Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line.

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

Can fibroblast growth factor (FGF)-23 circulating levels suggest coronary artery abnormalities in children with Kawasaki disease?

OBJECTIVES: Kawasaki disease (KD) is an acute self-limited panvasculitis, primarily affecting young children, with an outstanding risk of cardiovascular complications. Fibroblast Growth Factor-23 (FGF23) is the latest member of the FGF family, acting on phosphate metabolism, which has been shown to display a potential role in the vascular remodelling. The aim of our study was to test the hypothesis that circulating serum levels of FGF23 might be related to the occurrence of coronary artery abnormalities (CAA) in children with KD. METHODS: Serum of 109 consecutive KD patients (median age 30.5 months) were collected for the evaluation of intact FGF23 by ELISA test. Sixty sex/age-matched healthy children were studied as controls, after having excluded rheumatic, endocrinological and chronic renal diseases. In all these subjects a familiar predisposition to atherosclerosis was excluded. RESULTS: FGF23 levels resulted significantly higher in patients with KD than in controls (72±40 pg/ml vs. 12.3±3.2 pg/ml; p=0.01). Twenty-eight/109 KD patients having developed CAA (aneurysms or dilatations) presented significantly higher FGF23 levels than those without any coronary artery damage (120±40 pg/ml vs. 38.2±5 pg/ml; p<0.0001). Multiple logistic regression analysis showed that only serum FGF23 levels, among different general clinical and biochemical variables, were suggestive of coronary artery damage (OR=4.86). CONCLUSIONS: Based on this preliminary investigation, high serum FGF23 levels would seem suggestive of the potential occurrence of cardiac vascular complications in children with KD.

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology.

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

Fibroblast growth factor 23 (FGF23) gene polymorphism in children with Kawasaki syndrome (KS) and susceptibility to cardiac abnormalities.

BACKGROUND: Fibroblast Growth Factor (FGF) 23 influences endothelial integrity and few reports have studied the association between FGF23 and Kawasaki syndrome (KS), a childhood vasculitis displaying a high risk of subsequent cardiac abnormalities (CaA). AIM: To investigate the genetic variation in the FGF23 gene in a cohort of KS children and its association with serum FGF23 levels and eventual development of CaA, including both coronary artery dilatations and aneurysms. PATIENTS AND METHODS: 84 Italian KS children were recruited; 24/84 (28.6%) developed CaA. Each patient underwent evaluation of serum FGF23 levels and FGF23 genotype: the frequency of the c.212-37insC (rs3832879) polymorphism in intron 1 was examined and compared with sex, age at disease onset, fever duration, laboratory data, and occurrence of CaA. Univariate statistical analysis of categorical parameters was performed by the Pearson's Chi-square test or Fisher's exact test as appropriate. Parametric variables were assessed by Student's t-test for unpaired data. Independent predictors of disease were studied by a logistic regression model. RESULTS: 28/84 patients carried the FGF23 polymorphism (33.3%) and had higher serum FGF23 levels (p < 0.01). FGF23 polymorphism was significantly associated with CaA compared to wild type FGF23 children (respectively, p = 0.03 and p = 0.05). The comparison with demographical, clinical or laboratory data was not significant. CONCLUSIONS: The prevalent segregation of the c.212-37insC polymorphism in children with CaA advocates a possible functional FGF23 role in the predisposition to higher serum levels of FGF23 and potential occurrence of any coronary artery abnormalities in KS.

A novel germline inactivating mutation in the CASR gene in an Italian kindred affected by familial hypocalciuric hypercalcemia.

OBJECTIVE: Familial hypocalciuric hypercalcemia (FHH) syndrome is a rare benign condition, inherited as an autosomal dominant trait, in which inactivating mutations of the calcium-sensing receptor (CASR) gene affects the body's ability to regulate calcium homeostasis. Its outcome is featured by increased levels of serum calcium, moderate hypophosphatemia, and inadequately normal or elevated circulating parathyroid hormone levels. Affected patients are mostly asymptomatic and do not benefit from surgical resection of their mildly enlarged parathyroids. DESIGN: We evaluated for hypercalcemia an Italian family that was identified via a young adult male proband referred to our center for parathyroidectomy. METHODS: The patients and the family members were evaluated both biochemically and genetically as suspected FHH subjects. An in vitro functional study was performed by site-directed mutagenesis, and CASR activity was monitored by measuring intracellular calcium ([Ca(2)(+)](i)). RESULTS: The patient had a novel germline heterozygous CASR mutation (c.361_364GATT; p.D121del/fsX122). The mutation caused a premature stop codon at codon 122, exiting a truncated protein. The biochemical phenotype of all family members carrying the heterozygous deletion was concordant with classic FHH syndrome. CONCLUSIONS: Our findings confirm the role of CASR gene mutational analysis to offer a valuable addition for the recognition of FHH in hypercalcemic patients not yet characterized for a positive familial history of hypercalcemia, the only condition that identifies CASR gene mutations in hypercalcemia.

Neprilysin levels in plasma and synovial fluid of juvenile idiopathic arthritis patients.

OBJECTIVE: Neprilysin (neutral endopeptidase, 3:4:24:11, CD10) (NEP) is a Zn metallopeptidase linked to controlling inflammation through the degradation of neuropeptides involved in neurogenic inflammation of chronic rheumatic diseases. The aim of our study was to evaluate circulating activity and cellular expression of NEP in the plasma of 58 children with juvenile idiopathic arthritis (JIA) and 52 controls. In 20 subjects requiring local steroid injection, NEP was measured in synovial fluid. METHODS: Plasma and synovial NEP were evaluated using a fluorimetric technique. Neprilysin, expressed as the antigen CD10, was determined on circulating and synovial fluid cells as mean fluorescence intensity (MFI) and as percentage of positive cells by two-color immunofluorescence. RESULTS: Circulating NEP levels were lower in JIA patients than in controls (42.0+/-16.6 vs 76.5+/-24 pmol/ml per min, P<0.001), while synovial fluid NEP values were higher than circulating levels (241.4+/-86.2 vs 40+/-15.3 pmol/ml per min, P<0.001). In monocytes, the percentage of CD10-positive circulating cells and the MFI in JIA were lower than in controls (11.6+/-5.2% vs 41.4+/-13%, P<0.001 and 18.1+/-7.5 vs 31.2+/-5.4, P<0.05, respectively). On synovial monocytes, the percentage of CD10-positive cells and the MFI were higher than on circulating monocytes (35.2+/-14.6% vs 9.1+/-2.4%, P<0.001 and 66.4+/-5.4 vs 22.8+/-14.7, P<0.001, respectively). CONCLUSIONS: The downregulation of CD10 expression in monocytes and the reduction in NEP activity may be linked to the enzyme's role in the control of peptides involved in the inflammation. The increased levels of NEP, MFI, and CD10-positive monocytes in synovial fluid, even though in plasma, might reflect a reactive effort to control synovial proliferation.