Fast method for skeletal tissue gene expression analysis

open9Several chronic diseases have been associated with bone alteration in the last few years. Despite the wealth of information provided by the analysis of the transcriptome in affected tissues, only a limited number of studies evaluated gene expression in bone tissue due to the difficulty to obtain high quality RNA. Therefore, skeletal pathologies have been often associated to a defective maturation process that occurs during recruitment of progenitor stem cells. In order to explore the possibility of analysing the gene expression during osteogenic differentiation in skeletal tissue, a single-step method to extract well-preserved RNA from bone specimens was performed. A comparison between this technique and a traditional method was made by analysing the quality and yield of RNA obtained. In addition, RNAs were assayed by reverse transcription-quantitative polymerase chain reaction to analyse the expression levels of the bone genes associated with the differentiation process in a mouse model. The present data showed that good quality RNA can be obtained from bone tissue by a simple single-step method allowing the expression analysis of the genes encoded by skeletal tissue. In conclusion, the present study allows the possibility to easily obtain good quality RNA from bone tissue that is suitable for gene expression studies of bone diseases.openDalle Carbonare, Luca; Vilei, Maria Teresa; Stranieri, Chiara; Innamorati, Giulio; Rosato, Antonio; Boldrin, Elisa; Sella, Stefania; Giannini, Sandro; Valenti, Maria TeresaDALLE CARBONARE, LUCA GIUSEPPE; Vilei, MARIA TERESA; Stranieri, Chiara; Innamorati, Giulio; Rosato, Antonio; Boldrin, Elisa; Sella, Stefania; Giannini, Sandro; Valenti, Maria Teres

The effect of risedronate on osteogenic lineage is mediated by cyclooxygenase-2 gene upregulation.

Introduction: The purpose of this study was to evaluate the effects of risedronate (Ris) in the modulation of bone formation in rats with glucocorticoid (GC)-induced osteoporosis by histomorphometric, immunohistochemical and gene expression analyses. Methods: We analyzed structure, turnover and microarchitecture, cyclooxygenase 2 (COX-2) levels and osteocyte apoptosis in 40 female rats divided as follows: 1) vehicle of methylprednisolone (vGC) + vehicle of risedronate (vRis); 2) Ris 5 \u3bcg/Kg + vGC; 3) methylprednisolone (GC) 7 mg/Kg + vRis; 4) GC 7 mg/Kg +Ris 5 \u3bcg/Kg. In addition, we evaluated cell proliferation and expression of COX-2 and bone alkaline phosphatase (b-ALP) genes in bone marrow cells and MLO-y4 osteocytes treated with Ris alone or in co-treatment with the selective COX-2 inhibitor NS-398 or with dexametasone. Results: Ris reduced apoptosis induced by GC of osteocytes (41% vs 86%, P < 0.0001) and increased COX-2 expression with respect to controls (Immuno-Hystochemical Score (IHS): 8.75 vs 1.00, P < 0.0001). These positive effects of Ris in bone formation were confirmed by in vitro data as the viability and expression of b-ALP gene in bone marrow cells resulted increased in a dose dependent manner. Conclusions: These findings suggest a positive effect of Ris in bone formation and support the hypothesis that the up-regulation of COX-2 could be an additional mechanism of anabolic effect of Ris

Serum and bile bilirubin pigments in the differential diagnosis of Crigler-Najjar disease

OBJECTIVE: To differentiate between Crigler-Najjar (CN) disease types 1 and 2. DESIGN: The patterns of serum bilirubins, bile pigment composition, and phenobarbital response were studied. PATIENTS: Three infants, affected by high serum unconjugated bilirubin concentrations, previously classified as type 1 CN. METHODS: Serum and bile bilirubin pigment composition, both before and after phenobarbital (PB) treatment, were determined by alkaline methanolysis and high-pressure liquid chromatography. PB was given for at least 3 weeks by oral administration (5 mg/kg bw per day). RESULTS: No diconjugated bilirubin was found either before or after PB treatment in the serum of the three studied infants. In two patients traces of monoconjugated bilirubin were detected before PB therapy, and the ratio of conjugated/total bilirubin (percent) was increased by the PB response. In the third patient, traces of monoconjugated bilirubin appeared only after PB administration. However, the serum unconjugated bilirubin concentration decreased significantly only in the second patient, following the second cycle of PB treatment, leading to the diagnosis of type 2 CN. The analysis of the methyl ester derivatives of bile pigments was also performed on bile samples obtained in two patients by Entero-Test (R) both before and after PB treatment. An absolute increment in monoesterified bilirubin concentration was found after PB administration, although the percent concentration increased in one case and decreased in the other. No diesterified bilirubin was detected in the bile samples. CONCLUSIONS: The present results show that in types 1 and 2 CN disease it is possible to detect traces of monoconjugated but not diconjugated bilirubin both in serum and in bile. Whereas PB treatment is effective in slightly increasing the serum monoconjugated bilirubin concentration even in type 1 CN disease, the diagnosis of type 1 or 2 is based on finding a substantial decrease of serum unconjugated bilirubin following PB administration

The future of stem cells in liver diseases.

Preliminary experience with clinical hepatocyte transplantation during the past decade has provided proof of concept that cell therapy can be effective for the treatment of some liver diseases. Recent progress in cell biology resulting in the isolation and characterization of hepatic stem cells and progenitor cells further increased the expectation for a new approach to the treatment of genetic and chronic liver disease. Several potential sources have been identified of hepatic stem/ progenitor cells exhibiting both differentiation towards the hepatic lineage in vitro and hepatic parenchymal repopulation with liver-specific metabolic activity in liver-injured animal models. However, a few of these results proved to be poorly reproducible in different laboratories, and it was recognized that some initial optimistic conclusions were drawn from incorrect interpretation of experimental data or from insufficient knowledge of the mechanisms involved in tissue regeneration. Moreover, only modest results have emerged so far from ongoing clinical experience involving the use of putative stem cells in liver disease. There is much need for a joined effort to concentrate the resources on a specific cell population, in order to better characterize its function, to assess its safety and to develop better focused clinical trials. In conclusion, while the biological features of stem cells still justify the hope for future clinical applications, hepatic stem cell therapy has still a long way to go from bench to bedside