Effect of Electrode Distance in Grid Electrode: Numerical Models and In Vitro Tests.

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.

Notch pathway is active during osteogenic differentiation of human bone marrow mesenchymal stem cells induced by pulsed electromagnetic fields.

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.

Distinct gene expression profiles associated with Notch ligands Delta-like 4 and Jagged1 in plaque material from peripheral artery disease patients: a pilot study.

BACKGROUND: The lack of early diagnosis, progression markers and effective pharmacological treatment has dramatic unfavourable effects on clinical outcomes in patients with peripheral artery disease (PAD). Addressing these issues will require dissecting the molecular mechanisms underlying this disease. We sought to characterize the Notch signaling and atherosclerosis relevant markers in lesions from femoral arteries of symptomatic PAD patients. METHODS: Plaque material from the common femoral, superficial femoral or popliteal arteries of 20 patients was removed by directional atherectomy. RNA was obtained from 9 out of 20 samples and analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: We detected expression of Notch ligands Delta-like 4 (Dll4) and Jagged1 (Jag1), of Notch target genes Hes1, Hey1, Hey2, HeyL and of markers of plaque inflammation and stability such as vascular cell adhesion molecule 1 (VCAM1), smooth muscle 22 (SM22), cyclooxygenase 2 (COX2), Bcl2, CD68 and miRNAs 21-5p, 125a-5p, 126-5p,146-5p, 155-5p, 424-5p. We found an "inflamed plaque" gene expression profile characterized by high Dll4 associated to medium/high CD68, COX2, VCAM1, Hes1, miR126-5p, miR146a-5p, miR155-5p, miR424-5p and low Jag1, SM22, Bcl2, Hey2, HeyL, miR125a-5p (2/9 patients) and a "stable plaque" profile characterized by high Jag1 associated to medium/high Hey2, HeyL, SM22, Bcl2, miR125a and low Dll4, CD68, COX2, VCAM1, miR126-5p, miR146a-5p, miR155-5p, miR424-5p (3/9 patients). The remaining patients (4/9) showed a plaque profile with intermediate characteristics. CONCLUSIONS: This study reveals the existence of a gene signature associated to Notch activation by specific ligands that could be predictive of PAD progression.

MTHFR gene polymorphisms and methotrexate toxicity in adult patients with hematological malignancies: a meta-analysis.

BACKGROUND: MTHFR gene polymorphisms has been shown to be associated with methotrexate (MTX) toxicity in adult hematological malignancies; however, the results remain inconclusive. MATERIALS & METHODS: To examine the role of common MTHFR variants in MTX toxicity prediction, we performed a meta-analysis via identifying relevant studies for quantitative data pooling. RESULTS: Our results showed a significant association between MTHFR C677T polymorphism and increased risk of MTX-induced all-grade (grade 1-4) and severe (grade 3-4) hepatic and gastrointestinal toxicities in Caucasian independent of MTX dosage. MTHFR 677T allele increased risk of severe mucositis and all-grade hematological toxicity. MTHFR A1298C polymorphism was not significantly associated with hepatic and hematological toxicity, whereas perhaps having a protective effect on mucositis and gastrointestinal toxicity. CONCLUSION: MTHFR C677T polymorphism may be a good predictor for MTX toxicity in adult hematological malignancies.

Characterization of Notch Signaling During Osteogenic Differentiation in Human Osteosarcoma Cell Line MG63.

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.

Experimentally induced cartilage degeneration treated by pulsed electromagnetic field stimulation; an in vitro study on bovine cartilage.

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 1ß (IL1ß, 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 IL1ß or not (control), in terms of cartilage structure, cellularity and proteoglycans, glycosaminoglycans, collagen II and transforming growth factor ß1 synthesis by using histology, histomorphometry and immunohistochemistry. RESULTS: Making a comparison with control cartilage, IL1ß-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 IL1ß. 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.

Enhancement of melphalan activity by buthionine sulfoximine and electroporation in melanoma cells.

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.

Electromagnetic fields counteract IL-1ß activity during chondrogenesis of bovine mesenchymal stem cells.

Osteoarthritis (OA) is a common joint disease associated with articular cartilage degeneration. To improve the therapeutic options of OA, tissue engineering based on the use of mesenchymal stem cells (MSCs) has emerged. However, the presence of inflammatory cytokines, such as interleukin-1ß (IL-1ß), 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-1ß 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-1ß. Pellets of MSCs were differentiated for 3 and 5 weeks with transforming growth factor-ß3 (TGFß3), in the absence and presence of IL-1ß and exposed or unexposed to EMFs. Biochemical, quantitative real-time RT-PCR and histological results showed that EMFs alone or in the presence of TGFß3 play a limited role in promoting chondrogenic differentiation. Notably, in the presence of IL-1ß and TGFß3 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-1ß-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.

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

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

Bleomycin electrochemotherapy in elderly metastatic breast cancer patients: clinical outcome and management considerations.

PURPOSE: To evaluate the efficacy and toxicity of electrochemotherapy (ECT) in elderly metastatic breast cancer (BC) patients. METHODS: Retrospective analysis of 55 patients with superficial metastases who underwent ECT according to the European Standard Operative Procedures of electrochemotherapy. Treatment schedule consisted of intravenous or intratumoral bleomycin followed by locally delivered electric pulses. Statistical comparisons were performed between two groups: the patients aged <70 years (n = 27) and those ≥70 years (n = 28). Treatment outcomes were as follows: complete response (CR) rate, local progression-free survival (LPFS), new lesions-free survival (NLFS), toxicity and patient compliance. RESULTS: Patient groups were comparable for clinical-pathological features, except for the number of comorbidities (P < .001). The median follow-up was 32 months (range 6-53). Overall, CR rate was 40 % and was significantly higher in elderly patients (57 vs. 26 %, P = .023) and in patients with better performance status (PS = 0-1, 53 vs. PS = 2, 21 %, P = .048), although local tumor control showed a trend for lower values (2-year LPFS, 67 vs. 93 % among elderly and young patients, respectively; P = .061). Older women seemed less likely to progress outside the ECT field (2-year NLFS, 39 vs. 30 %, P = .075), but discontinued treatment more frequently due to impaired performance status (P = .002). Local pain was graded ≥3, according to a 10-point visual analog scale, by 16/28 (57.1 %) and 8/28 (28.6 %) elderly patients at 4 and 8 weeks, respectively. Wound debridement was required in 5/28 (18 %) older women, due to G3 skin ulceration. CONCLUSIONS: Elderly BC patients are highly responsive to ECT and achieve durable local tumor control. Physicians should be aware of possible debilitating side effects, such as pain and skin toxicity. Performance status and frailty screening could be a helpful addition to improve patient selection.

Functional tissue engineering in articular cartilage repair: is there a role for electromagnetic biophysical stimulation?

Hyaline cartilage lesions represent an important global health problem. Several approaches have been developed in the last decades to resolve this disability cause, including tissue engineering, but to date, there is not a definitive procedure that is able to promote a repair tissue with the same mechanical and functional characteristics of native cartilage, and to obtain its integration in the subchondral bone. The need of resolutive technologies to obtain a "more effective" tissue substitutes has led Butler to propose the "Functional Tissue Engineering" (FTE) paradigm, whose principles are outlined in a so-called FTE road map. It consists of a two-phase strategy: in vitro tissue engineering and clinically surgery evaluation. The first phase, based on construct development, should take into account not only the chondrocyte biology, as their sensitivity to biochemical and physical stimuli, the risk of dedifferentiation in culture, and the ability to produce extracellular matrix, but also the features of suitable scaffolds. The in vivo phase analyzes the inflammatory microenvironment where the construct will be placed, because the cytokines released by synoviocytes and chondrocytes could affect the construct integrity, and, in particular, cause matrix degradation. The use of pulsed electromagnetic fields (PEMFs) represents an innovative therapeutic approach, because it is demonstrated that this physical stimulus increases the anabolic activity of chondrocytes and cartilage explants with consequent increase of matrix synthesis, but, at the same time, PEMFs limit the catabolic effects of inflammatory cytokines, reducing the construct degradation inside the surgical microenvironment. PEMFs mediate an up-regulation of A2A adenosine receptors and a potentiation of their anti-inflammatory effects.

Chondroprotective effects of pulsed electromagnetic fields on human cartilage explants.

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

Human leukocyte antigen-G molecules are constitutively expressed by synovial fibroblasts and upmodulated in osteoarthritis.

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.

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

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

Can tumor necrosis factor receptor II gene 676T>G polymorphism predict the response grading to anti-TNFalpha therapy in rheumatoid arthritis?

In this study we analyzed whether the polymorphisms 676T>G in the tumor necrosis factor receptor (TNFR) II gene and -308G>A 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 -308G>A 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.

Time- and dose-dependent effects of chronic wound fluid on human adult dermal fibroblasts.

BACKGROUND: Wound healing is a biologic process that is altered in patients affected by chronic venous ulcers. The wound microenvironment is reflected in the chronic wound fluid (CWF), an exudate containing serum components and tissue-derived proteins. OBJECTIVES: We investigated the effects of increasing doses of CWF collected from patients suffering from chronic venous ulcers on human adult dermal fibroblasts cultured in vitro and the relationship among CWF effects and treatment length. METHODS: Fibroblasts were treated with 60, 240, and 720 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.

Methylenetetrahydrofolate reductase C677T and A1298C gene variants in adult non-Hodgkin's lymphoma patients: association with toxicity and survival.

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.

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

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

Effects of physical stimulation with electromagnetic field and insulin growth factor-I treatment on proteoglycan synthesis of bovine articular cartilage.

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.