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Osteoarthritis is a chronic inflammatory disease that affects all of the joints, especially those of the elderly. Aging is a natural and irreversible biological process implicated in the pathophysiology of many chronic diseases, such as osteoarthritis. Inflammation and oxidative stress are the main factors involved in osteoarthritis and aging, respectively, with the production of several pro-inflammatory cytokines such as Interleukin 1β (IL1β) and reactive oxygen species. The aim of the study was to set-up an in vitro model of osteoarthritis and aging, focusing on the sex differences by culturing male and female fibroblast-like synoviocytes (FLSs) with IL1β, hydrogen peroxide (H2O2), IL1β+H2O2 or a growth medium (control). IL1β+H2O2 reduced the cell viability and microwound healing potential, increased Caspase-3 expression and reactive oxygen species and IL6 production; IL1β increased IL6 production more than the other conditions did; H2O2 increased Caspase-3 expression and reactive oxygen species production; Klotho expression showed no differences among the treatments. The FLSs from female donors demonstrated a better response capacity in unfavorable conditions of inflammation and oxidative stress than those from the male donors did. This study developed culture conditions to mimic the aging and osteoarthritis microenvironment to evaluate the behavior of the FLSs which play a fundamental role in joint homeostasis, focusing on the sex-related aspects that are relevant in the osteoarthritis pathophysiology.

Background Diagnostic biomarkers for detecting chronic obstructive pulmonary disease (COPD) in acute coronary syndrome (ACS) patients are not available. SERPINA1, coding for the most potent circulating anti-inflammatory protein in the lung, has been found to be differentially methylated in blood cells from COPD patients. This study aimed to investigate the methylation profile of SERPINA1 in blood cells from ACS patients, with (COPD+) or without COPD (COPD−). Methods Blood samples were from 115 ACS patients, including 30 COPD+ and 85 COPD− according to lung function phenotype, obtained with spirometry. DNA treated with sodium bisulfite was PCR-amplified at SERPINA1 promoter region. Methylation analysis was carried out by sequencing the PCR products. Lymphocytes count in ACS patients was recorded at hospital admission and discharge. Results SERPINA1 was hypermethylated in 24/30 (80%) COPD+ and 48/85 (56.5%) COPD− (p SERPINA1 hypermethylated (1.98 × 103 ± 0.6 cell/µl) than in COPD− carrying SERPINA1 hypomethylated (1.7 × 103 ± 0.48 cell/µl) (p Conclusions SERPINA1 is hypermethylated in blood cells from COPD+ patients. COPD− carrying SERPINA1 hypermethylated and high lymphocytes count may be at risk of COPD development. Therefore, SERPINA1 hypermethylation may represent a potential biomarker for predicting COPD development in ACS patients.

MicroRNA (miR)‑29b has been reported to play a controversial role in breast cancer, particularly triple‑negative breast cancer (TNBC). Based on our previous data revealing that the PU.1‑mediated expression of miR‑29b in cells from acute myeloid leukemia is sustained by Vav1, the potential role of this multidomain protein in modulating miR‑29b levels in breast tumor cells, in which Vav1 is ecstopically expressed and shows a nuclear accumulation, was investigated. Breast cancer cell lines with various phenotypes and patient‑derived xenograft‑derived TNBC cells were subjected to Vav1 modulation and reverse transcription quantitative PCR of miR‑29b levels. The recruitment of CCAAT enhancer binding protein α (CEBPα) to miR‑29b promoters was investigated by quantitative chromatin immunoprecipitation assays. It was found that Vav1 was essential for the recovery of mature miR‑29b in breast cancer cell lines, and that it promoted the expression of the miRNA in TNBC cells of the mesenchymal molecular subtype by sustaining the transcription of the miR‑29b1/a cluster mediated by CEBPα. The present results suggest that Vav1 is a crucial modulator of miR‑29b expression in breast tumor cells, and this finding may help identify strategies that may be useful in the management of TNBC by targeting the Vav1/miR‑29b axis, as there is a lack of molecular‑based treatments for TNBC.

Mesenchymal stem cells (MSCs) have been identified in many adult tissues and they have been closely studied in recent years, especially in view of their potential use for treating diseases and damaged tissues and organs. MSCs are capable of self-replication and differentiation into osteoblasts and are considered an important source of cells in tissue engineering for bone regeneration. Several epigenetic factors are believed to play a role in the osteogenic differentiation of MSCs, including microRNAs (miRNAs). MiRNAs are small, single-stranded, non-coding RNAs of approximately 22 nucleotides that are able to regulate cell proliferation, differentiation and apoptosis by binding the 3′ untranslated region (3′-UTR) of target mRNAs, which can be subsequently degraded or translationally silenced. MiRNAs control gene expression in osteogenic differentiation by regulating two crucial signaling cascades in osteogenesis: the transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) and the Wingless/Int-1(Wnt)/β-catenin signaling pathways. This review provides an overview of the miRNAs involved in osteogenic differentiation and how these miRNAs could regulate the expression of target genes.

Mesenchymal stem cells (MSCs) are the main cell players in tissue repair and thanks to their self-renewal and multi-lineage differentiation capabilities, they gained significant attention as cell source for tissue engineering (TE) approaches aimed at restoring bone and cartilage defects. Despite significant progress, their therapeutic application remains debated: the TE construct often fails to completely restore the biomechanical properties of the native tissue, leading to poor clinical outcomes in the long term. Pulsed electromagnetic fields (PEMFs) are currently used as a safe and non-invasive treatment to enhance bone healing and to provide joint protection. PEMFs enhance both osteogenic and chondrogenic differentiation of MSCs. Here, we provide extensive review of the signaling pathways modulated by PEMFs during MSCs osteogenic and chondrogenic differentiation. Particular attention has been given to the PEMF-mediated activation of the adenosine signaling and their regulation of the inflammatory response as key player in TE approaches. Overall, the application of PEMFs in tissue repair is foreseen: (1) in vitro: to improve the functional and mechanical properties of the engineered construct; (2) in vivo: (i) to favor graft integration, (ii) to control the local inflammatory response, and (iii) to foster tissue repair from both implanted and resident MSCs cells.

Background Killian polyp (KP) is a benign lesion that arises from the maxillary sinus. The etiology of KP is unknown. The aim of this study was to investigate the potential involvement of human papilloma- (HPV) and polyoma-viruses (HPyV) infections in the onset of KP. Methods DNA from antral (n = 14) and nasal (n = 14) KP fractions were analyzed for HPV and HPyV sequences, genotypes, viral DNA load and physical status along with expression of viral proteins and p16 cellular protein. Results The oncogenic HPV16 was detected in 3/14 (21.4%) antral KPs, whilst nasal KPs tested HPV-negative (0/14). The mean HPV16 DNA load was 4.65 ± 2.64 copy/104 cell. The whole HPV16 episomal genome was detected in one KP sample, whereas HPV16 DNA integration in two KPs. P16 mRNA level was lower in the KP sample carrying HPV16 episome than in KPs carrying integrated HPV16 and HPV- negative KPs (p Conclusions A fraction of KP tested positive for the oncogenic HPV16. HPV16 detection in the KP antral portion may be consistent with HPV16 infection derived from the maxillary sinus. HPV16 DNA integration represents a novel finding. Altogether, these data improve our knowledge on the association between KP and HPV infection, whereas it indicates that the KP onset is heterogeneous.

Despite the high intrinsic ability of bone tissue to regenerate, bone healing fails in some pathological conditions and especially in the presence of large defects. Due to the strong relationship between bone development and vascularization during in vivo bone formation and repair, strategies promoting the osteogenic-angiogenic coupling are crucial for regenerative medicine. Increasing evidence shows that miRNAs play important roles in controlling osteogenesis and bone vascularization and are important tool in medical research although their clinical use still needs to optimize miRNA stability and delivery. Pulsed electromagnetic fields (PEMFs) have been successfully used to enhance bone repair and their clinical activity has been associated to their ability to promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs). In this study we investigated the potential ability of PEMF exposure to modulate selected miRNAs involved in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). We show that, during in vitro hBMSC differentiation, PEMFs up-modulate the expression of miR-26a and miR-29b, which favor osteogenic differentiation, and decrease miR-125b which acts as an inhibitor miRNA. As PEMFs promote the expression and release of miRNAs also involved in angiogenesis, we conclude that PEMFs may represent a noninvasive and safe strategy to modulate miRNAs with relevant roles in bone repair and with the potential to regulate the osteogenic-angiogenic coupling.

Pulsed electromagnetic fields (PEMFs) are clinically used with beneficial effects in the treatment of bone fracture healing. This is due to PEMF ability to favor the osteogenic differentiation of mesenchymal stem cells (MSCs). Previous studies suggest that PEMFs enhance the osteogenic activity of bone morphogenetic protein-2 (BMP2) which is used in various therapeutic interventions. This study investigated the molecular events associated to the synergistic activity of PEMFs and BMP2 on osteogenic differentiation. To this aim, human MSCs (hMSCs) were exposed to PEMFs (75 Hz, 1.5 mT) in combination with BMP2, upon detection of the minimal dose able to induce differentiation. Changes in the expression of BMP signaling pathway genes including receptors and ligands, as well as in the phosphorylation of BMP downstream signaling proteins, such as SMAD1/5/8 and MAPK, were analyzed. Results showed the synergistic activity of PEMFs and BMP2 on osteogenic differentiation transcription factors and markers. The PEMF effects were associated to the increase in BMP2, BMP6, and BMP type I receptor gene expression, as well as SMAD1/5/8 and p38 MAPK activation. These results increase knowledge concerning the molecular events involved in PEMF stimulation showing that PEMFs favor hMSCs osteogenic differentiation by the modulation of BMP signaling components.

Pulsed electromagnetic fields (PEMFs) have been used to treat bone diseases, particularly nonunion healing. Although it is known that PEMFs promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs), to date PEMF molecular mechanisms remain not clearly elucidated. The Notch signalling is a highly conserved pathway that regulates cell fate decisions and skeletal development. The aim of this study was to investigate if the known PEMF-induced osteogenic effects may involve the modulation of the Notch pathway. To this purpose, during in vitro osteogenic differentiation of bone marrow hMSCs in the absence and in the presence of PEMFs, osteogenic markers (alkaline phosphatase activity, osteocalcin and matrix mineralization), the messenger ribonucleic acid expression of osteogenic transcription factors (Runx2, Dlx5, Osterix) as well as of Notch receptors (Notch1-4), their ligands (Jagged1, Dll1 and Dll4) and nuclear target genes (Hes1, Hes5, Hey1, Hey2) were investigated. PEMFs stimulated all osteogenic markers and increased the expression of Notch4, Dll4, Hey1, Hes1 and Hes5 in osteogenic medium compared to control. In the presence of DAPT and SAHM1, used as Notch pathway inhibitors, the expression of the osteogenic markers, including Runx2, Dlx5, Osterix, as well as Hes1 and Hes5 were significantly inhibited, both in unexposed and PEMF-exposed hMSCs. These results suggest that activation of Notch pathway is required for PEMFs-stimulated osteogenic differentiation. These new findings may be useful to improve autologous cell-based regeneration of bone defects in orthopaedics.

Osteogenic differentiation is a multi-step process controlled by a complex molecular framework. Notch is an evolutionarily conserved intercellular signaling pathway playing a prominent role in cell fate and differentiation, although the mechanisms by which this pathway regulates osteogenesis remain controversial. This study aimed to investigate, in vitro, the involvement of Notch pathway during all the developmental stages of osteogenic differentiation in human osteosarcoma cell line MG63. Cells were cultured in basal condition (control) and in osteoinductive medium (OM). Notch inhibitors were also added in OM to block Notch pathway. During osteogenic differentiation, early (alkaline phosphatase activity and collagen type I) and late osteogenic markers (osteocalcin levels and matrix mineralization), as well as the gene expression of the main osteogenic transcription factors (Runx2, Osterix, and Dlx5) increased. Time dependent changes in the expression of specific Notch receptors were identified in OM versus control with a significant reduction in the expression of Notch1 and Notch3 receptors in the early phase of differentiation, and an increase of Notch2 and Notch4 receptors in the late phase. Among Notch nuclear target genes, Hey1 expression was significantly higher in OM than control, while Hes5 expression decreased. Osteogenic markers were reduced and Hey1 was significantly inhibited by Notch inhibitors, suggesting a role for Notch through the canonical pathway. In conclusion, Notch pathway might be involved with a dual role in osteogenesis of MG63, through the activation of Notch2, Notch4, and Hey1, inducing osteoblast differentiation and the depression of Notch1, Notch3, and Hes5, maintaining an undifferentiated status. J. Cell. Physiol. 231: 2652-2663, 2016. © 2016 Wiley Periodicals, Inc.

Electrochemotherapy is an emerging local treatment for the management of superficial tumors and, among these, also chest wall recurrences from breast cancer. Generally, the treatment of this peculiar type of tumor requires the coverage of large skin areas. In these cases, electrochemotherapy treatment by means of standard small size needle electrodes (an array of 0.73 cm spaced needles, which covers an area of 1.5 cm2) is time-consuming and can allow an inhomogeneous coverage of the target area. We have previously designed grid devices suitable for treating an area ranging from 12 to 200 cm2. In this study, we propose different approaches to study advantages and drawbacks of a grid device with needles positioned 2 cm apart. The described approach includes a numerical evaluation to estimate electric field intensity, followed by an experimental quantification of electroporation on a cell culture. The electric field generated in a conductive medium has been studied by means of 3-dimensional numerical models with varying needle pair distance from 1 to 2 cm. In particular, the electric field evaluation shows that the electric field intensity with varying needle distance is comparable in the area in the middle of the 2 electrodes. Differently, near needles, the electric field intensity increases with the increasing electrode distance and supply voltage. The computational results have been correlated with experimental ones obtained in vitro on cell culture. In particular, electroporation effect has been assessed on human breast cancer cell line MCF7, cultured in monolayer. The use of 2-cm distant needles, supplied by 2000 V, produced an electroporation effect in the whole area comprised between the electrodes. Areas of cell culture where reversible and irreversible electroporation occurred were identified under microscope by using fluorescent dyes. The coupling of computation and experimental results could be helpful to evaluate the effect of the needle distance on the electric field intensity in cell cultures in terms of reversible or irreversible electroporation.

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.

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.

Alcohol abuse and smoking habits have adverse effects on bone health and are a risk factor for osteoporosis, fractures and impaired fracture repair. Osteointegration processes around implanted biomaterials involve a coordinated cascade of complex events that are very similar to those occurring during fracture repair and require a suitable microenvironment and the coordinated action of cells and signal molecules. Therefore, diseases and harmful lifestyles that impair the normal bone healing process can reduce the success of implant surgery and may negatively influence the osteointegration of prostheses and implant devices for fracture fixation such as screws, nails and plates. Understanding the effects of harmful lifestyles on bone implant osteointegration is important for successful implant therapy, orthopedic reconstructive surgery and tissue-engineered-based therapies. However, the mechanisms by which smoking and alcoholism affect bone metabolism, bone mass and the balance of bone resorption and formation, also in the presence of an orthopedic implant, are not completely understood and remain inadequately elucidated. This review aims to analyze in vitro and in vivo studies regarding orthopedic implant integration in the presence of tobacco smoking and alcohol consumption with a focus on pathophysiology and local or systemic mechanisms of action on bone.

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

Background and Objectives Common methylenetetrahydrofolate reductase gene variants (MTHFR C677T and A1298C) have been described to have opposite effects on cancer patients. They may reduce cancer susceptibility and increase drug-related toxicity when folate antagonists (e.g. methotrexate) are utilized. We analyzed 110 patients with high-grade non-Hodgkin’s lymphoma (NHL), 68 of whom were eligible for a chemotherapy combination containing methotrexate (MACOP-B) and 42 for chemotherapy without methotrexate (CHOP).Design and Methods Patients were genotyped by polymerase chain reaction and stratified by MTHFR variants. These data were related to the toxicity (WHO grade GO-4) that the patients suffered and their survival. Overall 64 cases (58.2%) developed some form of toxicity and 23 (20.9%) had grade 3/4 toxicity.Results When considering toxicity of any grade (grade 1–4), the 677TT genotype was significantly over-represented among cases with mucositis (OR=4.85; 95%CI, 1.47–15.97; p=0.009) and those with hepatic toxicity (OR=3.43; 95%CI, 0.99–11.86; p=0.052). Sub-analyses in the group treated with MACOP-B showed a slight increase in the risk of developing mucositis (OR=5.22; 95%CI, 1.20–27.27; p=0.03), and a strong increase in the risk of hepatic toxicity (OR=7.08; 95%CI, 1.38–36.2; p=0.019) and thrombocytopenia (OR=7.69, 95%CI 1.0–58.94; p=0.05). Interestingly, compared to the risk of developing toxicity of any grade, the risk of developing severe (grade 3/4) mucositis was almost doubled in the whole group of cases with 677TT (OR=8.13; 95%CI 1.61–41.04; p=0.011) and dramatically increased in the MACOP-B-treated cases with this gene variant (OR=24.6; 95%CI 2.49–87.41; p=0.001). There were significant results for 1298CC cases exclusively for mucositis (any grade, OR=5.33; 95%CI, 1.25–22.70; p=0.023 and OR=9.15; 95%CI, 1.14–73.41; p=0.037; for the whole group and the MACOP-B-treated group, respectively). Similarly, the risk of 1298CC patients developing severe mucositis increased (OR=9.24; 95%CI, 1.47–58.0; p=0.017 and OR=11.53; 0.93–143.18; p=0.057; in the whole group and in the MACOP-B-treated group, respectively). Event-free survival analysis revealed a lower probability of event-free survival at 5 years for 677T-carriers (log-ranks, p=0.05 and p=0.07 in the whole group and in the MACOP-B-treated group, respectively). More significant results were obtained when 1298CC cases were excluded from the reference group (log-ranks, p=0.03 and p=0.04, respectively). No significant associations were found in the CHOP-treated group.Interpretation and Conclusions Our data suggest that MTHFR gene variants play a critical role in NHL outcome, possibly by interfering with the action of methotrexate with significant effects on toxicity and survival. Genotyping of folate pathway gene variants might be useful to enable reduction of chemotherapy toxicity and/or to improve survival by indicating when dose adjustments or alternative treatments are necessary.

It has been demonstrated recently that coagulation factor XIII (FXIII) plays an extraordinary role in myocardial healing after infarction, improving survival in a mouse model. Common FXIII gene variants (i.e. FXIIIA-V34L and FXIIIB-H95R) significantly influence the molecular activity. To evaluate whether there is a relationship between the two FXIII gene variants and survival in patients after myocardial infarction (MI), V34L and H95R were PCR-genotyped in a cohort of 560 MI cases and follow-up was monitored. Cases with ST-segment elevation MI (STEMI) were 416 (74.3%) and 374 of these were treated with primary percutaneous coronary intervention (PCI) (89.9%). The remaining 144 patients showed non-ST-segment elevation MI (NSTEMI) at enrollment. The combined endpoint was the occurrence of death, re-infarction, and heart failure. Kaplan-Meier analysis at one year yielded an overall rate for adverse events of 24.5% with a lower incidence in the L34-carriers (28.8% vs 17.1%; log-rank, P = 0.00025), similar to that of the 416 STEMI (23.8%) being (28.0% and 16.9%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Primary PCI-group had a slight lower incidence (22.9%) of adverse events (26.8% and 17.1%; VV34- and L34-carriers respectively; log-rank, P = 0.009). During hospitalization, 506 patients received PCI (374 primary PCI and 132 elective PCI). Significance was conserved also in the overall PCI-group (28.6% and 17.8%; VV34- and L34-carriers respectively; log-rank, P = 0.001). Similar findings were observed at 30 days follow-up. Cases carrying both FXIII variants had improved survival rate (log-rank, P = 0.019). On the other hand, minor bleeding complications were found increased in L34-carriers (P = 0.0001) whereas major bleeding complications were not. Finally, more direct evidence on the role of FXIII molecule on survival might come from the fact that despite significant FXIII antigen reductions observed in cases after MI, regardless the FXIII genotype considered, L34-carriers kept almost normal FXIII activity (VV34- vs L34-carriers; P < 0.001). We conclude that FXIII L34-allele improves survival after MI in all the groups analyzed, possibly through its higher activity associated with assumable positive effects on myocardial healing and recovered functions. Genetically determined higher FXIII activity might influence post-MI outcome. This paves the way for using FXIII molecules to improve myocardial healing, recovery of functions, and survival after infarction.

Background Osteoarthritis (OA) is the final result of progressive alterations to articular cartilage structure, composition and cellularity, followed by an increase in the concentration of pro-inflammatory cytokines in joint synovial fluid. Even though the effect of pulsed electromagnetic field (PEMF) stimulation in counteracting OA progression and inflammation is of increasing interest, because of its anabolic and anti-inflammatory properties, the present study aimed to improve the knowledge on cartilage extracellular matrix (ECM) and chondrocyte changes related to the exposure of PEMF, from a histological and histomorphometric point of view. Methods An in vitro OA model was realized, culturing bovine cartilage explants with a high dose of interleukin 1β (IL1β, 50 ng/ml) at different experimental times (24 h, and 7 and 21 days). The effects of PEMFs (75 Hz, 1.5 mT) were evaluated in cartilage explants treated with IL1β or not (control), in terms of cartilage structure, cellularity and proteoglycans, glycosaminoglycans, collagen II and transforming growth factor β1 synthesis by using histology, histomorphometry and immunohistochemistry. Results Making a comparison with control cartilage, IL1β-treated explants showed a decrease in cartilage matrix, structure and cellularity parameters. PEMFs were able to counteract the progression of OA acting on both cartilage cellularity and ECM in cartilage previously treated with IL1β. Normal distribution (Kolmogroc-Smirnov test) and homoscedasticity (Levene test) of data were verified, then, the non-parametric Kruskal Wallis test followed by Mann-Whiteny U test for pairwise comparisons were performed. The p-value was adjusted according to the Dunn-Sidak correction. Conclusions These results, obtained by culturing and treating cartilage explants from two different joints, confirmed that PEMF stimulation can be used as adjuvant therapy to preserve cartilage from detrimental effects of high inflammatory cytokine levels during OA.

Melphalan represents the reference drug for locoregional chemotherapy of melanoma; nevertheless, treatment failure may occur because of resistance to chemotherapy. Refractory melanoma cells show either an increased capability of drug inactivation, which is known to be associated with elevated intracellular levels of glutathione (GSH), or a decreased melphalan uptake. The aim of this study was to explore a biochemical and a biophysical strategy, and their combination, to overcome melphalan resistance in melanoma cells. The biochemical strategy was based on the treatment of melanoma cells with DL-buthionine (S,R)-sulfoximine (BSO) to deplete the GSH levels, thus reducing melphalan inactivation. In the biophysical strategy, cell membrane electroporation was used to increase melphalan uptake. The SK-MEL 28-resistant human melanoma cell line was pretreated with 50 μmol/l BSO for 24 h and then treated with increasing melphalan doses, with or without electroporation. Spectrophotometric quantification of cell viability was used to determine melphalan cytotoxicity. Intracellular total GSH was measured using a kinetic enzymatic assay. BSO induced 3.50-fold GSH depletion in untreated cells and a similar reduction was also maintained in melphalan-treated cells. BSO pretreatment produced a 2.46-fold increase in melphalan cytotoxicity. Electroporation increased melphalan cytotoxicity 1.42-fold. The combination of both BSO pretreatment with melphalan plus electroporation led to a 4.40-fold increase in melphalan cytotoxicity compared with melphalan alone. Pretreatment with BSO and cell membrane permeabilization by electroporation enhanced the cytotoxic activity of melphalan in melanoma cells. Their rational combination deserves further investigation and may improve the efficacy of locoregional chemotherapy of melanoma.

Summary Objective To investigate the single and combined effects of electromagnetic field (EMF) exposure and the insulin growth factor-I (IGF-I) on proteoglycan (PG) synthesis of bovine articular cartilage explants and chondrocytes cultured in monolayers. Design Bovine articular cartilage explants and chondrocyte monolayers were exposed to EMF (75Hz; 1.5mT) for 24h in the absence and in the presence of both 10% fetal bovine serum (FBS) and IGF-I (1–100ng/ml). PG synthesis was determined by Na 2 – 35 SO 4 incorporation. PG release into culture medium was determined by the dimethylmethylene blue (DMMB) assay. Results In cartilage explants, EMF significantly increased 35 S-sulfate incorporation both in the absence and in the presence of 10% FBS. Similarly, IGF-I increased 35 S-sulfate incorporation in a dose-dependent manner both in 0% and 10% FBS. At all doses of IGF-I, the combined effects of the two stimuli resulted additive. No effect was observed on medium PG release. Also in chondrocyte monolayers, IGF-I stimulated 35 S-sulfate incorporation in a dose-dependent manner, both in 0% and 10% FBS, however, this was not modified by EMF exposure. Conclusions The results of this study show that EMF can act in concert with IGF-I in stimulating PG synthesis in bovine articular cartilage explants. As this effect is not maintained in chondrocyte monolayers, the native cell–matrix interactions in the tissue may be fundamental in driving the EMF effects. These data suggest that in vivo the combination of both EMF and IGF may exert a more chondroprotective effect than either treatment alone on articular cartilage.

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

Cyclosporin A is a powerful immunosuppressive drug that has had a major impact on transplant therapy. It apparently links to different enzymatic pathways, and affects multiple enzymatic systems. Transforming growth factor β induces the deposition of glycosaminoglycans, proteoglycans, and collagen fibers in the extracellular matrix. The aim of this study of normal and hypertrophic human gingival fibroblast cultures was to evaluate the cytoskeletal and extracellular changes in glycosaminoglycan secretion due to the presence of cyclosporin A and transforming growth factor β. The results showed that there is an increase in total and individual classes of extracellular glycosaminoglycans in the presence of cyclosporin A and transforming growth factor β, but the action of the latter was significantly greater. Immunohistochemical analysis of the cytoskeleton did not reveal any morphological differences between treated and control cells. Our data suggest that the biochemical changes in the extracellular matrix are caused more by cytokine, and that cyclosporin A does not induce any morphological changes in fibroblast cultures derived from hypertrophic and normal gingiva.

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.

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 P2X(1) and P2X(3) receptors implicated in the modulation of inflammatory processes in joint diseases.mRNA, western blotting, saturation and competition binding experiments were used to characterize purinergic receptors. From a functional point of view nuclear factor kappaB (NF-kappaB) activation, tumour necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6) and prostaglandin E(2) (PGE(2)) production were evaluated by means of enzyme-linked immunosorbent assays.P2X(1) and P2X(3) receptors were present with high affinity and density. Selected purinergic agonists and antagonists exhibited a different thermodynamic behavior. P2X(1) receptors showed an anti-inflammatory effect reducing NF-kappaB activation and TNF-alpha release whilst P2X(3) receptors mediated opposite response. No effect was mediated by P2X(1) and P2X(3) receptors on IL-6 and PGE(2) production.SFs from OA patients and SW 982 cells similarly express P2X(1) and P2X(3) 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.

Human leukocyte antigen (HLA)-G molecules are nonclassical HLA class I antigens expressed as membrane bound and soluble isoforms (sHLA-G) with a restricted tissue distribution and anti-inflammatory functions. Because inflammation is involved in the pathogenesis of osteoarthritis (OA), we have analyzed the expression and production of HLA-G molecules in in vitro cultured synovial fibroblasts (SFs) from OA patients and control subjects. We have analyzed the levels of sHLA-G1 and HLA-G5 isoforms by immunoenzymatic assay (enzyme-linked immunosorbent assay) in the SF culture supernatants from six OA patients and six control subjects in 70-day in vitro cultures and after the addition of lipopolysaccharide or recombinant interleukin (IL)-10 (rIL-10). We have confirmed HLA-G modulation by cytofluorimetry and immunofluorescence. The results have demonstrated the spontaneous production of sHLA-G1 molecules by both OA and control SFs. The expression was confirmed by cytofluorimetry and immunofluorescence. OA SFs produce both sHLA-G1 and HLA-G5 molecules during the first 23 days of culture and higher levels of sHLA-G1 during the first 40 days of in vitro culture and after lipopolysaccharide or rIL-10 activation compared with control SFs. The production of HLA-G1 molecules, constitutively expressed by control and OA SFs, is significantly increased in OA, suggesting a possible mechanism to counteract the inflammation of the synovial joints.

Folate and its derivatives are pivotal for cell cycle and proliferation. They facilitate the crosstalk between DNA synthesis and methylation crucial processes in cancer establishment [1]. Dietary folate or supplements (e.g., folic acid) must be fully reduced by dihydrofolate reductase (DHFR) before entering cell metabolism [1]. DHFR is responsible for dihydrofolate (DHF) to tetrahydrofolate (THF) conversion, as well as for assisting the generation of additional partially reduced folates (i.e., methylene-THF and formyl-THF), which are then transformed into the fully active folate (i.e., methyl-THF) with the help of methylenetetrahydrofolate reductase (MTHFR). As the main folate isoforms involved in DNA synthesis and methylation are handled by these two key enzymes, alterations in DHFR and/or in MTHFR functions may have detrimental effects on DNA stability and cancer susceptibility [2-5].

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.

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 microg/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, alpha-smooth muscle actin expression by immunofluorescence, and senescence-associated beta-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.

In this study we analyzed whether the polymorphisms 676TG in the tumor necrosis factor receptor (TNFR) II gene and -308GA in the TNFalpha promoter gene may influence the response grading to anti-TNFalpha therapy in rheumatoid arthritis. We enrolled and genotyped 105 RA patients treated with etanercept (n = 55), infliximab (n = 40) and adalimumab (n = 10) for 1 year. The clinical response was evaluated according to the ACR criteria every 3 months. Patients with TNFRII 676TG genotype was significantly associated with lower ACR response compared with 676TT genotype, at 3 (OR 3.78 95% CI 1.07-13.31) and 12 months (OR 4.30 95% CI 1.16-15.99) of treatment. No significant association between TNFalpha -308GA polymorphism and the clinical response was found. TNFRII 676TG genotype is associated with a lower response to anti-TNFalpha therapy, independently from the specific agent used. This polymorphism could become a useful genetic marker for predicting the different response grading to anti-TNFalpha therapy.

ObjectiveRole of superficial venous surgery in reducing the time it takes for ulcers to heal is still controversial, although all studies confirm a significant reduction in ulcer recurrences. Recently, the HFE-C282Y and FXIII-V34L gene variants demonstrated a role in the risk of venous ulceration in primary chronic venous disorder (CVD) and in modulating lesion size in chronic venous ulcer (CVU), respectively. This study was conducted to investigate the role of HFE-C282Y and FXIII (V34L and P564L) gene variants in ulcer healing time after superficial venous surgery, by assessing the outcome of a cohort of homogeneous CVU patients.MethodsThe study selected 91 patients affected by primary CVU (CEAP C6, Ep, Asp, Pr), with the exclusion of any other comorbidity factor involved in delayed healing process, who underwent surgery. We assessed the ulcer area and the healing time. Patients were genotyped by polymerase chain reaction for FXIII (V34L and P564L) and for HFE-C282Y substitutions.ResultsGlobally, CVU cases had a postoperative mean healing time of 8.5 ± 5.7 weeks. For the subset of cases above and below the median value (M = 8.0 weeks), FXIII-V34L genotype distribution significantly differed (P < .0001). In addition, Kaplan-Meier analysis yielded specific healing time profiles for the different FXIII-V34L classes of genotype (P = .00001), with an increased risk of delayed healing for the FXIII-VV genotype (hazard ratio, 4.14; 95% confidence interval, 2.1 to 8.2; P = .00005). Although FXIII-P54L genotype distributions did not differ, homozygous 564LL cases (P = .005) and double carriers for both FXIII variants (P < .0001), had a significantly reduced healing time vs wild types. No differences in healing time were observed between carriers and noncarriers of the HFE-C282Y variant, whereas when these cases were stratified by FXIII-V34L genotypes, the L34 carriers had a significantly shorter healing time, irrespective of the HFE genotype.ConclusionThe FXIII-34L variant was significantly associated with shorter healing time after superficial venous surgery, suggesting a role in the healing and tissue regeneration phases. Conversely, HFE-C282Y, despite its role in ulcer establishment, did not affect the postoperative healing time. In perspective, the identification of patients with a poor prognosis may give clinicians the opportunity to modify management and to target tailored therapies in the view of a new and alternative concept of treatment based on pharmacogenomics.

Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.

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 cm(2,)+/- 23.6; VL = 6.1 cm(2,)+/- 6.9; LL = 4.1 cm(2,)+/- 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

Primary cultures of cortical neurons were employed to investigate the modulatory effects of neurotensin on glutamate excitotoxicity and the possible neuroprotective actions of the neurotensin receptor antagonist SR48692. NT(1-13) and its biologically active fragment NT(8-13) at 10 nM (30 min) increased endogenous glutamate levels. The inactive fragment NT(1-7) (10-100 nM; 30 min) was ineffective. SR48692, applied 20 min before NT and maintained in contact with cells during NT exposure as well as a low calcium medium (from the onset of the experiment) prevented the NT(1-13)-induced increase in extracellular glutamate levels. The addition of NMDA (0.01-10 micro M; 10 min) to the medium concentration-dependently increased extracellular glutamate levels. When 0.1 nM NT(1-13) was added in combination with 0.01 micro M NMDA, in concentrations by themselves ineffective, a significant increase in glutamate levels was observed. SR48692 at 100 nM counteracted the increase in glutamate levels induced by 0.1 nM NT(1-13) plus 0.01 micro M NMDA. The inhibitor of the protein kinase C (PKC) calphostin C (0.1 micro M; 10 min before NT) prevented the increase in glutamate levels induced by the combined treatments. The morphological analysis indicated that 10 nM NT(1-13) enhanced the glutamate (10 min)-induced apoptosis. The peptide was added 30 min prior to glutamate and maintained in contact with cells during the glutamate exposure. The presence of 100 nM SR48692 (20 min before NT) antagonized this effect of NT(1-13). These findings support the view of a pathophysiological role for NT in the cerebral cortex. Thus, under pathological conditions NT by enhancing glutamate outflow and by amplifying the NMDA-mediated glutamate signaling may be involved in increasing the degeneration of cortical neurons.

Cyclosporin A is a powerful immunosuppressive drug that has had a major impact on transplant therapy. It apparently links to different enzymatic pathways, and affects multiple enzymatic systems. Transforming growth factor beta induces the deposition of glycosaminoglycans, proteoglycans, and collagen fibers in the extracellular matrix. The aim of this study of normal and hypertrophic human gingival fibroblast cultures was to evaluate the cytoskeletal and extracellular changes in glycosaminoglycan secretion due to the presence of cyclosporin A and transforming growth factor beta. The results showed that there is an increase in total and individual classes of extracellular glycosaminoglycans in the presence of cyclosporin A and transforming growth factor beta, but the action of the latter was significantly greater. Immunohistochemical analysis of the cytoskeleton did not reveal any morphological differences between treated and control cells. Our data suggest that the biochemical changes in the extracellular matrix are caused more by cytokine, and that cyclosporin A does not induce any morphological changes in fibroblast cultures derived from hypertrophic and normal gingiva.