J.P. Dillon

Regulation of epidermal homeostasis through P2Y2 receptors

Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. Reverse transcriptase‐polymerase chain reaction (RT–PCR) revealed expression of mRNA for the G‐protein‐coupled, P2Y2 receptor in primary cultured human keratinocytes. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPγS were found to stimulate the proliferation of keratinocytes. Using a real‐time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Release and interconversion of P2 receptor agonists by human osteoblast-like cells

Nucleotides, acting as agonists at P2 receptors, are important extracellular signaling molecules in many tissues. In bone they affect both bone‐forming osteoblast and bone‐resorbing osteoclast cell activity. The presence of nucleotides in the extracellular microenvironment is largely determined by their release from cells and metabolism by ecto‐enzymes, both of which have scarcely been studied in bone. We have investigated adenosine 5′‐triphosphate (ATP) release from SaOS‐2 osteoblastic cells and the activities of cell surface ecto‐enzymes on ATP metabolism. ATP, but not LDH, was detected in SaOS‐2 cell conditioned medium, suggesting these cells were actively releasing ATP. Introduction of ADP resulted in increased ATP concentrations in the medium, which was found not to be receptor mediated. Nucleotide inhibition and substrate specificity studies revealed an ecto‐nucleoside diphosphokinase (ecto‐NDPK) was responsible for the ADP→ATP conversion; PCR and immunocytochemistry confirmed its presence. Analysis of ATP metabolism over time demonstrated overall ATP degradation was increased by inhibiting ecto‐NDPK activity; confirming that the combined action of multiple osteoblast‐expressed ecto‐enzymes affected extracellular nucleotide concentration. The data establish the coexistence of ATP‐consuming, and for the first time, ATP‐generating activities on the osteoblast cell surface, the discovery of which has significant implications for studies involving P2 receptor subtypes in bone. FASEB J. 17, 1401–1410 (2003)

Isolation and culture of human osteoblasts

The skeleton is a dynamic organ that is constantly active throughout life. The highly coordinated actions of bone cells early in life determine the bodys shape and form, whilst the constant remodelling (bone resorption followed by an equal amount of bone formation) during adulthood helps to maintain skeletal mass and repair microdamage. When the balance of bone resorption and bone formation becomes unequal, bone diseases, such as osteoporosis, occur. In order to develop drugs to combat bone disease, it is important to know the regulatory systems involved in normal bone formation and resorption. In this chapter, we concentrate on bone formation, providing a detailed guide to isolating and culturing primary human osteoblasts in bone explant cultures, as well as the methodology used to characterise and monitor the function of osteoblasts. In combination, these methods provide a powerful tool in bone cell biology and in the development of new novel treatments for bone disease.

Digesta flow and mineral absorption in lambs before and after weaning

Four young lambs fitted with cannulas at the pylorus and at the ileo-caecal junction were used to study the effects of weaning on digesta flow and mineral absorption. Prior to weaning when milk alone was fed the small intestine was the major site for dry matter disappearance and for the absorption of Ca, P, Na and K. Both the small and the large intestine were, however, important sites for Mg absorption at this time. After weaning, when the lambs were fed a concentrate diet, at least two-thirds of the digested dry matter disappeared within the reticulo-rumen. The small intestine, however, still remained the major site for the absorption of Ca, P, Na and K. Most Mg absorption, in contrast, now occurred anterior to the pylorus and it would appear that the move from hindgut to foregut as a site for Mg absorption was directly associated with the introduction of dry feed and the development of fermentative digestion within the reticulo-rumen. Both before and after weaning the faeces was the major route for the excretion of Ca surplus to requirement and the faeces also became increasingly important as a pathway for P and Mg excretion once dry feed was given. The urine, in contrast, was consistently the major pathway for the excretion of Na and K.

Parathyroid hormone‐related protein in the aetiology of fibrous dysplasia of bone in the McCune Albright syndrome

Fibrous dysplasia, observed in bone lesions in the McCune Albright syndrome (MAS), is thought to result from abnormalities in cells of the osteogenic lineage associated with over‐activation of the cAMP signalling pathway in affected cells. The aim of this study was to investigate the role of parathyroid hormone‐related protein (PTHrP) in the aetiology of MAS, and to determine a possible therapeutic role for 1,25‐dihydroxy vitamin D3 (1,25(OH)2D3).

Primary Human Osteoblast Cultures

Osteoblast cultures can be used to investigate the mechanisms of bone formation, to probe the cellular and molecular basis of bone disease, and to screen for potential therapeutic agents that affect bone formation. Here, we describe the methods for establishing and characterising primary human osteoblast cultures.

On fragmenting, densely mineralised acellular protrusions into articular cartilage and their possible role in osteoarthritis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26‐μm resolution high contrast X‐ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re‐imaged with 6‐μm resolution X‐ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self‐healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

Cartilage biomarkers in the osteoarthropathy of alkaptonuria reveal low turnover and accelerated ageing

Objective. Alkaptonuria (AKU) is a rare autosomal recessive disease resulting from a single enzyme deficiency in tyrosine metabolism. As a result, homogentisic acid cannot be metabolized, causing systemic increases. Over time, homogentisic acid polymerizes and deposits in collagenous tissues, leading to ochronosis. Typically, this occurs in joint cartilages, leading to an early onset, rapidly progressing osteoarthropathy. The aim of this study was to examine tissue turnover in cartilage affected by ochronosis and its role in disease initiation and progression. Methods. With informed patient consent, hip and knee cartilages were obtained at surgery for arthropathy due to AKU (n = 6; 2 knees/4 hips) and OA (n = 12; 5 knees/7 hips); healthy non-arthritic (non-OA n = 6; 1 knee/5 hips) cartilages were obtained as waste from trauma surgery. We measured cartilage concentrations (normalized to dry weight) of racemized aspartate, GAG, COMP and deamidated COMP (D-COMP). Unpaired AKU, OA and non-OA samples were compared by non-parametric Mann–Whitney U test. Results. Despite more extractable total protein being obtained from AKU cartilage than from OA or non-OA cartilage, there was significantly less extractable GAG, COMP and D-COMP in AKU samples compared with OA and non-OA comparators. Racemized Asx (aspartate and asparagine) was significantly enriched in AKU cartilage compared with in OA cartilage. Conclusions. These novel data represent the first examination of cartilage matrix components in a sample of patients with AKU, representing almost 10% of the known UK alkaptonuric population. Compared with OA and non-OA, AKU cartilage demonstrates a very low turnover state and has low levels of extractable matrix proteins.

Nitisinone arrests ochronosis and decreases rate of progression of Alkaptonuria: Evaluation of the effect of nitisinone in the United Kingdom National Alkaptonuria Centre

QUESTION Does Nitisinone prevent the clinical progression of the Alkaptonuria? FINDINGS In this observational study on 39 patients, 2 mg of daily nitisinone inhibited ochronosis and significantly slowed the progression of AKU over a three-year period. MEANING Nitisinone is a beneficial therapy in Alkaptonuria. BACKGROUND Nitisinone decreases homogentisic acid (HGA), but has not been shown to modify progression of Alkaptonuria (AKU). METHODS Thirty-nine AKU patients attended the National AKU Centre (NAC) in Liverpool for assessments and treatment. Nitisinone was commenced at V1 or baseline. Thirty nine, 34 and 22 AKU patients completed 1, 2 and 3 years of monitoring respectively (V2, V3 and V4) in the VAR group. Seventeen patients also attended a pre-baseline visit (V0) in the VAR group. Within the 39 patients, a subgroup of the same ten patients attended V0, V1, V2, V3 and V4 visits constituting the SAME Group. Severity of AKU was assessed by calculation of the AKU Severity Score Index (AKUSSI) allowing comparison between the pre-nitisinone and the nitisinone treatment phases. RESULTS The ALL (sum of clinical, joint and spine AKUSSI features) AKUSSI rate of change of scores/patient/month, in the SAME group, was significantly lower at two (0.32 ± 0.19) and three (0.15 ± 0.13) years post-nitisinone when compared to pre-nitisinone (0.65 ± 0.15) (p < .01 for both comparisons). Similarly, the ALL AKUSSI rate of change of scores/patient/month, in the VAR group, was significantly lower at one (0.16 ± 0.08) and three (0.19 ± 0.06) years post-nitisinone when compared to pre-nitisinone (0.59 ± 0.13) (p < .01 for both comparisons). Combined ear and ocular ochronosis rate of change of scores/patient/month was significantly lower at one, two and three years post-nitisinone in both VAR and SAME groups compared with pre-nitisinone (p < .05). CONCLUSION This is the first indication that a 2 mg dose of nitisinone slows down the clinical progression of AKU. Combined ocular and ear ochronosis progression was arrested by nitisinone.