi non considerano il danno d’organo subclinico con un possibile errore tra il rischio cardiovascolare calcolato e la reale estensione della malattia aterosclerotica in quel soggetto. Scopo di questo studio è definire la prevalenza dei fattori di rischio tradizionali e valutare la presenza di danno d’organo subclinico in soggetti asintomatici a basso rischio cardiovascolare. Disegno e metodi: Abbiamo reclutato 244 soggetti volontari asintomatici (168 maschi e 76 femmine), di età compresa tra i 40 e i 64 anni; abbiamo individuato i loro fattori di rischio cardiovascolari e abbiamo calcolato il loro rischio cardiovascolare globale utilizzando le linee guida del “Progetto Cuore”. Infine abbiamo ricercato il danno d’organo subclinico con l’ecocolordoppler delle Carotidi, il calcolo dell’indice ABI, l’ecocardiogramma transtoracico e l’elettrocardiogramma. Risultati: Abbiamo trovato un’elevata prevalenza di fattori di rischio modificabili. Abbiamo anche rilevato una scarsa consapevolezza del rischio cardiovascolare nella popolazione, con un’alta prevalenza di patologie misconosciute (ipertensione: 44% dei maschi e delle femmine%; ipercolesterolemia: 34% dei maschi e 25% delle femmine). Quasi tutti i pazienti (94% dei maschi e 100% delle femmine) avevano un rischio cardiovascolare inferiore al 10% (rischio di avere un evento acuto cardiovascolare nei successivi 10 anni). Persino tra questi soggetti a rischio relativamente basso, abbiamo trovato un’alta prevalenza di danno vascolare (14% dei maschi e 4% delle femmine avevano lo spessore intima-media> 0,9 mm, il 22% dei maschi e il 5% delle femmine avevano almeno una placca a livello carotideo, il 4% dei maschi e il 5% delle femmine avevano un indice caviglia-braccio patologico) e d’iniziale rimodellamento cardiaco (nel 12% dei maschi lo spessore del setto cardiaco era superiore a 1,1 cm). Conclusioni: Il nostro risultato principale è che perfino i soggetti con un rischio cardiovascolare basso (secondo il calcolo del rischio individuale) hanno un’alta prevalenza di danno d’organo subclinico; è quindi nostra opinione che la ricerca del danno d’organo potrebbe essere utile per ridefinire il rischio cardiovascolare per ottenere il calcolo di un punteggio realmente individuale.ROLE OF ORGAN DAMAGE MARKERS IN GLOBAL EVALUATION OF CARDIOVASCULAR RISK IN LOW RISK PATIENTS. Objective: Current guidelines for the assessment of cardiovascular risk suggest the use of a global risk score based on traditional risk factors for all asymptomatic adults. Actually these scores don’t consider target organ damage leading to a possible mismatch between individual risk score and the real atherosclerotic burden of a subject. The aim of this study is to assess the prevalence of traditional risk factors and to evaluate the presence of target organ damage, focusing attention on low risk patients. Design and method: We recruited 244 asymptomatic volunteers (168 males and 76 females) aged between 40 and 64, we assessed their traditional risk factors and we calculated the cardiovascular risk score based on the Italian “Progetto Cuore” guidelines. Eventually, we evaluated target organ damage with Carotid Ultrasound, Ankle-Brachial Index, Transthoracic Echocardiography and Electrocardiography. Results: We found a high prevalence of avoidable risk factors. We also noticed remarkably poor awareness of cardiovascular risk in the population, with a high prevalence of undiagnosed conditions (hypertension: 44% of men and 44% of women; hypercholesterolemia: 34% of men and 25% of women). Almost all patients (94% of males and 100% of females) had a cardiovascular risk score 0.9 mm, 22% of men and 5% of women had at least a carotid plaque, 4% of men and 5% of women had pathological ankle-brachial index) and starting cardiac remodeling (in 12% of men cardiac septum was >1.1 cm). Conclusions: Our main finding is that even subjects with low cardiovascular risk (according to individual risk score) have a high prevalence of target organ damage, so the research of organ damage could be useful to reclassify cardio-vascular risk in an assessment of a real “individual-based” risk-score

Hepatitis A Virus among Drug Users and the Role of Vaccination: A Review

In countries with advanced economies better health and hygiene conditions, along with the introduction, in some cases, of global vaccination, have relegated most viral hepatitis to marginal social groups and, in particular, drug users (DUs). The availability of safe and effective vaccines for hepatitis A virus (HAV) and B (HBV) may play a major role in combating this phenomenon. Despite the availability of a safe and effective vaccine for over a decade and the recommendations of international health organizations, vaccinations against HAV among DUs are not as widely known and available as are HBV vaccinations. The purpose of this review article is to present the most significant data in the literature on the prevalence of HAV among DUs and the role of targeted vaccination. To our knowledge, the present article is the first to solely deal with vaccination against HAV in DUs. Immunization after the administration of anti-HAV vaccine has been demonstrated in DUs even if they have responded significantly less than either the general population or carriers of chronic liver disease. All the vaccines were well tolerated and adherence to the vaccine schedule was good. Further studies are needed to optimize the timing and doses of vaccine to be administered to DUs, especially to assess adherence and antibody persistence. Vaccination campaigns are feasible among DUs and have proven to be highly cost–effective

Dynamic Regulation of TWIST1 Expression during Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Human bone marrow-derived mesenchymal stem cells (BMSCs) are clinically promising to repair damaged articular cartilage. This study investigated TWIST1, an important transcriptional regulator in mesenchymal lineages, in BMSC chondrogenesis. We hypothesized that downregulation of TWIST1 expression is required for in vitro chondrogenic differentiation. Indeed, significant downregulation of TWIST1 was observed in murine skeletal progenitor cells during limb development (_N_ = 3 embryos), and during chondrogenic differentiation of culture-expanded human articular chondrocytes (_N_ = 3 donors) and isolated adult human BMSCs (_N_ = 7 donors), consistent with an inhibitory effect of TWIST1 expression on chondrogenic differentiation. Silencing of TWIST1 expression in BMSCs by siRNA, however, did not improve chondrogenic differentiation potential. Interestingly, additional investigation revealed that downregulation of TWIST1 in chondrogenic BMSCs is preceded by an initial upregulation. Similar upregulation is observed in non-chondrogenic BMSCs (_N_ = 5 donors); however, non-chondrogenic cells fail to downregulate TWIST1 expression thereafter, preventing their chondrogenic differentiation. This study describes for the first time endogenous TWIST1 expression during in vitro chondrogenic differentiation of human BMSCs, demonstrating dynamic regulation of TWIST1 expression whereby upregulation and then downregulation of TWIST1 expression are required for chondrogenic differentiation of BMSCs. Elucidation of the molecular regulation of, and by, TWIST1 will provide targets for optimization of BMSC chondrogenic differentiation culture

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Serum Oxidative Stress-Induced Repression of Nrf2 and GSH Depletion: A Mechanism Potentially Involved in Endothelial Dysfunction of Young Smokers

Background: Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs). Methods and Results: 52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers ’ serum but not to non-smokers ’ serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamatecysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found i

Leucocyte Abnormalities in Synovial Fluid of Degenerative and Inflammatory Arthropathies

Genome damage has been related to the induction of autoimmune processes, chronic inflammation, and apoptosis. Recent studies suggest that some rheumatological diseases are associated with overall genomic instability in the T cell compartment. However, no data regarding leucocyte abnormalities in synovial fluid (SF) and their relationship with inflammation are available. The aim of this study was to investigate cellular phenotypes in SF collected from patients with different inflammatory arthropathies, including rhematoid arthritis (RA), psoriatic arthritis (PsA), crystal-induced arthritis (CIA), and non-inflammatory arthropathies, such as osteoarthritis (OA). We found high percentage of micronuclei in SF from CIA compared to the other groups and a high frequency of pyknotic cell in RA and CIA patients. A correlation between pyknosis and immature polymorphonuclear cells with local inflammatory indices was observed. The study of the apoptosis process revealed an increased BAX expression in CIA and RA compared to OA and PsA, while Bcl-2 was higher in CIA. Caspase-3 activity was increased in SF from RA patients and correlates with inflammatory and anti-inflammatory cytokines. In conclusion, our results showed that inflammatory SF is associated with genomic instability and abnormal cell subset