A novel ex vivo organotypic culture model of alkaptonuria-ochronosis.

OBJECTIVES: Alkaptonuria (AKU) is an orphan disease that has an estimated prevalence of 0.3/100,000. The disease is caused by the lack of activity of homogentisic acid oxidase (HGO), an enzyme involved in tyrosine and phenylalanine metabolism. To date, there is only one drug, the nitisinone, with orphan designation authorised by both Food and Drug Administration (FDA) and European Medical Agency (EMA) for AKU. A clinical trial on AKU patients using nitisinone has recently been completed but it needs further investigation for long-term therapy. In recent years our group has developed a series of AKU in vitro models using cell lines, primary chondrocytes and human plasma in order to test the efficacy of new substances, mainly antioxidant compounds, for AKU therapy. Herein, we report the optimisation of an ex vivo reproducible culture method exploiting cartilage slices in order to investigate the deposition of ochronotic pigment in this kind of connective tissue. METHODS: Human normal cartilage slices, obtained after surgery for prosthesis replacement, were cultured for several days in the presence of a sublethal concentration of homogentisic acid (HGA). RESULTS: After two months of incubation with HGA, the peculiar melanin-like ochronotic pigmentation can be observed into the cartilage tissue. CONCLUSIONS: This novel organo-typic ex vivo model could be extremely useful to investigate the efficacy of substances able to ameliorate the conditions of AKU patients. Moreover, it could be used for genetic and proteomic investigations to better define AKU pathophysiology.

Evaluation of the effect of Bosentan treatment on proinflammatory cytokine serum levels in patients affected by Systemic Sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease characterized by vascular and fibrotic changes in the skin and in internal organs. Endothelin-1 (ET-1) is a peptide that has a role in promoting both vascular injury and the fibrotic process in SSc; indeed, patients with systemic sclerosis have higher levels of ET-1 compared with healthy subjects. Moreover, ET-1 enhances expression of pro-inflammatory cytokines in animal model. Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension and digital ulcers in scleroderma patients. In animal models and in vitro models, after treatment with Bosentan, a significant reduction of cytokine (TNF α, IFN γ,IL-8, IL-4) levels was observed. The aim of the study is to verify whether Bosentan treatment in SSc patients can reduce circulating cytokines levels. We enrolled 10 patients affected by SSc with digital ulcers and/or pulmonary hypertension, treated with Bosentan 125 mg twice daily. Patients were tested for cytokines and ET-1 level before treatment and after 12 months. The cytokines tested were IL-10, IL-2, IL-4, IL-5, IL-6, IL-8, GM-CSF, IFN-γ and TNF. Levels of ET-1, IL-10, IL-4, IL-5, GM-CSF and TNFalpha did not show consistent modification during treatment with Bosentan in respect to baseline, while IL-2, IL-6, IL-8 and IFN-γ were significantly decreased. Bosentan significantly reduced IL-2, IL-6, IL-8 and IFN- γ levels in SSc patients, probably slowing progression to fibrosis and vascular damage. This is the first report showing a decrease of profibrotic and proinflammatory cytokines levels in humans during treatment with Bosentan.

Role of apoptosis in osteoporosis induced by glucocorticoids.

Glucocorticoid (GC)-induced osteoporosis (GIO) is a common and serious complication of prolonged systemic GC use. Bone loss with risk of fractures resulting from GC therapy is a relatively common disorder, and is the most prevalent form of secondary osteoporosis. It is generally accepted that GC can cause a rapid bone loss, decreasing bone formation and increasing bone resorption in vitro as well as in vivo. The decrease in bone formation has been mainly attributed to GC effects on osteoblastogenesis and osteocyte apoptosis, while the increase in bone resorption has been referred to an extension of the life-span of pre-existing osteoclasts. This article focuses on newer molecular aspects regarding the apoptotic mechanisms involved in the pathogenesis of GIO and is based on a presentation that was held at the 3rd Congresso Nazionale in Osteoporosi Secondarie e Endocrinopatie, in Ancona, Italy, October 2007.

Effect of chlorhexidine digluconate on different cell types: a molecular and ultrastructural investigation.

Although several studies have shown that chlorhexidine digluconate (CHX) has bactericidal activity against periodontal pathogens and exerts toxic effects on periodontal tissues, few have been directed to evaluate the mechanisms underlying its adverse effects on these tissues. Therefore, the aim of the present study was to investigate the in vitro cytotoxicity of CHX on cells that could represent common targets for its action in the surgical procedures for the treatment of periodontitis and peri-implantitis and to elucidate its mechanisms of action. Osteoblastic, endothelial and fibroblastic cell lines were exposed to various concentrations of CHX for different times and assayed for cell viability and cell death. Also analysis of mitochondrial membrane potential, intracellular Ca2+ mobilization and reactive oxygen species (ROS) generation were done in parallel, to correlate CHX-induced cell damage with alterations in key parameters of cell homeostasis. CHX affected cell viability in a dose and time-dependent manners, particularly in osteoblasts. Its toxic effect consisted in the induction of apoptotic and autophagic/necrotic cell deaths and involved disturbance of mitochondrial function, intracellular Ca2+ increase and oxidative stress. These data suggest that CHX is highly cytotoxic in vitro and invite to a more cautioned use of the antiseptic in the oral surgical procedures.

Sphingosine 1-phosphate induces myoblast differentiation through Cx43 protein expression: a role for a gap junction-dependent and -independent function.

Although sphingosine 1-phosphate (S1P) has been considered a potent regulator of skeletal muscle biology, acting as a physiological anti-mitogenic and prodifferentiating agent, its downstream effectors are poorly known. In the present study, we provide experimental evidence for a novel mechanism by which S1P regulates skeletal muscle differentiation through the regulation of gap junctional protein connexin (Cx) 43. Indeed, the treatment with S1P greatly enhanced Cx43 expression and gap junctional intercellular communication during the early phases of myoblast differentiation, whereas the down-regulation of Cx43 by transfection with short interfering RNA blocked myogenesis elicited by S1P. Moreover, calcium and p38 MAPK-dependent pathways were required for S1P-induced increase in Cx43 expression. Interestingly, enforced expression of mutated Cx43(Delta130-136) reduced gap junction communication and totally inhibited S1P-induced expression of the myogenic markers, myogenin, myosin heavy chain, caveolin-3, and myotube formation. Notably, in S1P-stimulated myoblasts, endogenous or wild-type Cx43 protein, but not the mutated form, coimmunoprecipitated and colocalized with F-actin and cortactin in a p38 MAPK-dependent manner. These data, together with the known role of actin remodeling in cell differentiation, strongly support the important contribution of gap junctional communication, Cx43 expression and Cx43/cytoskeleton interaction in skeletal myogenesis elicited by S1P.

[Anabolic effects and inhibition of interleukin 6 production induced by neridronate on human osteoblasts]. / Effetti anabolici e di inibizione della produzione di interleuchina 6 indotti dal neridronato in colture di osteoblasti umani.

UNLABELLED: Bisphosphonates (BPs) are pharmacological compounds widely used in the treatment of a variety of bone-related diseases, particularly where the bone-turnover is skewed in favour of osteolysis. The mechanisms by which BPs reduce bone-resorption directly acting on osteoclasts (OCs) are now largely clarified even at molecular level. The researches concerning the BPs effects on osteoblasts (OBs) have instead shown variable results. OBJECTIVES: We have investigated the efficacy of neridronate (NER), an amino-BP, as anabolic agent on human OBs. Moreover, we have tried to verify if NER is able to negatively modulate the production of IL-6 on OBs stimulated or not by the pro-inflammatory cytokine IL-1beta. METHODS: We have tested if different concentrations of NER (from 10-11 M to 10-3 M), added to primary human OB cultures, could affect the cells number, the endogenous cellular alkaline phosphatase (ALP) activity, the collagen I (COL-I) synthesis, the formation of mineralized nodules and the IL-6 production. Our experimental approach was performed testing a wide range of NER concentrations because, under physiological conditions, OBs seems to be exposed to variable and transient levels of the drug. RESULTS: Our results show that NER doesn't negatively affect in vitro the viability, proliferation and cellular activity of human OBs, even after 20 days of exposure to concentrations < or =10-5 M (therapeutic dose). In addition, NER seems to enhance the differentiation of cultured OBs in mature bone-forming cells. A maximum increase of COL-I synthesis (+25% after 4 days; p < 0.05), ALP activity (+50% after 10 days; p < 0.01) and mineralized nodules (+48% after 20 days; p < 0.05) was observed in cultures treated with NER 10-8 M. A maximal reduction of IL-6 secretion (-24% on IL-1beta stimulated cultures and -29% on unstimulated cultures) was observed for NER 10-9 M. CONCLUSIONS: These results encourage the use of neridronate in therapy of demineralizing metabolic bone disorders.

Effect of hyaluronic acid (MW 500-730 kDa) on proteoglycan and nitric oxide production in human osteoarthritic chondrocyte cultures exposed to hydrostatic pressure.

OBJECTIVE: This study investigated the in vitro effects of hyaluronic acid (HA) of molecular weight (MW) 500-730 kDa on human articular chondrocytes cultivated for 48 h in the presence of interleukin-1beta (IL-1beta) with and without hydrostatic cyclical pressure. DESIGN: The effects of 10 and 100 microg/ml HA with and without IL-1beta were assessed in the culture medium of cells exposed to pressurization cycles in the form of sinusoidal waves (minimum pressure 1MPa, maximum pressure 5MPa) at a frequency of 0.25Hz for 3h, by the immunoenzymatic method on microplates for the quantitative measurement of human proteoglycans (PG) and by the Griess method for nitrites (NO). Morphological analyses were performed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). RESULTS: The presence of IL-1beta determines a significant decrease in PG and a significant increase in NO concentrations measured in the culture medium. When the cells are cultured in the presence of IL-1beta and HA at the two concentrations, a statistically significant restoration of PG and a decrease in NO levels are observed. Under pressurization conditions, we observed that the PG concentration in the medium of cells presented a very significant increase in all the conditions used in the study, except for IL-1beta alone. NO production decreased very significantly in the presence of IL-1beta+HA 10 and IL-1beta+HA 100. The results of metabolic evaluation are confirmed by morphological findings obtained by TEM and SEM. CONCLUSIONS: These in vitro studies confirm both the protective role of HA (MW 500-730 kDa), which counteracts the IL-1beta-induced effects, and the importance of pressure on chondrocyte metabolism and morphology.

Simvastatin reduces MMP-3 level in interleukin 1beta stimulated human chondrocyte culture.

OBJECTIVES: Matrix metalloproteinases (MMPs) produced by chondrocytes play a role in the development of cartilage degradation in joint diseases. Moreover, inhibition of MMP secretion by macrophages accumulating in arteriosclerotic plaques would account for the plaque stabilising activity of statins in cardiovascular patients. Recently, simvastatin has been shown to inhibit both developing and established collagen induced arthritis in a murine model. We thus decided to investigate the effect of simvastatin on the production of MMP-3 from cultured interleukin (IL)1 stimulated human chondrocytes. METHODS: Cells from human cartilage, obtained from eight subjects with osteoarthritis undergoing surgery for total hip prostheses, were cultured in the presence of different concentrations of simvastatin (5, 10, and 50 micromol/l) with and without IL1beta (5 ng/ml). MMP-3 level was measured in the culture medium after 48 h of incubation. RESULTS: IL1beta stimulation of chondrocytes increased MMP-3 concentration in the cultures (from 0.69 (0.09) to 1.94 (0.12) ng/microg protein). Incubation with simvastatin was associated with a dose dependent reduction in MMP-3 increase, both in the presence (-15%, -17%, and -26% with 5, 10, and 50 micromol/l, respectively) and in the absence (-32% with 50 micromol/l) of IL1beta. The inhibiting effect of simvastatin was completely reversed by the addition of mevalonate (100 micromol/l) or farnesol (10 micromol/l). CONCLUSIONS: Our data show that simvastatin, by blocking HMGCoA-reductase and interfering in the prenylation processes, is able to inhibit MMP-3 production from cultured human chondrocytes that have been either unstimulated or stimulated with IL1beta, thus suggesting a possible additional mechanism for statins in counteracting chronic joint disease related cartilage damage.

Clinical determinants of bone mass and bone ultrasonometry in patients with systemic sclerosis.

OBJECTIVE: The aim of this study was to evaluate bone mass and bone ultrasonometry in patients affected with systemic sclerosis (SSc). METHODS: Fifty-five patients (mean age 54.1 +/- 14.1 years; 25 premenopausal, and 30 postmenopausal women) affected with SSc (in a limited, intermediate or diffused form) and 60 age-matched healthy controls (30 premenopausal, and 30 postmenopausal women) were studied for Bone Mineral Density (BMD) measured by fan-beam x-ray densitometry, Stiffness Index (SI) measured by ultrasonometry of the heel, inflammation indices (erithrocyte sedimentation rate, C-reactive protein), and autoantibodies (ANA, ENA). Examinations were also carried out in order to determine any internal organ involvement. None of the patients had previously received steroid treatment. RESULTS: BMD was significantly lower in the SSc group than in the control group, whether it was expressed in g/cm2 (lumbar spine: 0.980 vs 1.241, p < 0.01; femoral neck: 0.832 vs 0.955, p < 0.05; total body 1.050 vs 1.168, p < 0.01) or by T- and Z-score (lumbar spine: T = -2.48; Z = -1.10; femoral neck: T = -1.69; Z = -0.55; total body: T = -1.11; Z = -0.48). SI was also altered (75.8 vs 96.2, p < 0.01; T = -2.10, Z = -1.12). BMD and SI were lower in women with the diffuse form of skin involvement. BMD and SI were lower in women in whom one or more internal organs were involved. CONCLUSION: SSc patients had reduced BMD and SI that was more marked in the diffuse form and in those with internal organ involvement and that became more marked with age and estrogen deficiency. This demineralisation was not related to the inflammation indices, disease duration, or to the immunological pattern.

Effects of S1P on myoblastic cell contraction: possible involvement of Ca-independent mechanisms.

Sphingosine-1-phosphate (S1P) is a lipid mediator, which affects many essential processes such as cell proliferation, differentiation and contraction in many cell types. We have previously demonstrated that the lipid mediator elicits Ca(2+) transients in a myoblastic cell line (C2C12) by interacting with its specific receptors (S1PR(s)). In the present study, we wanted to correlate the Ca(2+) response with activation of myoblastic cell contractility. C2C12 cells were first investigated for the expression and cellular organization of cytoskeletal proteins by immunoconfocal microscopy. We found that myoblasts exhibited a quite immature cytoskeleton, with filamentous actin dispersed as a web-like structure within the cytoplasm. To evaluate intracellular Ca(2+) mobilization, the cells were loaded with a fluorescent Ca(2+) indicator (Fluo-3), stimulated with S1P and simultaneously observed with differential interference contrast and fluorescence optics. Exogenous S1P-induced myoblastic cell contraction was temporally unrelated to S1P-induced intracellular Ca(2+) increase; cell contraction occurred within 5-8 s from stimulation, whereas intracellular Ca(2+) increase was evident only after 15-25 s. To support the Ca(2+) independence of myoblastic cell contraction, the cells were pretreated with a Ca(2+) chelator, BAPTA/AM, prior to stimulation with S1P. In these experimental conditions, the myoblasts were still able to contract, whereas the S1P-induced Ca(2+) transients were completely abolished. On the contrary, when C2C12 cells were induced to differentiate into skeletal myotubes, they responded to S1P with a rapid cell contraction concurrent with an increase in the intracellular Ca(2+). These data suggest that Ca(2+)-independent mechanism of cell contraction may be replaced by Ca(2+)-dependent ones during skeletal muscle differentiation.

Evidence of H beta 58, a gene involved in mammalian placental development, in the three-toed skink, Chalcides chalcides (Squamata: Scincidae), a viviparous placentotrophic reptile.

The H beta 58 gene, whose disruption in mice causes reabsorption of the embryo at 9.5 days post-conception, is believed to be essential for development of the placenta. Although the H beta 58 gene is well conserved in some Amniota, nothing is known about its presence in reptiles, some species of which have developed a chorioallantoic placenta. In this work, we investigated the expression of H beta 58 mRNA and protein in the three-toed skink, Chalcides chalcides. H beta 58 protein expression was found in the uterine epithelium beginning from the peri-ovulatory stage. However, it increased strongly at the moment of placental formation, when a high level of expression of mRNA and protein was also observed in the extra-embryonic membranes. The expression of H beta 58 mRNA and protein was maintained, although to a lesser degree, in the placenta during late pregnancy. It was also present in the early embryo. Finally, cloning and sequencing of a gene fragment revealed strong homology of the reptile gene with that of mammals. The high degree of conservation of the gene in amniote vertebrates and its presence in a viviparous squamate reptile (as in mammals) indicates an important role of this gene in the chorioallantoic placenta formation and development.

Correlation between DNA content and p53 deletion in colorectal cancer.

OBJECTIVE: To find out whether tumour DNA content correlates with allelic loss of p53 and other pathological features in primary colorectal carcinomas. DESIGN: Ongoing prospective study. SETTING: University hospital, Italy. SUBJECTS: 128 patients who had undergone radical resections for colorectal carcinoma. INTERVENTIONS: Flow cytometric measurement of tumour DNA content and detection of allelic loss on the short arm of chromosome 17 by Southern blot (restriction fragment length polymorphism) analysis in fresh tumour specimens. MAIN OUTCOME MEASURES: Correlation between DNA ploidy and deletion of p53, as well as between these two genetic events and clinicopathological variables. RESULTS: Interpretable DNA histograms were obtained for 122 tumour specimens. Forty-three tumours (35%) were diploid and 79 (65%) aneuploid. The diploid tumours were significantly more common in the proximal colon (from the caecum to the splenic flexure) than in the distal colon (from the descending colon to the rectum) (p = 0.002). The allelic state on the short arm of chromosome 17 was evaluated in 80 heterozygous patients. Forty-four tumour specimens (55%) showed deletion of 17p. Allelic loss of p53 was significantly more common in the distal and rectal tumours than in the proximal ones (p < 0.0001). Aneuploidy was more common among those tumours which had shown deletion of p53 than in those that had not (p = 0.0008). CONCLUSIONS: DNA aneuploidy was significantly associated with the deletion of the p53 gene. This suggests that the functional loss of p53 may favour the growth and establishment of an aneuploid cell population within tumours. Tumours of the proximal and distal colon differ in their genetic nature.