Giordano Stabellini

C677T variant form at the MTHFR gene and CL/P: A risk factor for mothers?

Maternal folic acid supplementation in early pregnancy has been suggested to play a role in the prevention of nonsyndromic orofacial cleft, i.e., cleft lip with or without cleft palate (CL/P). Moreover, some authors demonstrated association of the C-->T mutation (C677T), converting an alanine to a valine residue in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, with other congenital anomalies such as neural tube defects (NTDs). Because of MTHFRs involvement in the metabolism of folate, we investigated 64 CL/P patients and their parents for C677T MTHFR mutation. No linkage disequilibrium was found using the transmission disequilibrium test (TDT). However, a significantly higher mutation frequency was detected in mothers of CL/P patients compared to controls. The odds ratios calculated for mothers having CT or TT genotype, compared to the normal CC genotype, were 2.75 (95% confidence interval 1.30-5.57) and 2.51 (1.00-6.14), respectively. These results support the involvement of the folate pathway in the etiology of CL/P, and indicate an effect of the maternal genotype, rather than influence of the embryos genotype.

Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants.

Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of interleukin-1 g (IL-1 g ) in the culture medium. Proteoglycan synthesis and the residual PG tissue content resulted significantly higher in EMF-exposed explants than in controls, whereas no effect was observed on PG release and nitric oxide (NO) production. IL-1 g induced both a reduction in PG synthesis and an increase in PG release, related to a strong stimulation of NO production, which resulted in a net loss of tissue PG content. In IL-1 g -treated explants, EMF increased PG synthesis, whereas in spite of a slight stimulation of NO production EMF did not modify PG release. This resulted in the residual PG tissue content being maintained at the control level. In both experimental conditions, the effects of EMF were associated with an increase in lactate production. The results of our study show that EMFs are able to promote anabolic activities and PG synthesis in bovine articular cartilage explants. This effect also is maintained in the presence of IL-1 g , thus counteracting the catabolic activity of the cytokine. Altogether, these data suggest that EMF exposure exerts a chondroprotective effect on articular cartilage in vitro.

Effects of Pulsed Electromagnetic Fields on Human Articular Chondrocyte Proliferation

Low-energy, low-frequency pulsed electromagnetic fields (PEMFs) can induce cell proliferation in several cell culture models. In this work we analysed the proliferative response of human articular chondrocytes, cultured in medium containing 10% FBS, following prolonged exposure to PEMFs (75 Hz, 2.3 mT), currently used in the treatment of some orthopaedic pathologies. In particular, we investigated the dependence of the proliferative effects on the cell density, the availability of growth factors and the exposure lengths. We observed that PEMFs can induce cell proliferation of low density chondrocyte cultures for a long time (6 days), when fresh serum is added again in the culture medium. In the same conditions, in high density cultures, the PEMF-induced increase in cell proliferation was observed only in the first three days of exposure. The data presented in this study show that the availability of growth factors and the environmental constrictions strongly condition the cellular proliferative response to PEMFs.

CD44 as prognostic factor in oral and oropharyngeal squamous cell carcinoma.

This retrospective case control study was conducted to assess the prognostic value of some patient-, tumor-, treatment-related variables, and to correlate markers of primary tumor with survival and cervical metastases. Twenty-five patients with histologically proven squamous cell carcinoma of the oral cavity and oropharynx were analyzed. Patients were never treated before and had a minimum follow-up review of 45 months.Results show that T-stage is the most important clinical prognostic parameter. Regarding immunohistochemical markers (Ki67 and CD44), only CD44 seems to be significantly correlated with prognosis but this value showed a multicollinear effect with N upon survival. Decreased expression of CD44 correlates with a decreased survival, although increased CD44 expression was consistent with a longer survival. Therefore, it was assessed that a loss of cell adhesion, related to decreased expression of CD44, may be determinant of survival in these patients.

Basic fibroblast growth factor autocrine loop controls human osteosarcoma phenotyping and differentiation.

BackgroundWe focused on the phenotype of non-mineralizing MG63 and mineralizing TE85 human osteosarcoma cells and investigated the role of bFGF in modulating their differentiative responses. Basic FGF expression and bFGF effects on osteocalcin, runt-related transcription factor-2 (RUNX2), matrix molecular production and bFGF receptors, were evaluated.Materials and MethodsOsteocalcin and RUNX2 gene expression were studied by RT-PCR analysis. We evaluated cell proliferation by DNA content and performed differentiation studies on glycosaminoglican (GAG), collagen and proteoglican (PG) synthesis by using radiolabelled precursors and Northern blotting. BFGF receptors were quantified by bFGF receptor binding assay.ResultsOsteocalcin is expressed in MG63 and TE65. RUNX2 RNA is differentially spliced in the two cell lines. BFGF elicits the effects of differentially splicing RUNX2. Proliferation, GAG synthesis, bFGF and proteoglycan mRNA expression, high and low affinity bFGF receptors, were more marked in MG63 and differently affected by bFGF. Procollagen expression and alkaline phosphatase activity were significantly reduced. BFGF increased TE85 cell proliferation and reduced TE85 procollagen and osteocalcin production.ConclusionsThe different splice variants in RUNX2 gene in the two cell lines might be related to their different phenotypes. The less differentiated stage of MG63 could also be related to bFGF over-production and more bFGF receptors. The consequent increase in bFGF-bFGF receptor binding could explain the bFGF differentiative effects on MG63. We suggest an autocrine role of bFGF endogenous release in controlling the different osteosarcoma phenotypes.

Glycosaminoglycan Metabolism and Cytokine Release in Normal and Otosclerotic Human Bone Cells Interleukin-1 Treated

Glycosaminoglycans (GAGs), normal components of the extracellular matrix (ECM), and the glycosidases, that degrade them, play a key role in the bone remodelling process. The effects of interleukin-1 alpha (IL-1 alpha) on GAG metabolism in normal and otosclerotic human bone cells as well as its capacity to modulate IL-1 alpha, IL-1 beta and IL-6 secretion in both populations was analyzed. The amount of radiolabeled GAGs was lower in otosclerotic than in normal bone cells. IL-1 alpha reduced newly synthesized cellular and extracellular GAGs in normal cells, but only those of the cellular compartment in otosclerotic bone cells. It depressed heparan sulphate (HS) more in normal cells and chondroitin sulphate (CS) more in otosclerotic bone cells. The HA/total sulphated GAG ratio was shifted in favour of the latter in otosclerotic cells, whereas the opposite effect was seen after IL-1 alpha treatment. There was little difference in the beta-D-glucuronidase levels of the normal and pathological cells, while beta-N-acetyl-D-glucosaminidase was significantly increased in otosclerotic bone cells. As the activity of neither enzyme was modified by treatment with IL-1 alpha, the cytokine seems to exert its influences on GAG synthesis rather than on the degradation process. IL-1 alpha, IL-1 beta and IL-6 secretion was markedly higher in otosclerotic cells. IL-1 alpha modulated the secretion of each interleukin differently, thus resulting in a cytokine cascade that may act in autocrine/paracrine manner on target cells. The authors suggest that changes in the cytokine network may have a specific, yet still unknown, role during normal and pathological osteogenesis.

Epithelial-mesenchymal interactions and lung branching morphogenesis. Role of polyamines and transforming growth factor ß1

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.

Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism†

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine‐treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age‐matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real‐time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF‐beta, retinoic acid (RA), and GABA‐ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin α2, and MMP13 genes were concordantly modulated, while integrin β5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition. J. Cell. Physiol. 222: 748–756, 2010.

Effects of interleukin-1β on chondroblast viability and extracellular matrix changes in bovine articular cartilage explants

Osteoarthritis is a degenerative disease of joint cartilage, characterized by the progressive and permanent degeneration of cartilage due to an imbalance in normal extracellular matrix turnover. Interleukin-1 beta is a proinflammatory agent, which is present in an elevated amount in osteoarthritic cartilage, and is thought to play a decisive role in osteoarthritis. Interleukin-1 beta acts as an important mediator of extracellular matrix changes where its activity is regulated by glycosaminoglycan composition. The aim of this study was to investigate the extracellular matrix changes in bovine cartilage explants following interleukin-1 beta treatment by morphological, histochemical and biochemical methods. Interleukin-1 beta stimulated the release of matrix sulfated proteoglycans in the culture medium, and significantly inhibited sulfated proteoglycan synthesis. These events were associated to a strong stimulation of nitric oxide production. Interleukin-1 beta-treated cartilage showed evident collagen fibers around the chondrocytes, together with diminished glycosaminoglycan sulfate content in the extracellular matrix of the explants. Moreover, the ultrastructure and viability of cells did not change in treated cartilage. Our data show that interleukin-1 beta modifies the ECM turnover without toxic effect on chondrocytes.

Bronchial Branching Correlates with Specific Glycosidase Activity, Extracellular Glycosaminoglycan Accumulation, TGFβ2, and IL-1 Localization During Chick Embryo Lung Development

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), β-N-acetyl-d-glucosaminidase activity, the presence of transforming growth factor β2 (TGFβ2), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of β-N-acetyl-d-glucosaminidase with biochemical methods. Our data show that β-N-acetyl-d-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that β-N-acetyl-d-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.