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Oral cavity and oropharynx cancer associated with human papil loma virus infection, particularly in young people who are continuously exposed to this virus, is a serious public health problem worldwide, especially for high-risk strains that are most associated with premalignant lesions and tumors. These neoplasms remain asymptomatic for a long time and, when they occur, they are already at an advanced stage. If diagnosed and treated early, oral cancer induced by human papilloma virus allows for high survival, as it often has a more favorable prognosis than oral cancers not directly related to viral infection. In this study, the presence of different high-risk and low-risk HPV strains was investigated to assess the epidemiological status in a population of healthy individuals. Two types of samples, one from the tonsils and one from the base of the tongue, were collected from 2015 healthy individuals of different sex and age. A total of 1885 DNA samples belonging to 1285 patients were tested for the presence of 11 high-risk HPV types plus 4 low-risk HPV types using real-time PCR. Of the patients’ DNA samples screened for 15 HPV types, only four samples were positive, all of which were taken from male smokers. These results indicate that newly acquired oral oncogenic HPV infections in the healthy population are rare and, in many cases, controversial. Therefore, more studies are needed to ensure fewer variations in outcomes and a greater clarification of HPV infection and its prevalence in the oropharynx of the healthy population, and to guide efforts to prevent the development of this infection which, if undiagnosed, can lead to the onset of malignant tumors in the oral cavity.

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.

Background and Aims Cardiovascular disease is the main cause of death worldwide, but the collective efforts to prevent this pathological condition are directed exclusively to individuals at higher risk due to hypercholesterolemia, hypertension, obesity, diabetes. Recently, vitamin D deficiency was identified as a risk factor for cardiovascular disease in healthy people, as it predisposes to different vascular dysfunctions that can result in plaque development and fragility. In this scenario, the fundamental aim of the study was to reproduce a disease model inducing vitamin D deficiency and atheromatosis in ApoE-/- mice and then to evaluate the impact of this vitamin D status on the onset/progression of atheromatosis, focusing on plaque formation and instability. Methods and Results In our murine disease model, vitamin D deficiency was achieved by 3 weeks of vitamin D deficient diet along with intraperitoneal paricalcitol injections, while atheromatosis by western-type diet. Under these experimental conditions, vitamin D deficient mice developed more unstable atheromatous plaques with reduced or absent fibrotic cap. Since calcium and phosphorus metabolism and also cholesterol and triglycerides systemic concentration were not affected by vitamin D level, our results highlighted the role of vitamin D deficiency in the formation/instability of atheromatous plaque and, although further studies are needed, suggested a possible intervention with vitamin D to prevent or delay the atheromatous disease. Conclusions The data obtained open the question about the potential role of the vitamins in the pharmacological treatments of cardiovascular disorders as coadjutant of the primary drugs used for these pathologies., Graphical abstract Image 1, Highlights • The vitamin D deficiency represents a risk factor for cardiovascular disease. • The vitamin D deficiency has been correlated to atheromatosis progression. • A model of vitamin D deficiency was fundamental to evaluate the role of the vitamin D the onset/progression of atheromatosis. • The vitamin D deficiency was clearly involved in an early formation of atherosclerotic lesions. • The vitamins play a potential role in the pharmacological treatments of cardiovascular disorders.

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.

Electrochemotherapy (ECT) is a local anticancer treatment based on the combination of chemotherapy and short, tumor-permeabilizing, voltage pulses delivered using needle electrodes or plate electrodes. The application of ECT to large skin surface tumors is time consuming due to technical limitations of currently available voltage applicators. The availability of large pulse applicators with few and more spaced needle electrodes could be useful in the clinic, since they could allow managing large and spread tumors while limiting the duration and the invasiveness of the procedure. In this article, a grid electrode with 2-cm spaced needles has been studied by means of numerical models. The electroporation efficiency has been assessed on human osteosarcoma cell line MG63 cultured in monolayer. The computational results show the distribution of the electric field in a model of the treated tissue. These results are helpful to evaluate the effect of the needle distance on the electric field distribution. Furthermore, the in vitro tests showed that the grid electrode proposed is suitable to electropore, by a single application, a cell culture covering an area of 55 cm2. In conclusion, our data might represent substantial improvement in ECT in order to achieve a more homogeneous and time-saving treatment, with benefits for patients with cancer.

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.

In this paper, the electric field generated in a conductive medium by needles pairs with inter-needle distance of 1 or 2 cm has been studied by means of 3D numerical models. The computational results have been correlated with experimental ones obtained in vitro on cultured cells. In particular, electroporation effect has been assessed on human osteosarcoma cell line MG63 cultured in monolayer. These results are helpful to evaluate the effect of the needle distance on the electric field distribution in cultured cells in terms of occurrence of reversible or irreversible electroporation.

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.

Coating characteristics of dental implants such as composition and topography regulate cell response during implant healing. The aim of this study was to assess how surface topography can affect osteogenic differentiation of mesenchymal stem cells (MSCs) by analyzing the expression levels of bone-related genes and MSCs marker. Thirty disk-shaped, commercially pure Grade 2 titanium samples (10 × 2 mm) with 3 different surface topographies (DENTSPLY-Friadent GmbH, Mannheim, Germany) were used in the present study: 10 Ti machined disks (control), 10 Ti sandblasted and acid-etched disks (DPS(®)) and 10 sandblasted and acid-etched disks at high temperature (Plus(®)). Samples were processed for real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. By comparing machined and Plus(®) disks, quantitative real-time RT-PCR showed a significant reduction of the bone-related genes osteocalcin (BGLAP) and osteoblast transcriptional factor (RUNX2). The comparison between DPS(®) and Plus(®) disks showed a slight induction of all the genes examined (RUNX2, ALPL, COL1A1, COL3A1, ENG, FOSL1, SPP1, and SP7); only the expression of BGLAP remained stable. The present study, demonstrated that implant surface topography affects osteoblast gene expression. Indeed, Plus(®) surface produces an effect on MSCs in the late differentiation stages.

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 A(2A) and A(3) 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 A(2A) and A(3) adenosine agonists and antagonists showed that EMFs inhibit the release of prostaglandin E(2) (PGE(2)) 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 A(2A) and A(3) 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 A(2A) and A(3) AR activation. Taken together, these results open new clinical perspectives to the control of inflammation associated to joint diseases.

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

Summary Objective To investigate the role of adenosine analogs and electromagnetic field (EMF) stimulation on prostaglandin E 2 (PGE 2 ) release and cyclooxygenase-2 (COX-2) expression in bovine synovial fibroblasts (SFs). Methods SFs isolated from synovia were cultured in monolayer. Saturation and binding experiments were performed by using typical adenosine agonists: N 6-cyclohexyladenosine (CHA, A 1 ), 2-[ p -(2-carboxyethyl)-phenetyl-amino]-5′- N -ethylcarboxamidoadenosine (CGS 21680, A 2A ), 5′- N -ethylcarboxamidoadenosine (NECA, non-selective), N 6-(3-iodobenzyl)2-chloroadenosine-5′- N -methyluronamide (Cl-IB-MECA, A 3 ). SFs were treated with TNF-α (10ng/ml) and lipopolysaccharide (LPS) (1μg/ml) to activate inflammatory response. Adenosine analogs were added to control and TNF-α- or LPS-treated cultures both in the absence and in the presence of adenosine deaminase (ADA) which is used to deplete endogenous adenosine. Parallel cultures were exposed to EMFs (75Hz, 1.5mT) during the period in culture (24h). PGE 2 release was measured by immunoassay. COX-2 expression was evaluated by RT-PCR. Results TNF-α and LPS stimulated PGE 2 release. All adenosine agonists, except for Cl-IB-MECA, significantly inhibited PGE 2 production. EMFs 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 and NECA was stronger than in the absence of ADA and the EMF-inhibitory effect was lost. Changes in PGE 2 levels were associated to modification of COX-2 expression. Conclusions This study supports anti-inflammatory activities of A 1 and A 2A adenosine receptors and EMFs in bovine SFs. EMF activity appears mediated by an EMF-induced up-regulation of A 2A receptors. Biophysical and/or pharmacological modulation of adenosine pathways may play an important role to control joint inflammation.

Summary Objective The aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X 1 and P2X 3 purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE 2 ) release. Methods Saturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X 1 and P2X 3 purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X 1 and P2X 3 purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE 2 release) the potency of purinergic agonists and antagonists was evaluated. Results Bovine chondrocytes expressed P2X 1 and P2X 3 purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5′-triphosphate (ATP) and α,β-methyleneATP were able to increase NO and PGE 2 release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists. Conclusions These data demonstrate for the first time the presence of functional P2X 1 and P2X 3 purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE 2 release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption.

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.

Summary Objective To investigate the role of pulsed electromagnetic field (PEMF) exposure parameters (exposure length, magnetic field peak amplitude, pulse frequency) in the regulation of proteoglycan (PG) synthesis of bovine articular cartilage explants. Methods Bovine articular cartilage explants were exposed to a PEMF (75Hz; 2mT) for different time periods: 1, 4, 9, 24h. Then, cartilage explants were exposed for 24h to PEMFs of different magnetic field peak amplitudes (0.5, 1, 1.5, 2mT) and different frequencies (2, 37, 75, 110Hz). PG synthesis of control and exposed explants was determined by Na 2 - 35 SO 4 incorporation. Results PEMF exposure significantly increased PG synthesis ranging from 12% at 4h to 17% at 24h of exposure. At all the magnetic field peak amplitude values, a significant PG synthesis increase was measured in PEMF-exposed explants compared to controls, with maximal effect at 1.5mT. No effect of pulse frequency was observed on PG synthesis stimulation. Conclusions The results of this study show the range of exposure length, PEMF amplitude, pulse frequency which can stimulate cartilage PG synthesis, and suggest optimal exposure parameters which may be useful for cartilage repair in in vivo experiments and clinical application.

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.

Normal bone tissue is characterised by a balancing of osteoblast and osteoclast activity. The activity and differentiation of these cells are regulated by vitamins, hormones and cytokines. The action of these factors on bone tissue cells depends on the composition and mineralisation of extracellular bone matrix. In particular, transforming growth factor beta 1 (TGFbeta1) acts on collagen fibres, glycosaminoglycan secretion and on the enzymes correlated to the turnover of glycosaminoglycans. The normal functions of bone tissue also depend on its mineralisation, which is highly altered in the process of uraemia.In this study, we analysed in vitro the effect of transforming growth factor beta on osteoblast proliferation, collagen synthesis and glycosaminoglycan secretion with 3H-thymidine, 3H-proline or 3H-glucosamine incorporation, and on enzymes, such as beta-N-acetyl-D-glucosaminidase and beta-glucuronidase, involved in extracellular matrix turnover. Moreover, phosphatase alkaline activity and osteocalcin related to mineralisation of extracellular matrix were determined.Our data show that TGFbeta1 significantly decreases 3H-thymidine and 3H-proline incorporation and increases (por = 0.01) extracellular sulphated glycosaminoglycan synthesis. It also increases osteocalcin levels, phosphatase alkaline, beta-N-acetyl-D-glucosaminidase and beta-glucoronidase activities.TGFbeta1 changes the synthesis of extracellular matrix components by osteoblasts. These variations favour the action of cytokine and osteoclasts. Since the TGFbeta1 accumulates in bone tissue and increases during uraemia, with due limitations this action leads to an imbalance between synthesis and degradation and could explain bone alterations in uraemic patients.

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.

Summary Aim During uraemia, an increase of middle molecules and acetylpolyamines occurs. In vitro the middle molecules produce cell toxicity, while the acetylpolyamines stimulate cell proliferation and differentiation. These phenomena are related to protein and extracellular glycosaminoglycan production. The aim of the present study was to evaluate the activity of dialysate, dialysate fluid and the chromatographic peaks of dialysate fractionated by G-15 Sephadex column on chick embryo skin development. Methods We evaluated the effects of protein and glycosaminoglycan synthesis using monolayer and organotypic cultures. Results Our data show that dialysate, chromatographic peak II, and 2×10 28 M N 1 -acetylspermine cause inhibition of chick embryo skin culture development. On the contrary, 10 −8 M N -acetylornithine and dialysate fluid increase protein and extracellular glycosaminoglycan synthesis, whereas chromatographic peak I does not reveal differences when compared to controls. Conclusions These extracelluar changes are related to cell proliferation and feather formation in chick embryo organotypic culture. Moreover, the pH changes of culture medium do not influence the biological action of acetylpolyamines and dialysate fluid on protein and extracellular glycosaminoglycan synthesis. Cell death in the presence of N 1 -acetylspermine, dialysate and peak II appears unrelated to the apoptotic process. The data show that acetylpolyamines, dialysis fluid, dialysate and chromatographic peaks act on fibroblasts, and are able to modify glycosaminoglycan synthesis. The organotypic cultures of chick embryo back skin could represent a model for studying the modifications of the extracellular matrix induced by these substances.

Objectives This study aims to study how Bio-Oss® can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells markers using real time Reverse Transcription-Polymerase Chain Reaction. Methods PB-hMSCs stem preparations were obtained for gradient centrifugation from peripheral blood of healthy anonymous volunteers, using the Acuspin System-Histopaque 1077. The samples were then cultured for 7 days for RNA processing, and the expression was quantified using real time PCR. Results Bio-Oss® caused an induction of osteoblast transcriptional factor like RUNX2 and of bone related genes; SPP1 and FOSL1. In contrast, the expression of ENG was significantly decreased in stem cells treated with Bio-Oss® with respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. Conclusion The results obtained can be relevant to enhance the understanding of the molecular mechanism of bone regeneration and can act as a model for comparing other materials with similar clinical effects.

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.

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.

We have studied the effects of low-energy, low-frequency pulsed electromagnetic fields (PEMF) on cell proliferation, in both human osteoblast-like cells obtained from bone specimens and in human MG-63 osteosarcoma cell line. Assessment of osteoblastic phenotype was performed both by immunolabeling with antiosteonectin antibody and by verifying the presence of parathyroid hormone receptors. The cells were placed in multiwell plates and set in a tissue culture incubator between a pair of Helmholtz coils powered by a pulse generator (1.3 ms, 75 Hz) for different periods of time. [3H]Thymidine incorporation was used to evaluate cell proliferation. Since it had previously been observed that the osteoblast proliferative response to PEMF exposure may also be conditioned by the presence of serum in the medium, experiments were carried out at different serum concentrations. [3H]Thymidine incorporation increases in osteoblast-like cells, when they are exposed to PEMF in the presence of 10% fetal calf serum (FCS). The greatest effect is observed after 24 hours of PEMF exposure. No effects on cell proliferation are observed when osteoblast-like cells are exposed to PEMF in the presence of 0.5% FCS or in a serum-free medium. On the other hand, PEMF-exposed MG-63 cells show increased cell proliferation either at 10% FCS, 0.5% FCS and in serum-free medium. Nevertheless, the maximum effect of PEMF exposure on MG-63 cell proliferation depends on the percentage of FCS in the medium. The higher the FCS concentration, the faster the proliferative response to PEMF exposure. Our results show that, although MG-63 cells display some similarity with human bone cells, their responses to PEMF's exposure are quite different from that observed in normal human bone cells.

Bio-engineered scaffolds used in orthopedic clinical applications induce different tissue responses after implantation. In this study, non-stoichiometric Mg2+ ions and stoichiometric apatites, which are used in orthopedic surgery as bone substitutes, have been assayed in vitro with human adult mesenchymal stem cells (hMSC) to evaluate cytocompatibility and osteoconductivity. hMSCs from the bone marrow aspirates of orthopedic patients were isolated and analyzed by flow cytometry for the surface markers Stro1, CD29, CD44, CD71, CD73, CD90, CD105 (positive) and CD45, CD235 (negative). The hMSC were analyzed for self-renewal capacity and for differentiation potential. The hMSC, which were grown on different biomaterials, were analyzed for (i) cytotoxicity by AlamarBlue metabolic assay, (ii) osteoconductivity by ELISA for activated focal adhesion kinase, (iii) cytoskeleton organization by fluorescence microscopy, and (iv) cell morphology which was investigated by scan electron microscopy (SEM). Results indicate that isolated cell populations agree with minimal criteria for defining hMSC cultures. Non-stoichiometric Mg2+ and stoichiometric apatites, in granular form, represent a more favorable environment for mesenchymal stem cell adhesion and growth compared to the non-stoichiometric Mg2+ apatite, in nano-structured paste form. This study indicates that different forms of biomaterials modulate osteoconductivity and cellular growth by differential activation focal adhesion kinase. J. Cell. Physiol. 228: 1229–1237, 2013. © 2012 Wiley Periodicals, Inc.

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.

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.

Summary Aims Morphogenetic processes during palate development are related to extracellular matrix composition. The cell–extracellular matrix relation plays a role in cell activity and in gene expression. We studied the effect of diphenylhydantoin, a teratogen known to induce cleft palate in human newborns, on extracellular matrix production. We investigated whether diphenylhydantoin treatment caused any differences in glycosaminoglycans, collagen synthesis and gene expression in human normal palate fibroblasts. Methods Human palate fibroblasts were maintained for 24 hours in serum-free 199 medium containing 5 µg/mL 3 H-glucosamine or 3 H proline hydrochloride. Collagen and glycosaminoglycan classes were then measured using biochemical methods, gene expression with microarray analysis and cytoskeleton components with immunofluorescent antibodies and computer analysis. Results In normal fibroblasts diphenylhydantoin reduced collagen and glycosaminoglycan synthesis with a marked effect on sulphated glycosaminoglycans. There were also substantial decreases in tubulin, vimentin and alpha-actin staining and an increase of vinculin compared to controls. Diphenylhydantoin acted on several genes related to the synthesis of cytoskeleton and adhesion membrane proteins. It inhibited caderin, caveolin, RTK and alpha-actin, and increased nectin, cytoplasmatic FRG vinculin, ITGA, ITGB extracellular matrix ligand and EDG2 gene expression. DNA binding gene expression, which plays a role in cell growth and senescence, was activated. Conclusions Since cell activity is dependent on the cell morphology and extracellular matrix composition, these findings indicate that in human normal palate fibroblasts diphenylhydantoin can modify cytoskeletal components and extracellular matrix–cell adhesion, with consequent effects on gene expression. These changes might be related to anomalous palate development.

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.

In uremic patients during chronic hemodialysis an increase in the volume of red blood cells is observed. Contemporaneously there is an increase in intraerythrocytic ornithynedecarboxylase activity beyond the normal content (P0.01), a high level of seric and plasmatic polyamines (P0.01) and a decrease in seric osmolality (P0.01) with pH improvement. The trends of osmolality, ornithynedecarboxylase, mean cell volume and pH are significantly related. Our data support the hypothesis that, during hemodialysis, red blood cell volume changes and increased ornithynedecarboxylase activity are dependent on the general improvement of plasma tonicity. Moreover, the absence of inhibition of ornithynedecarboxylase activity by high levels of putrescine is noted.

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.

Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

Implanting an expander in the subcutaneous layer causes gradual expansion and provides additional tissue for reconstruction of tissular defects. The force applied remodels the connective tissue and modifies dermis contractibility in additional tissue. Other authors confirm that parameters such as mitosis and hyaluronan influence the system in the tissue regeneration processes. We studied histochemical and morphological variations of tissue expanders before and 6 months after transplant. Our histochemical data do not show any changes in dermis glycosaminoglycans of the expanded and transplant-expanded skin when compared to controls. Morphological data demonstrate reorganization of connective fibers and disappearance of the papillar layer. The latter is not yet formed in the expanded skin 6 months after transplant. This suggests that a long time is required for biological reconstruction of epidermal-dermal interactions after transplant.

We have previously shown that feather formation in chick embryonic skin depends on accumulation of sulphated glycosaminoglycans in the underlying mesenchyme, and that addition of spermidine to chick embryo fibroblasts increases the extracellular sulphated glycosaminoglycans. In the present work, using histological, histochemical and biochemical procedures, we have investigated the effects on glycosaminoglycan accumulation and on epithelial differentiation of adding spermidine and bis-cyclohexylammonium sulphate, a spermidine inhibitor, to embryonic chick skin cultures. Our results demonstrate that spermidine induces an accumulation of sulphated glycosaminoglycan and an increase in feather formation, suggesting that the morphogenetic effect of spermidine may be dependent on specific glycosaminoglycan accumulation.

Administration of di(2-ethylhexyl) phthalate (DEHP) to primary cultures of chick embryo fibroblasts brought about a decrease in cell proliferation rate after 48 h and an inhibition of both DNA and protein synthesis measured by [3H]thymidine and [3H]leucine, respectively, after 48h. The growth of chick embryo lung rudiments in vitro was also depressed by DEHP treatment. Lung rudiment were smaller in DEHP-treated embryos after 6 days' treatment. These results indicate that DEHP has a cytostatic effect on embryonic cells and tissues. L'administration de di(2-éthylhexyl) phthalate (DEHP) à des cultures primaires de fibroblastes d'embryon de poulet a provoqué une diminution du taux de prolifération cellulaire après 48 heures et une inhibition de la synthèse à la fois d'ADN et de protéines mesurée, respectivement, par [3H]thymidine et [3H]leucine après 48 h. La croissance des ébauches pulmonaires de l'embryon de poulet in vitro a également été abaissée par le traitement DEHP. Les ébauches pulmonaires étaient plus petites dans les embryons traités au DEHP après un traitement de 6 jours. Ces résultats indiquent que le DEHP a un effet cytostatique sur les cellules et tissus embryonnaires.

The administration of bis-cyclohexylammonium sulfate (BCHS), a spermidine synthase inhibitor, to in vitro cultures of chick embryo fibroblasts caused a decrease in cellular spermidine levels and an increase in putrescine and spermine. Cell proliferation rate and DNA synthesis were also inhibited. As protein synthesis did not change, it would seem that low levels of cellular spermidine inhibit cell growth depressing DNA synthesis.