NEW INSIGHTS AND POSSIBLE THERAPEUTIC IMPLICATIONS OF ADENOSINE ANALOGS AND PULSED ELECTROMAGNETIC FIELDS (PEMFs) IN OSTEOARTICULAR PATHOLOGIES

Objective: The aim of this study was to characterize adenosine receptors in synovial fibroblasts (SFs) and to investigate the potential link between adenosine pathway and pulsed electromagnetic fields (PEMFs). In particular, we articulated our study and we pointed our efforts to: 1) characterize, by a pharmacological point of view, the presence of adenosine receptors subtypes (A1, A2A, A2B and A3) in two cell models: bovine and human SFs; 2) verify the effect of PEMFs on affinity and density parameters of the adenosine receptors characterized ; 3) investigate the functionality of adenosine receptor subtypes in the presence and in the absence of PEMFs through the analysis of cAMP release; 4) investigate if adenosine receptor agonists and PEMF biophysical stimulation, alone or combined, may modulate pro-inflammatory parameters (PGE-2 and IL-6 release; COX-2 expression) in SFs treated with known inflammatory stimuli. Methods: SFs isolated from bovine synovial fluids or from human synovial OA pannus were cultured in monolayer. Western blotting analysis was done to confirm the expression of adenosine receptors in bovine and human SFs. Moreover, competition binding experiments in the absence and in the presence of PEMFs on the adenosine receptors were also performed. In the adenylate cyclase assays, the cAMP levels modulated by typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs were evaluated in both cell types. Further, bovine SFs were treated with TNF-a (10 ng/ml) to activate inflammatory response. Adenosine analogs (CHA for A1 receptors, NECA non-selective agonist, CGS 21680 for A2A receptors and Cl-IB-MECA for A3 receptors) were added to control and TNF-α-treated bovine cultures both in the absence and in the presence of adenosine deaminase (ADA), which is used to deplete endogenous adenosine. Parallel cultures of bovine SFs were exposed to PEMFs (75 Hz, 1.5 mT) during the period in culture (24 hours). PGE-2 release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. In addition, human SFs were treated with f IL-1β (50 ng/ml) to activate inflammatory response. Parallel cultures of human SFs were exposed to PEMFs (75 Hz, 1.5 mT) during the period in culture (24 hours). PGE-2 and IL-6 release was measured by immunoassays. Results: Bovine and human SFs expressed all adenosine receptors (A1, A2A, A2B, A3). PEMFs evoked an up-regulation of A2A and A3 receptors in both cell types. In both PEMF-treated cell models, cAMP levels modulated by A2A or A3 agonists were significantly increased and decreased respectively, when compared with the untreated cells, both in human and in bovine SFs. Further, TNF-α significantly stimulated PGE-2 release in bovine SFs. All adenosine agonists, except for Cl- IB-MECA, significantly inhibited PGE-2 production. PEMFs inhibited PGE-2 production in the absence of adenosine agonists and increased the effects of CHA, CGS 21680 and NECA. In ADA, the inhibition on PGE-2 release induced by CHA, CGS 21680 and NECA was stronger than in the absence of ADA and the PEMF-inhibitory effect was lost. Changes in PGE-2 levels were associated to a modification of COX-2 expression. To what concern human SFs, IL-1β strongly increased both PGE-2 and IL-6 release. In parallel experiments, PEMF exposure significantly inhibited PGE-2 and IL-6 release. Conclusions: All adenosine receptors are present and have a similar pharmacological behaviour both in bovine and in human SFs. Further, in these cells PEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities. In addition, this study supports anti-inflammatory activities of A1 and A2A adenosine receptors and PEMFs in bovine SFs. PEMF activity appears to be mediated by a PEMF-induced up-regulation of A2A receptors. Finally, PEMF exposure seems exert anti-inflammatory activities in human SFs. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation, and further may open interesting perspectives to develop new therapeutic approaches in osteoarticular pathologies

Electromagnetic fields counteract IL-1β during chondrogenesis in synovial bovine mesenchymal progenitor cells

Objective. Mesenchymal stem cells (MSCs) isolated from synovium and from synovial fluid, have shown a chondrogenesis potential suggesting that synovium is an excellent source of MSCs for cartilage regeneration. Electromagnetic fields (EMFs) display several effects on cartilage: increase the synthesis of proteoglycans (PGs), prevents the catabolic effect of the pro-inflammatory cytokine interleukin-1β (IL-1β), appear useful for the treatment of osteoarthritis. Our goal was to evaluate if the chondrogenic differentiation of synovial bovine mesenchymal progenitor cells, may be influenced by EMFs. Further, as chondrogenic differentiation of MSCs could be altered in an inflammatory environment and EMFs can counteract IL-1β activity, we also evaluated the role of EMFs during chondrogenic differentiation in the presence of IL-1β. Design. Synovial fluid was aspirated from the metacarpophalangeal joints of bovine. Synovial cells at the 3rd passage were centrifuged to obtain pellet cultures. Pellets were cultured in chondrogenic medium alone (control) or supplemented with 10 ng/ml TGF-β3 and/or 50 ng/ml IL-1β. The pellets were unexposed or exposed to EMF (75 Hz, 1.5 mT) (Igea, Carpi, Italy), during the whole period in culture (34 days). Alcian blue for sulphated glycosaminoglycans and immunostaining for type II collagen, were performed. PG synthesis was measured by radioactive 35S-sulphate incorporation. Results. Pellets cultured in the presence of TGF-β3 exhibited positive staining for type II collagen and Alcian blue, compared to control, indicating chondrogenic differentiation of synovial bovine mesenchymal progenitor cells. In the presence of IL-1β, type II collagen and Alcian blue staining dramatically decreased compared to TGF-β3 treatment alone. When pellets treated with both TGF-β3 and IL-1β were exposed to EMF, the histochemical staining for type II collagen and Alcian blue increased compared to EMF-unexposed pellets, suggesting that EMF might counteract the IL-1β effect. Biochemical analysis on PG synthesis confirmed histochemical data. Conclusions. The presence of inflammatory cytokines, such as IL-1β in human joints, may explain why existing methods of cartilage engineering repair strategies, that rely on the in situ differentiation of MSCs, fail to provide a reliably successful. Results of this study support the hypothesis that EMF treatment may favour chondrogenic differentiation in inflammatory conditions, suggesting a possible strategy for improving the clinical outcome of cartilage repair procedures

The Middle Part of the Plucked Hair Follicle Outer Root Sheath Is Identified as an Area Rich in Lineage-Specific Stem Cell Markers

Hair follicle outer root sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E2 and cytokine production in human osteoarthritic synovial fibroblasts

Objective. Synovial fibroblasts (SFs) contribute to the development of osteoarthritis (OA) by the secretion of a wide range of pro-inflammatory mediators, including cytokines and lipid mediators of inflammation (1). Previous studies show that electromagnetic fields (EMFs) may represent a potential therapeutical approach to limit cartilage degradation and to control inflammation associated to OA, and that they may act through the adenosine pathway (2). On this basis the aim of this study was to investigate if EMFs might modulate inflammatory activities of human SFs derived from OA patients (OASFs) and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. Design. SFs obtained from OA patients, undergoing total hip joint replacement surgery, were exposed to EMFs (1.5 mT; 75 Hz) for 24 hours. In control and EMF-exposed cells, ARs were evaluated by western blotting, quantitative real-time RT-PCR and saturation binding experiments and cAMP levels were measured by a specific assay. In the absence and in the presence of interleukin-1β (IL-1β), used as a pro-inflammatory stimulus, prostaglandin E2 (PGE2), cytokine and matrix degrading enzyme production was evaluated in OASFs exposed to EMFs and treated with selective adenosine receptor agonists and antagonists. Results. EMF exposure induced a selective increase in A2A and A3 ARs. These increases were associated to changes in cAMP levels, indicating that ARs were functionally active in EMF-exposed cells. In IL-1β-treated OASFs, functional data obtained in the presence of  A2A and A3 adenosine agonists and antagonists showed that EMFs inhibit the release of (PGE2) and of the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), whilst stimulate the release of interleukin-10 (IL-10), an antinflammatory cytokine. Further, results show that these effects appear to be mediated by the EMF-induced upregulation of A2A and A3 ARs. No effects of EMFs or ARs have been observed on matrix degrading enzymes production. Conclusions: EMFs display anti-inflammatory effects in human OASFs and these EMF-induced .ffects are in part mediated by the adenosine pathway, specifically by the A2A and A3 ARs activation. Taken together, these results suggest that SFs could represent potential therapeutic targets cells for EMF treatment and open new clinical perspectives to the control of inflammation associated to joint diseases. 1. Martel-Pelletier J et al. Eklem Hastalik Cerrahisi. 2010; 21(1):2-14. 2. De Mattei M et al. Osteoarthritis Cartilage. 2009; 17(2):252-262

Autologous, Non-Invasively Available Mesenchymal Stem Cells from the Outer Root Sheath of Hair Follicle Are Obtainable by Migration from Plucked Hair Follicles and Expandable in Scalable Amounts

Background: Regenerative therapies based on autologous mesenchymal stem cells (MSC) as well as stem cells in general are still facing an unmet need for non-invasive sampling, availability, and scalability. The only known adult source of autologous MSCs permanently available with no pain, discomfort, or infection risk is the outer root sheath of the hair follicle (ORS). Methods: This study presents a non-invasively-based method for isolating and expanding MSCs from the ORS (MSCORS) by means of cell migration and expansion in air–liquid culture. Results: The method yielded 5 million cells of pure MSCORS cultured in 35 days, thereby superseding prior art methods of culturing MSCs from hair follicles. MSCORS features corresponded to the International Society for Cell Therapy characterization panel for MSCs: adherence to plastic, proliferation, colony forming, expression of MSC-markers, and adipo-, osteo-, and chondro-differentiation capacity. Additionally, MSCORS displayed facilitated random-oriented migration and high proliferation, pronounced marker expression, extended endothelial and smooth muscle differentiation capacity, as well as a paracrine immunomodulatory effect on monocytes. MSCORS matched or even exceeded control adipose-derived MSCs in most of the assessed qualities. Conclusions: MSCORS qualify for a variety of autologous regenerative treatments of chronic disorders and prophylactic cryopreservation for purposes of acute treatments in personalized medicine

Dupilumab in the treatment of severe uncontrolled chronic rhinosinusitis with nasal polyps (CRSwNP): A multicentric observational Phase IV real-life study (DUPIREAL)

Background Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with significant morbidity and reduced health-related quality of life. Findings from clinical trials have demonstrated the effectiveness of dupilumab in CRSwNP, although real-world evidence is still limited. Methods This Phase IV real-life, observational, multicenter study assessed the effectiveness and safety of dupilumab in patients with severe uncontrolled CRSwNP (n = 648) over the first year of treatment. We collected data at baseline and after 1, 3, 6, 9, and 12 months of follow-up. We focused on nasal polyps score (NPS), symptoms, and olfactory function. We stratified outcomes by comorbidities, previous surgery, and adherence to intranasal corticosteroids, and examined the success rates based on current guidelines, as well as potential predictors of response at each timepoint. Results We observed a significant decrease in NPS from a median value of 6 (IQR 5–6) at baseline to 1.0 (IQR 0.0–2.0) at 12 months (p < .001), and a significant decrease in Sino-Nasal Outcomes Test-22 (SNOT-22) from a median score of 58 (IQR 49–70) at baseline to 11 (IQR 6–21; p < .001) at 12 months. Sniffin' Sticks scores showed a significant increase over 12 months (p < .001) compared to baseline. The results were unaffected by concomitant diseases, number of previous surgeries, and adherence to topical steroids, except for minor differences in rapidity of action. An excellent-moderate response was observed in 96.9% of patients at 12 months based on EPOS 2020 criteria. Conclusions Our findings from this large-scale real-life study support the effectiveness of dupilumab as an add-on therapy in patients with severe uncontrolled CRSwNP in reducing polyp size and improving the quality of life, severity of symptoms, nasal congestion, and smell

Electromagnetic Fields Counteract IL-1β Activity during Chondrogenesis of Bovine Mesenchymal Stem Cells

Osteoarthritis (OA) is a common joint disease associated with articular cartilage degeneration. To improve the therapeutic options of OA, tissue engineering based on the use of mesenchymal stem cells (MSCs) has emerged. However, the presence of inflammatory cytokines, such as interleukin-1β (IL-1β), during chondrogenesis reduces the efficacy of cartilage engineering repair procedures by preventing chondrogenic differentiation. Previous studies have shown that electromagnetic fields (EMFs) stimulate anabolic processes in OA cartilage and limit IL-1β catabolic effects. We investigated the role of EMFs during chondrogenic differentiation of MSCs, isolated from bovine synovial fluid, in the absence and presence of IL-1β. Pellets of MSCs were differentiated for 3 and 5 weeks with transforming growth factor-β3 (TGFβ3), in the absence and presence of IL-1β and exposed or unexposed to EMFs. Biochemical, quantitative real-time RT-PCR and histological results showed that EMFs alone or in the presence of TGFβ3 play a limited role in promoting chondrogenic differentiation. Notably, in the presence of IL-1β and TGFβ3 a recovery of proteoglycan (PG) synthesis, PG content and aggrecan and type II collagen mRNA expression in the EMF-exposed compared to unexposed pellets was observed. Also, histological and immunohistochemical results showed an increase in staining for alcian blue, type II collagen and aggrecan in EMF-exposed pellets. In conclusion, this study shows a significant role of EMFs in counteracting the IL-1β-induced inhibition of chondrogenesis, suggesting EMFs as a therapeutic strategy for improving the clinical outcome of cartilage engineering repair procedures, based on the use of MSCs

The Angiogenic Potential of Mesenchymal Stem Cells from the Hair Follicle Outer Root Sheath

Neovascularization is regarded as a pre-requisite in successful tissue grafting of both hard and soft tissues alike. This study considers mesenchymal stem cells from hair follicle outer root sheath (MSCORS) as powerful tools with a neat angiogenic potential that could in the future have wide scopes of neo-angiogenesis and tissue engineering. Autologous MSCORS were obtained ex vivo by non-invasive plucking of hair and they were differentiated in vitro into both endothelial cells and vascular smooth muscle cells (SMCs), two crucial cellular components of vascular grafts. Assessment was carried out by immunostaining, confocal laser-scanning microscopy, gene expression analysis (qRT-PCR), quantitative analysis of anastomotic network parameters, and cumulative length quantification of immunostained α-smooth muscle actin-containing stress fibers (α -SMA). In comparison to adipose mesenchymal stem cells, MSCORS exhibited a significantly higher differentiation efficiency according to key quantitative criteria and their endothelial derivatives demonstrated a higher angiogenic potential. Furthermore, the cells were capable of depositing their own extracellular matrix in vitro in the form of a membrane-cell sheet, serving as a base for viable co-culture of endothelial cells and SMCs integrated with their autologous matrix. Differentiated MSCORS hereby provided a complex autologous cell-matrix construct that demonstrates vascularization capacity and can serve as a base for personalized repair grafting applications

Gene polymorphisms in folate metabolizing enzymes in adult acute lymphoblastic leukemia: effects on methotrexate-related toxicity and survival

Individual variations in response and/or toxicity to anti-cancer agents is common. The antifolate agent methotrexate is frequently used in maintenace therapy of acute lymphoblastic leukemia. The findings of this study suggest that genotyping of folate polymorphisms might be useful in adult acute lymphoblastic leukemia to optimize methotrexate therapy, reducing the associated toxicity with possible effects on survival

The Middle Part of the Plucked Hair Follicle Outer Root Sheath Is Identified as an Area Rich in Lineage-Specific Stem Cell Markers

Hair follicle outer root sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells