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Regulation of bone formation is important in the pathogenesis of many conditions such as osteoporosis, fracture healing, and loosening of orthopedic implants. We have recently identified a novel rat cDNA (best5) by differential display PCR that is regulated during osteoblast differentiation and bone formation in vitro and in vivo. Expression of best5 mRNA is induced in cultures of osteoblasts by both interferon-alpha (IFN-alpha) or IFN-gamma. Whereas IFN-alpha induced a rapid, transient induction of best5 expression peaking at 4-6 h poststimulation, IFN-gamma elicited a more prolonged induction of best5 expression, which remained elevated 48 h poststimulation. A polyclonal antibody generated to a peptide derived from the best5 coding region recognized a 27 kDa protein on Western blot analysis of osteoblast lysates. We localized BEST5 protein in osteoblast progenitor cells and mature osteoblasts in sections of rat tibiae and in sections of bones loaded in vivo to induce adaptive bone formation. Best5 may therefore be a fundamental intermediate in the response of osteoblasts to stimuli that modulate proliferation/differentiation, such as interferons or mechanical loading. These findings highlight the close interactions between the immune system and bone cells and may open new therapeutic avenues in modulating bone mass.

Ultrasound imaging of atheromatous plaques in patients with carotid artery disease has revealed a spectrum of lesions ranging from plaques with predominantly echolucent properties to those which are densely echogenic. Although echogenic (fibrous) plaques are essentially stable lesions, whereas echolucent (lipidladen) plaques are prone to intimal tearing (the commonest event initiating embolization and stroke) [I], it is still not possible to show any definite link between a specific plaque type and cerebrovascular events. Such an association is probably multifactorial and mediated by a combination of the degree of stenosis, plaque morphology and dynamic factors. A critical interaction of these factors will result in embolization or haemodynamic compromise on one occasion, but at other times may adually be beneficial and lead to stabilisation of the plaque [2]. It is possible that most of these events are preceded and accompanied by changes in the expression of specific genes in the arterial wall, although it is still unclear what precisely controls these processes. In recent years several genes have been identified and cloned which may play an important role in the pathogenesis of atherosclerosis [3].The aims of this study are to use differential displaypolymerase chain reaction @D PCR) technique [4,5] in order to identify genes that are differentially expressed in either the echogenic (fibrous) or the echolucent (lipid-laden) atherosclerotic carotid plaques obtained from surgical endarterectomy specimens and to correlate the up-or downregulation of these genes with the preoperative ultrasonic appearances of these plaques. The clinical application of carotid plaque characterisation lies in detecting patients at risk of developing the criteria for clinical intervention in order to prevent or reduce the risk of stroke. Carotid artery plaque specimens were collected from routine endarterectomy surgical operations at the Royal Bournemouth Hospital. The non-invasive ulhasonic appearance (i.e. echogenicity or echolucency) and the quantitative grading (i.e., Type I-IV) of these plaques were carried out preoperatively according to the criteria adopted by Gray-Weale et al. [6], using duplex scanning aided by computerised measurements.The samples were washed immediately with PBS in diethyl pyrocarbonate @EX)-treated water and were frozen by immersion in liquid nitrogen. RNA was extracted from the echogenic and the echolucent plaques, using an adaptation of a standard protocol [A.For reverse transcription, RNA samples were primed with 0.5 pg oligo (dT)15 (Promega) at 72°C for 10 min and 4°C for 3 min. cDNA synthesis of each sample was achieved using 200 units RNase H-reverse transcriptase in lOmM dithiothreitol and 1st strand buffer (50mM Tris-HC1 pH 8.3, 75mM MgCI,) with 0.5mM each of dATP, dCTP, dGTP and dlTP. Samples were mixed and incubated at 42°C for 1 hr and the reaction tenninated by heating to 95°C for 10 min. cDNA was diluted 1:20 by adding DEPC-treated water.cDNA was then amplified from further samples using nine...