cell-based-assay-protocol-for-prognostic-prediction-idiopathic

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

Sainte-Justine University Hospital Research Center

Manganese (Mn) and bilirubin (BR) induce intrahepatic cholestasis when injected sequentially. It was suggested that accumulation of newly synthesized cholesterol in the canalicular membrane is an initial step in the development of cholestasis. To clarify the involvement of cholesterol in the pathogenesis of Mn-BR-induced cholestasis, we examined biliary secretion and liver subcellular distribution of lipids in the cholestatic conditions (Mn-BR combination). We also examined hepatic metabolism of cholesterol under cholestatic and noncholestatic (Mn or BR given alone) conditions. The Mn-BR combination reduced bile flow by 50%, and bile acid, phospholipids, and cholesterol output by 42, 75, and 90%, respectively. There was a dramatic impairment of cholate excretion but not chenodeoxycholate excretion. Phosphatidylcholine species secreted into bile were unchanged, and microsomal total phospholipid content was significantly increased. Although there was no changes in liver membrane phospholipid content, the cholesterol/phospholipid ratio was increased by more than 50% in the canalicular fraction. Despite the increased concentration of cholesterol in canalicular membrane the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, key enzyme in cholesterol synthesis, and cholesterol 7alpha-hydroxylase, key enzyme in cholesterol conversion to bile acids, were significantly reduced. However, the injection of Mn alone significantly increased both enzymes, while BR alone inhibited cholesterol 7alpha-hydroxylase by 62%, without affecting HMG-CoA reductase. In conclusion, the critical cholestatic events in Mn-Br-induced cholestasis appear to be mediated through the synergistic effects of Mn and BR acting on synthesis and degradation of cholesterol.

Synergistic role of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7alpha-hydroxylase in the pathogenesis of manganese-bilirubin-induced cholestasis in rats.

The pathogenesis of manganese-bilirubin (Mn-BR) induced cholestasis has only been studied in rats and is associated with alteration in the hepatic homeostasis of cholesterol and phospholipids. Multidrug resistance-2 (mdr2) transporter, which mediates excretion of these lipids, is suggested to be involved in this phenomenon. The present study was undertaken to examine if Mn-BR induced cholestasis is reproducible in mice, then to clarify the role of mdr2 in its pathogenesis, using mice with disrupted mdr2 gene (mdr2 (-/-)). Results showed that Mn-BR combination decreased bile flow in mice. This reduction in bile flow was similar in mdr2 (-/-) and the wild type mdr2 (+/+). Furthermore, the change in biliary lipid excretion was comparable in both genotypes. These data indicate that Mn-BR induced cholestasis is reproducible in mice and provide evidence that mdr2 alteration is not a primary event in this form of cholestasis.

The role of mdr2 in manganese-bilirubin induced cholestasis in mice.

Immunor (IM28), an analog of dehydroepiandrosterone (DHEA), inhibits human immunodeficiency virus type-1 (HIV-1) by inhibiting reverse transcriptase. We assessed the ability of IM28 to inhibit the cell-cell fusion mediated by HIV envelope glycoprotein in an in vitro system. For this purpose, we co-cultured TF228.1.16, a T-cell line expressing stably HIV-1 glycoprotein envelopes, with an equal number of 293/CD4+, another T cell line expressing CD4, and with the SupT1 cell line with or without IM28.

Inhibition of human immunodeficiency virus type-1 (HIV-1) glycoprotein-mediated cell-cell fusion by immunor (IM28).

Bi-directional relationships operate between the hypothalamic-pituitary-gonadal axis and the immune system. Cytokines, peptide hormones and their shared receptors/ligands are used as a common biological language for communication within and between the immune and neuroendocrine systems. Such communication suggests an immunoregulatory role for the brain and a sensory function for the immune system. We used a radioimmunoassay to measure the concentrations of steroid hormones (cortisol, testosterone, estradiol and progesterone) and pituitary hormones [follicle stimulating hormone (FSH), luteinizing hormone (LH) human chorionic gonadotropin (HCG) and prolactin] in peripheral blood plasma from 78 young Gabonese women with chronic filarial infections. We used an enzyme-linked immunosorbent assay to determine the concentrations of four proinflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma), interleukin-1 (IL-1) and IL-6] in the same plasma samples. Progesterone was unchanged and all other steroid hormone plasma concentrations were lower in microfilaremic women than in amicrofilaremic women. The concentration of LH was higher in amicrofilaremic women, whereas the prolactin concentration was higher in microfilaremics. The plasma concentrations of TNF-alpha, IFN-gamma, IL-1 and IL-6 were higher in microfilaremic women. A strong negative correlation was found between the steroid and pituitary hormones and the pro-inflammatory cytokines. Conversely, a strong positive correlation was found between prolactin and the same cytokines. These data provide first evidence of immune system and hormonal system disturbance during chronic filarial infections and suggest that the observed imbalance should be taken into account in the diagnosis and treatment of filarial infections.

Hypothalamic-pituitary gonadal axis and immune response imbalance during chronic filarial infections.

A cell-based assay was developed to identify asymptomatic children at risk of developing idiopathic scoliosis (IS) and to stratify IS patients at an earlier stage in order to better predict their clinical outcome. Clinical validation of this assay was performed by testing IS patients at different stages, healthy control subjects, and asymptomatic offspring, born from at least one scoliotic parent, who are considered at risk of developing this disorder.

Cell-based screening test for idiopathic scoliosis using cellular dielectric spectroscopy.

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ÃŸarrestin. Receptor recruitment of ÃŸarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ÃŸarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ÃŸarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ÃŸarrestin1 or ÃŸarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ÃŸarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5min) followed by sustained activation for at least 60min. The early but not the sustained phase was predictably affected by inhibition of either GÎ±q/11 or GÎ±i/o, whereas loss of ÃŸarrestin2 but not ÃŸarrestin1 resulted in diminished prolonged ERK1/2 activation. ÃŸarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ÃŸarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ÃŸarrestin's role using the B2R-4A, which is deficient in ÃŸarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ÃŸarrestin's crucial role in the maintenance of proper B2R signalling.

Role of ÃŸarrestins in bradykinin B2 receptor-mediated signalling.

This chapter reviews the biochemical, hormonal, and hematological factors in the onset and development of adolescent idiopathic scoliosis (AIS), an orthopedic entity of unknown etiology. Briefly, AIS is defined as a lateral curvature of the spine combined with vertebral rotation that occurs in patients of 10 years of age or older until bone maturity (18-20 years of age). AIS is predominant in females. If untreated, the curvature could evolve with negative long-term prognosis including psychosocial impact, back pain, pulmonary compromise, cor pulmonale, and even death due to respiratory failure. Causes of the disease have been postulated to involve genetics, abnormal muscle, connective tissue and bone structures, and neuroendocrine disorders. Psychological pathways have also been studied. Little data, however, have been collected on bone turnover in these patients. Some studies demonstrated decreased bone mineral density which may be suggestive of increased osteoblast activity. Other studies suggested a correlation to abnormal platelet morphology. Alterations in the spinal muscle contractile function may be responsible for spinal curvature. Measurement of trace elements in serum revealed impaired zinc and selenium metabolism, probably secondary to hormonal deregulation. Subsequent endocrine studies suggested a role for leptin and growth hormone in AIS. Recently, a neuroendocrine hypothesis has been proposed. This theory involves a unique melatonin-signaling dysfunction and opens new frontiers in the elucidation of the pathologic mechanisms for onset and progression of this disease.

Marie-Yvonne Akoume

Viscogliosi Laboratory in Molecular Genetics of Musculoskeletal Diseases

Sainte-Justine University Hospital Research Center

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy