Federica Francesca Masieri

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. Background The antifolate agent methotrexate is an important component of maintenance therapy in acute lymphoblastic leukemia, although methotrexate-related toxicity is often a reason for interruption of chemotherapy. Prediction of toxicity is difficult because of inter-individual variability susceptibility to antileukemic agents. Methotrexate interferes with folate metabolism leading to depletion of reduced folates. Design and Methods The aim of this study was to investigate the influence of polymorphisms for folate metabolizing enzymes with respect to toxicity and survival in adult patients with acute lymphoblastic leukemia treated with methotrexate maintenance therapy. To this purpose, we evaluated possible associations between genotype and hematologic and non-hematologic toxicity and effects on survival at 2 years of follow-up in patients with acute lymphoblastic leukemia. Results Polymorphisms in the genes encoding for methylenetetrahydrofolate reductase (MTHFR 677C>T) and in dihydrofolate reductase (DHFR 19 bp deletion) significantly increased the risk of hepatotoxicity in single (odds ratio 5.23, 95% confidence interval 1.13–21.95 and odds ratio 4.57, 95% confidence interval 1.01–20.77, respectively) and in combined analysis (odds ratio 6.82, 95% confidence interval 1.38–33.59). MTHFR 677C>T also increased the risk of leukopenia and gastrointestinal toxicity, whilst thymidylate synthase 28 bp repeat polymorphism increased the risk of anemia (odds ratio 8.48, 95% confidence interval 2.00–36.09). Finally, patients with MTHFR 677TT had a decreased overall survival rate (hazard ratio 2.37, 95% confidence interval 1.46–8.45). Conclusions 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.

Electromagnetic fields (EMFs) and adenosine receptors modulate prostaglandin E(2) and cytokine release in human osteoarthritic synovial fibroblasts.

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. Previous studies suggest that electromagnetic fields (EMFs) may represent a potential therapeutic approach to limit cartilage degradation and control inflammation associated to OA, and that they may act through the adenosine pathway. Therefore, we investigated whether EMFs might modulate inflammatory activities of human SFs from OA patients (OASFs) treated with interleukin‐1β (IL‐1β), and the possible involvement of adenosine receptors (ARs) in mediating EMF effects. 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 also in EMF‐exposed cells. Functional data obtained in the presence of selective A2A and A3 adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E2 (PGE2) and the proinflammatory cytokines interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8), while stimulating the release of interleukin‐10 (IL‐10), an antinflammatory cytokine. These effects seem 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 enzyme production. In conclusion, this study shows that EMFs display anti‐inflammatory effects in human OASFs, and that these EMF‐induced effects are in part mediated by the adenosine pathway, specifically by the A2A and A3 AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases. J. Cell. Physiol. 227: 2461–2469, 2012.

Chondroprotective effects of pulsed electromagnetic fields on human cartilage explants

This study investigated the effects of pulsed electromagnetic fields (PEMFs) on proteoglycan (PG) metabolism of human articular cartilage explants from patients with osteoarthritis (OA). Human cartilage explants, recovered from lateral and medial femoral condyles, were classified according to the International Cartilage Repair Society (ICRS) and graded based on Outerbridge scores. Explants cultured in the absence and presence of IL-1β were treated with PEMF (1.5  mT, 75  Hz) or IGF-I alone or in combination for 1 and 7 days. PG synthesis and release were determined. Results showed that explants derived from lateral and medial condyles scored OA grades I and III, respectively. In OA grade I explants, after 7 days exposure, PEMF and IGF-I significantly increased (35) S-sulfate incorporation 49% and 53%, respectively, compared to control, and counteracted the inhibitory effect of IL 1β (0.01 ng/ml). The combined exposure to PEMF and IGF-I was additive in all conditions. Similar results were obtained in OA grade III cartilage explants. In conclusion, PEMF and IGF-I augment cartilage explant anabolic activities, increase PG synthesis, and counteract the catabolic activity of IL-1β in OA grades I and III. We hypothesize that both IGF-I and PEMF have chondroprotective effects on human articular cartilage, particularly in early stages of OA.

Time- and Dose-Dependent Effects of Chronic Wound Fluid on Human Adult Dermal Fibroblasts

BACKGROUND Wound healing is a biologic process that is altered in patients affected by chronic venous ulcers. The wound microenvironment is reflected in the chronic wound fluid (CWF), an exudate containing serum components and tissue-derived proteins. OBJECTIVES We investigated the effects of increasing doses of CWF collected from patients suffering from chronic venous ulcers on human adult dermal fibroblasts cultured in vitro and the relationship among CWF effects and treatment length. METHODS Fibroblasts were treated with 60, 240, and 720 μg/mL CWF for 3 and 7 days. We evaluated cell proliferation and viability by MTT and Trypan blue assay, cell morphology by light microscopy, F-actin microfilaments organization by tetramethylrhodamine B isothiocyanate-conjugated phalloidin, α-smooth muscle actin expression by immunofluorescence, and senescence-associated β-galactosidase activity. RESULTS CWF induced an increase in cell proliferation in the first 3 days of treatment. In contrast, at 7 days, a strong decrease in cell viability was observed. These changes were related to a cytoskeletal F-actin reorganization and not to fibroblast–myofibroblast differentiation nor to changes in cellular senescence. CONCLUSIONS This study shows a dose-dependent and biphasic effect of CWF on dermal fibroblasts, suggesting that a continuous exposure to chronic wounds microenvironment may induce late cellular dysfunctions possibly involved in the delayed wound healing.

P2X 1 and P2X 3 Purinergic Receptors Differentially Modulate the Inflammatory Response in Human Osteoarthritic Synovial Fibroblasts

Background/Aims: P2X receptors are membrane ion channels activated by extracellular adenosine 5’-triphosphate (ATP) which contribute to various physiological processes. The present study describes in synovial fibroblasts (SFs) obtained from osteoarthritis (OA) patients and in SW 982 cells derived from human synovial sarcoma a pharmacological characterization of P2X1 and P2X3 receptors implicated in the modulation of inflammatory processes in joint diseases. Methods: mRNA, western blotting, saturation and competition binding experiments were used to characterize purinergic receptors. From a functional point of view nuclear factor ĸB (NF-ĸB) activation, tumour necrosis factor-α (TNF-α), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) production were evaluated by means of enzyme-linked immunosorbent assays. Results: P2X1 and P2X3 receptors were present with high affinity and density. Selected purinergic agonists and antagonists exhibited a different thermodynamic behavior. P2X1 receptors showed an anti-inflammatory effect reducing NF-ĸB activation and TNF-α release whilst P2X3 receptors mediated opposite response. No effect was mediated by P2X1 and P2X3 receptors on IL-6 and PGE2 production. Conclusion: SFs from OA patients and SW 982 cells similarly express P2X1 and P2X3 receptors which are able to modulate in opposite way some functional responses closely associated with inflammation suggesting that purinergic receptors may represent a potential target in therapeutic anti-inflammatory joint interventions.

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.

Chondroprotective effects of low-frequency lowenergy pulsed electromagnetic fields on human cartilage explants

Objective To investigate the effects of low-frequency low-energy pulsed electromagnetic fields (PEMFs) on proteoglycans (PG) metabolism [1-2] of human articular cartilage explants derived from patients with different osteoarthritis (OA) grades in the presence and in the absence of anabolic and catabolic stimuli for cartilage. Design Human knee cartilage explants derived from lateral and medial femoral condyles of OA patients were exposed to PEMF (1.5 mT; 75 Hz) for 1 and 7 days with and without insulin-like growth factor-I (IGF-I) (50 ng/ml) and interleukin-1β (IL-1β) (0.01-50ng/ml). Histological analysis was used to classify cartilage according to the score developed by the International Cartilage Repair Society (ICRS). PG synthesis was determined by 35S-sulfate incorporation. PG release into culture media was determined by the dimethylmethylene blue assay. Results By histological evaluation, cartilage explants derived from lateral and medial condyles were classified as OA ICRS grade I and III, respectively. After 7 days treatment, in OA grade I cartilage explants, PEMF and IGF-I alone significantly increased 35S-sulfate incorporation respect to control and counteracted the effect induced by IL-1β (0.01 ng/ml) of 26% and 24% respectively. The combination of PEMF and IGF-I resulted additive in all conditions. Similar results were obtained in OA grade III cartilage, although basal 35S-sulfate incorporation was lower than in OA grade I. No effect was observed on medium PG release. Conclusions PEMF exerts chondroprotective effects on human articular cartilage. The biological responsiveness of chondrocyte to PEMF was similar in different OA grades cartilage, suggesting that human chondrocytes maintain susceptibility to PEMF during OA progression.

HLA-G molecules are costitutively expressed by human synovial fibroblasts and up-modulated in osteoarthritis

Objective HLA-G molecules are non classical HLA class I antigens expressed as membrane bound and soluble isoforms (sHLA-G) with a restricted tissue distribution and anti-inflammatory functions [1]. Osteoarthritis (OA) is a complex degenerative disease that affects articular cartilage components and causes damage to the entire joint structure including synovium [2]. Since inflammation is involved in the pathogenesis of OA, we have analyzed the expression and the production of HLA-G molecules in “in vitro” cultured synovial fibroblasts (SFs) from OA patients and control subjects. Design We have analyzed the levels of sHLA-G1 and HLA-G5 isoforms by immunoenzymatic assay (ELISA) in the SFs culture supernatants from six OA patients and six control subjects during a 70 day “in vitro” cultures in the presence and in the absence of lipopolysaccharide (LPS) or recombinant IL-10 (rIL-10). We have confirmed HLA-G modulation by cytofluorimetry and immunofluorescence. Results Data in ELISA have demonstrated the spontaneous production of sHLA-G molecules by both OA and control SFs. The expression has been confirmed by cytofluorimetry and immunofluorescence. OA SFs produce higher levels of sHLA-G1 and HLA-G5 molecules during the first 23 days of culture in comparison to control SFs. The sHLA-G1 levels further increase over the next 20 days of “in vitro” culture. Atfer LPS and rIL-10 treatments, sHLA-G secretion increases in a similar manner in both OA and control SFs. Conclusions The production of sHLA-G1 molecules, constitutively expressed by control and OA SFs, is significantly higher in OA than in control SFs, suggesting that these molecules represent a possible molecular target to counteract the synovial joints inflammation.