Alessia Ongaro

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.

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

Background and purpose:  Adenosine is an endogenous modulator, interacting with four G‐protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes.

Pulsed electromagnetic fields stimulate osteogenic differentiation in human bone marrow and adipose tissue derived mesenchymal stem cells

Pulsed electromagnetic fields (PEMFs) play a regulatory role on osteoblast activity and are clinically beneficial during fracture healing. Human mesenchymal stem cells (MSCs) derived from different sources have been extensively used in bone tissue engineering. Compared with MSCs isolated from bone marrow (BMSCs), those derived from adipose tissue (ASCs) are easier to obtain and available in larger amounts, although they show a less osteogenic differentiation potential than BMSCs. The hypothesis tested in this study was to evaluate whether PEMFs favor osteogenic differentiation both in BMSCs and in ASCs and to compare the role of PEMFs alone and in combination with the biochemical osteogenic stimulus bone morphogenetic protein (BMP)-2. Early and later osteogenic markers, such as alkaline phosphatase (ALP) activity, osteocalcin levels, and matrix mineralization, were analyzed at different times during osteogenic differentiation. Results showed that PEMFs induced osteogenic differentiation by increasing ALP activity, osteocalcin, and matrix mineralization in both BMSCs and ASCs, suggesting that PEMF activity is maintained during the whole differentiation period. The addition of BMP-2 in PEMF exposed cultures further increased all the osteogenic markers in BMSCs, while in ASCs, the stimulatory role of PEMFs was independent of BMP-2. Our results indicate that PEMFs may stimulate an early osteogenic induction in both BMSCs and ASCs and they suggest PEMFs as a bioactive factor to enhance the osteogenesis of ASCs, which are an attractive cell source for clinical applications. In conclusion, PEMFs may be considered a possible tool to improve autologous cell-based regeneration of bone defects in orthopedics.

Factor XIII V34L polymorphism modulates the risk of chronic venous leg ulcer progression and extension

Low Factor XIII (FXIII) activity has been reported in the blood of patients with chronic venous leg ulcer (CVU). In vivo studies have described increased wound healing in CVU patients treated with FXIII concentrate, and in vitro studies have shown increased regenerative capacity in FXIII‐treated fibroblasts. In addition, a common G‐to‐T polymorphism in the FXIIIA‐subunit gene (V34L) significantly increases the activity and modifies the cross‐linking properties of the FXIII molecule and this variant has been investigated as a protective factor against thrombosis, a recognized risk factor for CVU establishment. Therefore, the role of FXIII levels, FXIII V34L, FVR506Q, and FIIG20210A, common gene polymorphisms in the pathogenesis of CVU was investigated. Ninety‐one patients with CVU and 195 healthy controls (91 of them sex‐ and age‐matched) were PCR‐genotyped for the FXIIIV34L, FVR506Q, and FIIG20210A substitutions and FXIIIA‐subunit levels were determined by immuno‐electrophoresis. The extent of the venous ulcer surface in patients was measured by computer software. The allele frequency and the genotype distribution of the FXIII polymorphism did not show significant differences between the whole group of cases and controls as well as prothrombin variants did. On the contrary, the FVR506Q variant (FV Leiden) allele was more frequent in patients, yielding a significant OR value of 5.93 (95 percent CI, 1.83–19.17; p= 0.003). Considering only CVU cases secondary to a post–thrombotic syndrome (n= 24), FV Leiden yielded a greater OR value of 16.08 (95 percent CI, 4.33‐59.6; p < 0.0001). When the CVU cases were stratified by the three possible FXIII genotypes, a significant trend toward a lower mean value of the ulcerated area was clearly evident as the number of the polymorphic alleles (L34) increased in the genotype of patients (VV = 11.9 cm2,± 23.6; VL = 6.1 cm2,± 6.9; LL = 4.1 cm2,± 2.8; p= 0.01). On the other hand, FXIIIA antigen levels were similar between CVU cases and matched controls, but 11 percent of cases had FXIII deficiency (FXIIIA ≤ 0.65 U/ml; p= 0.003) and they showed a greater mean extension of the lesion if compared with the remaining cases without FXIIIA deficiency (14.5 cm2, ± 20.2 vs. 9.0 cm2, ± 6.3; p= 0.08). We conclude that FXIII antigen levels and FXIII V34L polymorphism may play a crucial role in the complex cascade of CVU pathophysiology, being significantly related to the CVU progression and extension because of the direct effects they have on the FXIII molecular activity.

In vivo effect of two different pulsed electromagnetic field frequencies on osteoarthritis

Osteoarthritis (OA) is a joint pathology characterized by fibrillation, reduced cartilage thickness and subchondral bone sclerosis. There is evidence that pulsed electromagnetic fields (PEMFs) counteract OA progression, but the effect of two different PEMF frequencies has not yet been shown. The aim of this study was to test the effectiveness of PEMFs at two different frequencies (37 and 75 Hz) in a late OA stage in 21‐month‐old Guinea pigs. After 3 months of 6 h/day PEMF stimulation, histological and histomorphometric analyses of the knees were performed. At both frequencies, PEMFs significantly reduced histological cartilage score, fibrillation index (FI), subchondral bone thickness (SBT) and trabecular number (Tb.N) and increased trabecular thickness (Tb.Th) and separation (Tb.Sp) in comparison to the not treated SHAM group. However, PEMFs at 75 Hz produced significantly more beneficial effects on the histological score and FI than 37 Hz PEMFs. At 75 Hz, PEMFs counteracted cartilage thinning as demonstrated by a significantly higher cartilage thickness values than either those of the SHAM or 37 Hz PEMF‐treated groups. Although in severe OA both PEMF frequencies were able to limit its progression, 75 Hz PEMF stimulation achieved the better results.

Factor XIII contrasts the effects of metalloproteinases in human dermal fibroblast cultured cells

Matrix metalloproteinases (MMPs) are overexpressed in venous leg ulcers, determining a breakdown of the main extracellular matrix (ECM) components owing mainly to collagenase activities, and so playing a crucial role in ulcer pathogenesis. The authors studied the effects of coagulation factor XIII (FXIII), which cross-links collagen and other ECM components, in human fibroblast cultured cells in the presence and in the absence of matrix metalloproteinases from Clostridium histolyticum collagenase. Clostridium collagenase at concentrations of 2.0, 1.0, and 0.5 mg/mL was added to normal human dermal fibroblasts cultured in the presence of 0.0, 1.0, and 5.0 U/mL of FXIII concentrate (Fibrogammin P, Aventis Behring). Cell counting and metabolically active fibroblast evaluation in the cultures were monitored for 72 hours, by means of trypan-blue dye and MTT test, respectively. The MTT test showed that at the highest collagenase concentration (2.0 mg/mL), the cell number decreased more than 95% in 72 hours of treatment and no significant differences were observed regardless of the FXIII concentrations utilized. At lower collagenase concentration (1.0 mg/mL), in absence or in presence of FXIII (1.0 U/mL), the cell number decreased by about 80% in 72 hours. In contrast, in the presence of higher FXIII levels (5.0 U/mL), cells suffered globally significantly less collagenase effects (p=0.011) and the gain was appreciable at each time tested. Finally, at 0.5 mg/mL of collagenase concentration, in the absence of FXIII, the cell number decreased by about 60% in 72 hours, whereas in presence of FXIII 1.0 U/mL and 5.0 U/mL, cells decreased significantly less, by about 35% and 20%, respectively (p<0.025 and p<0.01, respectively). These data were also confirmed by direct cell counting utilizing the trypan-blue test. Factor XIII contrasts effectively the detrimental action of Clostridium collagenases in human fibroblast cultured cells. These results support several in vivo reports about the effectiveness of its topical application in order to enhance the venous ulcer healing processes.

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.

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. Copyright