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Two cases of advanced alkaptonuria (AKU) with co-existing osteoporosis are described. Case 1 developed multiple non-vertebral fragility fractures, while Case 2 developed vertebral fragility fractures, both refractory to bisphosphonates. Difficulties in diagnosing osteoporosis in AKU complicated by extensive calcifying and ossifying spondylosis are discussed. Both patients continued to fracture despite nitisinone therapy for metabolic control of AKU, as well as bisphosphonate antiresorptive therapy for osteoporosis. Subsequently the patients were treated with teriparatide 20 μg subcutaneous injections daily for two years, leading to reduction in fractures soon after commencing therapy in both cases. Markers of bone remodelling P1NP and CTX were stimulated. No complications due hypercalcaemia or calcification were encountered in either case. We conclude that teriparatide is an effective adjunct in the treatment of AKU when bisphosphonates prove ineffective.

Ptprv is a member of the transmembrane tyrosine phosphatase gene family reported to be expressed in osteoblasts and gonads. To better define the developmental and tissue specificity of Ptprv expression, we generated knock-in mice expressing a nuclear localised β-galactosidase reporter under the control of resident Ptprv regulatory elements. Histochemical staining of Ptprv-nLacZ mice revealed that Ptprv expression is readily detectable in the foetal gonadal ridge of both sexes and in adult gonads where it is localised to Sertoli cells of the testis and celomic epithelial cells of the ovaries. During early limb development, Ptprv expression is prominent in the apical ectodermal ridge of the limb bud. At latter stages of development, Ptprv is predominantly expressed in the perichondrial and periosteal region of long bones, known as the bone collar. In contrast to previous indications from in vitro studies, there is little if any expression in mature osteoblasts in vivo. Analysis of Ptprv mRNA localisation by in situ hybridization in parallel with molecular markers of chondrocytes and osteoblasts confirmed the specific expression of Ptprv in immature bone collar cells. The specificity of Ptprv expression in these cells may be a useful tool to elucidate their role in the transition of skeletal elements from cartilage template to bone. Developmental Dynamics 229:826–834, 2004. © 2004 Wiley-Liss, Inc.

In the human breast cancer cell line MCF-7, the nucleotides ATPγS and UTP, acting extracellularly through the purinergic receptor P2Y2, lead to elevated intracellular calcium levels and increased proliferation. ATPγS and UTP treatment of MCF-7 cells activated transcription of the immediate early gene c-fos, an important component in the response to proliferative stimulation. c-fos induction was enhanced by co-treatment with ATPγS and a variety of proliferative agents including growth factors, tumour promoters and stress. Stimulation with ATPγS or epidermal growth factor (EGF) led to extracellular signal-regulated kinase (ERK) activation and phosphorylation of the transcription factors CREB and Elk-1. Co-stimulation synergistically activated fos expression and notably led to increased levels of ERK, CREB and EGF receptor phosphorylation, as well as hyperphosphorylation of ternary complex factor. Nevertheless, the ERK pathway does not fully account for this synergy, since fos induction was differentially sensitive to the MEK inhibitor U0126, indicating that these two agonists signal differently to this immediate early gene. Thus, extracellular nucleotides co-operate with growth factors to activate genes linked to the proliferative response in MCF-7 cells through activation of specific purinergic receptors, which thereby represent important potential targets for arresting the neoplastic progression of breast cancer cells.

Dentine slices were soaked in TSP-1, FN or buffer before carrying out resorption assays using bone cell suspensions prepared from the long bones of 18-20-day old pre-hatch chick embryos. Soaking dentine slices in FN resulted in a five-fold increase in resorption over controls, while TSP-1 gave a two-fold increase. However, whereas the increase in resorption on FN-soaked slices could be explained by a vast increase in cell adhesion to the slices, TSP-1-soaked wafers showed no increase in cell adhesion. TSP-1, therefore, appeared to promote resorption by a mechanism other than simply increasing adhesion of stromal cells to the dentine slices. In confirmation of this, TSP-1 was able to further increase resorption an FN-soaked slices where cell adhesion was already maximal. We conclude that the effect of TSP-1 is on resorption, and that osteoclast resorption can be regulated by the presence of TSP in the matrix.Copyright 1995, 1999 Academic Press, Inc.

A simple three-step assay has been developed for the analysis of nanomole quantities of glucose, fructose and sucrose from small plant samples in the presence of fructans. The assay of crude ethanol/water extracts proceeds by the sequential addition of the commercially available enzymes; hexokinase, phosphogluco-isomerase, and sucrose Phosphorylase. The assay was compared with conventional coupled assays for glucose and fructose, together with TLC and HPLC assays for total water-soluble carbohydrates. All assays showed similar results over the range of standards and plant samples tested. The assay has been developed for use on microtiter plate readers, allowing for the screening of large numbers of samples rapidly. Fructan content in the same plant samples can be calculated following the determination of total free and combined hexoses.

Parathyroid hormone-related peptide (PTHrP) is a local regulator of human bone turnover, which shares some sequence homology with the systemic hormone parathyroid hormone (PTH). The two proteins exert cellular effects through interaction with a common G protein-coupled PTH/PTHrP receptor on target cells. Whilst the PTH gene has a relatively simple structure the PTHrP gene is complex, alternative splicing of which can generate multiple mRNA species encoding PTHrP of 139, 141 and 173 amino acids. To date little is known regarding the extent to which PTH and PTHrP interact to modulate bone cell function. In this study we have used the quantitative technique of Real-Time polymerase chain reaction (PCR) to investigate the ability of PTH to induce PTHrP expression in SaOS-2 cells and in primary human osteoblasts. In addition, we have used the semi-quantitative techniques of PCR followed by Southern analysis and scanning densitometry to investigate the effects of PTH(1-34) on expression of mRNA species encoding the three PTHrP isoforms. We report a 50 fold increase in PTHrP mRNA expression 30 min after treatment with 100 ng/ml human recombinant PTH (1-34) in SaOS-2 cells, and a 38 fold rise in human osteoblasts 45-90 min post-PTH treatment. mRNA species encoding for PTHrP 1-139, 1-141 and 1-173 were all induced in human osteoblasts 45 min after exposure to PTH. Whilst the 1-139 mRNA species exhibited a sustained expression, both the 1-141 and 1-173 isoforms showed a biphasic induction with a second peak 6 hr post PTH treatment. These data demonstrate that PTH induces expression of the PTHrP gene in both SaOS-2 and primary human osteoblasts with the kinetics of an immediate early gene. Up-regulation of the PTHrP gene in response to PTH may be an important physiological mechanism by which this systemic factor effects a localised response in bone.

Combustion-modified polyurethane flexible foams can exhibit different flammability characteristics when covered with a fabric than when the foam alone is exposed to the same conditions. Recognizing this, regulatory and advisory agencies have turned to composite testing rather than component testing (e.g., the Boston Chair Test, California Technical Bulletin 133). It is doubtful that a small scale test could supplant the full scale tests; however, a method of screening composites would be very desirable.The Society of the Plastics Industry's Flexible Foam Combustibility Commit tee previously reported a classification system for foams alone using ASTM E 906 as the test procedure. Using piloted ignition and an imposed flux of 1.75 Watts/cm2, the foams could be partially classified. When the self-propagating flux values were added to the prior data, a classification system was developed.As a continuation of this project, five laboratories participated in a round robin wherein four generic classes of fabric were tested in triplicate over seven different types of foam at 1.75 Watts/cm2. The cumulative heat release at one minute intervals, the maximum rate of heat release, the time at which this oc curred and the percentage weight loss were the parameters studied.The variability of the inter-laboratory data averages was too large to permit the development of a definitive classification system. If the wool/nylon fabric group (which gave the largest inter-laboratory variation but the lowest heat release results) is eliminated from consideration, the remaining composites (save one) could be classified into three categories based on the heat release at both three and four minutes.This project indicates that it is possible to rank foam/fabric composites ac cording to their ability to resist combustion using ASTM E 906. However, fur ther work is necessary to define the causes of poor inter-laboratory agreement. Perhaps some other parameter not previously studied could aid in classifying foam/fabric composites.

We investigated the production of parathyroid hormone‐related protein (PTHrP) by cells derived from explants of human bone. Using an immunoradiometric assay (IRMA), PTHrP was detected in conditioned medium from cultures of bone‐derived cells from 6 of 7 patients investigated in this study. PTHrP mRNA was identified in human bone cells using reverse transcriptase‐linked polymerase chain reaction (RT‐PCR) and by Northern analysis. Transcripts for PTHrP were detected in a purified population of alkaline phosphatase positive cells isolated from human bone marrow cultures by flow cytometry, confirming the expression of PTHrP mRNA by cells of the osteoblastic lineage. Production of PTHrP was inhibited by 10−6M of the glucocorticoids, prednisolone and desacetylated deflazacort, in a dose‐dependent manner. In addition, RT‐PCR followed by Southern blot analysis detected a decrease in steady‐state PTHrP mRNA in cultures of human bone‐derived cells treated with 10−6M prednisolone.

Extracellular ATP acting through purinoceptors may be an important factor in the modulation of bone turnover. In this study we cloned and sequenced the P2Upurinoceptor from osteoclastoma, confirming the recently published human sequence. Furthermore, by the reverse transcriptase‐linked polymerase chain reaction (RT‐PCR) and Southern blotting we demonstrated expression of P2Ureceptor mRNA in bone, primary cultures of human bone‐derived cells, and two osteosarcoma cell lines, Saos2 and Te85. P2Ureceptor transcripts were identified in alkaline phosphatase‐positive human bone‐derived cells isolated by flow cytometry providing strong evidence for the expression of the P2Upurinoceptor in mature osteoblasts. P2Ureceptor transcripts were also detected in a purified giant cell population isolated from osteoclastoma, indicating that this receptor is also expressed by osteoclasts. These data suggest that purinergic agonists may play a role in the regulation of bone metabolism.

It has been proposed that Paget's disease of bone is caused by the infection of bone cells with one or several paramyxoviruses. In this study we used the polymerase chain reaction (PCR), which allows the detection of very low levels of a target nucleic acid sequence, to study cultures of pagetic bone cells and samples of pagetic bone. Oligonucleotide primers were designed to flank a sequence of the nucleocapsid genome of measles virus and canine distemper virus (CDV). Within this fragment there were contrasting restriction endonuclease sites specific to measles or CDV that allowed identification of the original template. We were unable to detect paramyxovirus RNA in four strains of human bone cells outgrown from pagetic bone and one strain derived from an uninvolved site of a patient with Paget's disease. Furthermore, paramyxovirus sequences were not detected in cDNA prepared from six samples of pagetic bone biopsies. The work presented here further questions the role of measles and CDV in the abnormal remodeling observed in Paget's disease.

We investigated the expression of cytokine transcripts in osteoblast‐like cells derived from explants of pagetic and normal bone. A reverse transcription‐linked PCR was used that allowed the simultaneous analysis of a range of cytokines. Normal osteoblast‐like cells were found to contain the transcripts for IL‐1β, IL‐6, and TGF‐β1. For the first time we detected in bone cells the two other mammalian isoforms of TGF‐β, β2, and β3. Furthermore, we have also identified mRNA for IL‐3 and the novel chemotactic factor, IL‐8. Using this sensitive technique it was not possible to detect mRNA for IL‐1α, IL‐2, IL‐4, IL‐5, IL‐7, TNF‐α, or interferon‐γ. The human osteosarcoma cell line Saos‐2 also showed a similar pattern of expression of these cytokines to primary osteoblast‐like cells, with the exception that TNF‐α was also identified. Cells isolated from pagetic bone showed essentially the same profile of cytokine expression as normal bone except that TNF‐α was also detected in two of four samples. The cytokine profile of successive populations of cells harvested from one explant culture at 9, 22, and 57 days showed a consistent pattern of cytokine expression, demonstrating the phenotypic stability of the osteoblast‐like cells in long‐term cultures.

A simple technique has been developed to identify the resorption lacunae excavated by avian osteoclasts in vitro. Briefly, devitalized bovine bone wafers, with cells in situ, are fixed, stained with toluidine blue, and then examined by reflected light microscopy. Resorption pits are clearly visible using a x 10 objective, even when the resorbed areas are covered by a confluent layer of cells. The technique can be used to quantify the plan area, depth, and volume of the excavations. Depth and volume are assessed using the x 50 objective lens. This technique has been used to investigate the effects of retinyl acetate and EHDP on bone resorption.

It has previously been reported that low extracellular pH stimulates the excavation of resorption lacunae by rodent osteoclasts in vitro. Using avian bone cells in a similar in vitro assay we have demonstrated that osteoclast activity is optimal at pH 7.20–7.40 and is inhibited at extremes of pH (<7.10 and >7.60). Over the first 24 h of incubation at low pH there may be an increase in osteoclastic resorption but to a lesser extent than that reported for rodent cells. However, after 24–30 h in culture there is little or no further increase in bone resorption, presumably due to a cytotoxic effect of low pH acting either on the osteoclast directly or via nonosteoclastic bone cells. In contrast to a previous report, in which preincubation of wafers for 24 h had no effect on bone resorption, we found that preincubation of bone substrates at pH 6.50 for longer periods enhances subsequent resorption at pH 7.20.

We have previously shown that the multifunctional platelet glycoprotein thrombospondin-1 (TSP-1) promotes resorption in an in vitroresorption assay. However, TSP-1 is one of a family of multifunctional TSP molecules, and the current study was undertaken to investigate whether it is TSP-1 or another TSP family member which may be involved in regulation of resorption in vivo.RT-PCR was performed on cultured human bone cells, cultured human chondrocytes, and three separate samples of human osteoclastoma tissue using primers specific for each TSP family member. mRNA for TSP-2 was detected in almost all samples, and significantly in all osteoclastomas in the above tissues, while TSP-1 was detected less frequently and was only seen in one of three osteoclastomas. TSP-3, -4, and COMP were detected only in a minority of cases. These results indicate that TSP-2 is the most common TSP family member found in skeletal tissues and that TSP-2, rather than TSP-1, may be the molecule responsible for promoting resorption in vivo.