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The optimal functionality of the native corneal stroma is mainly dependent on the well-ordered arrangement of extracellular matrix (ECM) and the pressurized structure. In order to develop an in vitro corneal model, it is crucial to mimic the in vivo microenvironment of the cornea. In this study, the influence of surface topography and mechanical strain on keratocyte phenotype and ECM formation within a biomimetic 3D corneal model is studied. By modifying the surface topography of materials, it is found that patterned silk fibroin film with 600 grooves mm(-1) optimally supports cell alignment and ECM arrangement. Furthermore, treatment with 3% dome-shaped mechanical strain, which resembles the shape and mechanics of native cornea, significantly enhances the expression of keratocyte markers as compared to flat-shaped strain. Accordingly, a biomimetic 3D corneal model, in the form of a collagen-modified, silk fibroin-patterned construct subjected to 3% dome-shaped strain, is created. Compared to traditional 2D cultures, it supports a significantly higher expression of keratocyte and ECM markers, and in conclusion better maintains keratocyte phenotype, alignment, and fusiform cell shape. Therefore, the novel biomimetic 3D corneal model developed in this study serves as a useful in vitro 3D culture model to improve current 2D cultures for corneal studies.

Stem cells have been widely used in tendon tissue engineering. The lack of refined and controlled differentiation strategy hampers the tendon repair and regeneration. This study aimed to find new effective differentiation factors for stepwise tenogenic differentiation. By microarray screening, the transcript factor Fos was found to be expressed in significantly higher amounts in postnatal Achilles tendon tissue derived from 1 day as compared with 7-days-old rats. It was further confirmed that expression of Fos decreased with time in postnatal rat Achilles tendon, which was accompanied with the decreased expression of multiply tendon markers. The expression of Fos also declined during regular in vitro cell culture, which corresponded to the loss of tendon phenotype. In a cell-sheet and a three-dimensional cell culture model, the expression of Fos was upregulated as compared with in regular cell culture, together with the recovery of tendon phenotype. In addition, significant higher expression of tendon markers was found in Fos-overexpressed tendon stem/progenitor cells (TSPCs), and Fos knock-down gave opposite results. In situ rat tendon repair experiments found more normal tendon-like tissue formed and higher tendon markers expression at 4 weeks postimplantation of Fos-overexpressed TSPCs derived nonscaffold engineering tendon (cell-sheet), as compared with the control group. This study identifies Fos as a new marker and functional driver in the early stage teno-lineage differentiation of tendon, which paves the way for effective stepwise tendon differentiation and future tendon regeneration.

Background/Aim. In a condition of pain in the Achilles tendon insertion there are multiple structures involved, such as the Achilles tendon itself, the retrocalcaneal bursa and a bony protrusion at the calcaneal tuberosity called Haglund’s deformity. The innervation patterns of these structures are scarcely described, and the subcutaneous calcaneal bursa is traditionally not considered to be involved in the pathology. This study aimed at describing the innervation patterns of the four structures described above to provide a better understanding of possible origins of pain at the Achilles tendon insertion. Methods. Biopsies were taken from 10 patients with insertional Achilles tendinopathy, which had pathological changes in the subcutaneous and retrocalcaneal bursae, a Haglund deformity and Achilles tendon tendinopathy as verified by ultrasound. The biopsies were stained using immunohistochemistry in order to delineate the innervation patterns in the structures involved in insertional Achilles tendinopathy. Results. Immunohistochemical examinations found that the subcutaneous bursa scored the highest using a semi-quantitative evaluation of the degree of innervation when compared to the retrocalcaneal bursa, the Achilles tendon, and the calcaneal bone. Conclusions. These findings suggest that the subcutaneous bursa, which is traditionally not included in surgical treatment, may be a clinically important factor in insertional Achilles tendinopathy.

Previous in vitro studies on human tendon cells (tenocytes) have demonstrated that the exogenous administration of substance P (SP) and acetylcholine (ACh) independently result in tenocyte proliferation, which is a prominent feature of tendinosis. Interestingly, the possible link between SP and ACh has not yet been explored in human tenocytes. Recent studies in other cell types demonstrate that both SP and ACh independently upregulate TGF-β1 expression via their respective receptors, the neurokinin 1 receptor (NK-1R) and muscarinic ACh receptors (mAChRs). Furthermore, TGF-β1 has been shown to downregulate NK-1R expression in human keratocytes. The aim of this study was to examine if TGF-β1 is the intermediary player involved in mediating the proliferative pathway shared by SP and ACh in human tenocytes. The results showed that exogenous administration of SP and ACh both caused significant upregulation of TGF-β1 at the mRNA and protein levels. Exposing cells to TGF-β1 resulted in increased cell viability of tenocytes, which was blocked in the presence of the TGFβRI/II kinase inhibitor. In addition, the proliferative effects of SP and ACh on tenocytes were reduced by the TGFβRI/II kinase inhibitor; this supports the hypothesis that the proliferative effects of these signal substances are mediated via the TGF-β axis. Furthermore, exogenous TGF-β1 downregulated NK-1R and mAChRs expression at both the mRNA and protein levels, and these effects were negated by simultaneous exposure to the TGFβRI/II kinase inhibitor, suggesting a negative feedback loop. In conclusion, the results indicate that TGF-β1 is the intermediary player through which the proliferative actions of both SP and ACh converge mechanistically. Originally published in manuscript form

In a condition of pain in the Achilles tendon insertion there are multiple structures involved, such as the Achilles tendon itself, the retrocalcaneal bursa and a bony protrusion at the calcaneal tuberosity called Haglund's deformity. The innervation patterns of these structures are scarcely described, and the subcutaneous calcaneal bursa is traditionally not considered to be involved in the pathology. This study aimed at describing the innervation patterns of the four structures described above to provide a better understanding of possible origins of pain at the Achilles tendon insertion.Biopsies were taken from 10 patients with insertional Achilles tendinopathy, which had pathological changes in the subcutaneous and retrocalcaneal bursae, a Haglund deformity and Achilles tendon tendinopathy as verified by ultrasound. The biopsies were stained using immunohistochemistry in order to delineate the innervation patterns in the structures involved in insertional Achilles tendinopathy.Immunohistochemical examinations found that the subcutaneous bursa scored the highest using a semi-quantitative evaluation of the degree of innervation when compared to the retrocalcaneal bursa, the Achilles tendon, and the calcaneal bone.These findings suggest that the subcutaneous bursa, which is traditionally not included in surgical treatment, may be a clinically important factor in insertional Achilles tendinopathy.

The loss of collagen organization is considered a hallmark histopathologic feature of tendinosis. At the cellular level, tenocytes have been shown to produce signal substances that were once thought to be restricted to neurons. One of the main neuropeptides implicated in tendinosis, substance P (SP), is known to influence collagen organization, particularly after injury. The aim of this study was to examine the influence of SP on collagen remodeling by primary human tendon cells cultured in vitro in three-dimensional collagen lattices. We found that SP stimulation led to an increased rate of collagen remodeling mediated via the neurokinin-1 receptor (NK-1 R), the preferred cell receptor for SP. Gene expression analysis showed that SP stimulation resulted in significant increases in MMP3, COL3A1 and ACTA2 mRNA levels in the collagen lattices. Furthermore, cyclic tensile loading of tendon cell cultures along with the administration of exogenous SP had an additive effect on MMP3 expression. Immunoblotting confirmed that SP increased MMP3 protein levels via the NK-1 R. This study indicates that SP, mediated via NK-1 R, increases collagen remodeling and leads to increased MMP3 mRNA and protein expression that is further enhanced by cyclic mechanical loading.

The histopathology of tendons with painful tendinopathy is often tendinosis, a fibrosis-like condition of unclear pathogenesis characterized by tissue changes including hypercellularity. The primary tendon cells (tenocytes) have been shown to express adrenoreceptors (mainly alpha-2A) as well as markers of catecholamine production, particularly in tendinosis. It is known that adrenergic stimulation can induce proliferation in other cells. The present study investigated the effects of an exogenously administered alpha-2 adrenergic agonist in an established in vivo Achilles tendinosis model (rabbit) and also in an in vitro human tendon cell culture model. The catecholamine producing enzyme tyrosine hydroxylase and the alpha-2A-adrenoreceptor (α(2A) AR) were expressed by tenocytes, and alpha-2 adrenergic stimulation had a proliferative effect on these cells, in both models. The proliferation was inhibited by administration of an α(2A) AR antagonist, and the in vitro model further showed that the proliferative alpha-2A effect was mediated via a mitogenic cell signaling pathway involving phosphorylation of extracellular-signal-regulated kinases 1 and 2. The results indicate that catecholamines produced by tenocytes in tendinosis might contribute to the proliferative nature of the pathology through stimulation of the α(2A) AR, pointing to a novel target for future therapies. The study furthermore shows that animal models are not necessarily required for all aspects of this research.

Background: Patellar tendinopathy (PT) is one of the most common reasons for sport-induced pain of the knee. Low ankle dorsiflexion range might predispose for PT because of load-bearing compensation in the patellar tendon.Purpose: The purpose of this 1-year prospective study was to analyze if a low ankle dorsiflexion range increases the risk of developing PT for basketball players.Study Design: Cohort study (prognosis); Level of evidence, 2.Methods: Ninety junior elite basketball players were examined for different characteristics and potential risk factors for PT, including ankle dorsiflexion range in the dominant and nondominant leg. Data were collected over a 1-year period and follow-up, including reexamination, was made at the end of the year.Results: Seventy-five players met the inclusion criteria. At the follow-up, 12 players (16.0%) had developed unilateral PT. These players were found to have had a significantly lower mean ankle dorsiflexion range at baseline than the healthy players, with a mean difference of −4.7° ( P = .038) for the dominant limb and −5.1° ( P = .024) for the nondominant limb. Complementary statistical analysis showed that players with dorsiflexion range less than 36.5° had a risk of 18.5% to 29.4% of developing PT within a year, as compared with 1.8% to 2.1% for players with dorsiflexion range greater than 36.5°. Limbs with a history of 2 or more ankle sprains had a slightly less mean ankle dorsiflexion range compared to those with 0 or 1 sprain (mean difference, −1.5° to −2.5°), although this was only statistically significant for nondominant legs.Conclusion: This study clearly shows that low ankle dorsiflexion range is a risk factor for developing PT in basketball players. In the studied material, an ankle dorsiflexion range of 36.5° was found to be the most appropriate cutoff point for prognostic screening. This might be useful information in identifying at-risk individuals in basketball teams and enabling preventive actions. A history of ankle sprains might contribute to reduced ankle dorsiflexion range.

PURPOSE. To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism. METHODS. Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-kappa B (NF-kappa B) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot. RESULTS. Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-kappa B RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage. CONCLUSIONS. Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.

The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt) and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway.

Background: The use of corticosteroids (e.g., dexamethasone) as treatment for tendinopathy has recently been questioned as higher risks for ruptures have been observed clinically. In vitro studies have reported that dexamethasone exposed tendon cells, tenocytes, show reduced cell viability and collagen production. Little is known about the effect of dexamethasone on the characteristics of tenocytes. Furthermore, there are uncertainties about the existence of apoptosis and if the reduction of collagen affects all collagen subtypes. Methods: We evaluated these aspects by exposing primary tendon cells to dexamethasone (Dex) in concentrations ranging from 1 to 1000 nM. Gene expression of the specific tenocyte markers scleraxis (Scx) and tenomodulin (Tnmd) and markers for other mesenchymal lineages, such as bone (Alpl, Ocn), cartilage (Acan, Sox9) and fat (Cebpα, Pparg) was measured via qPCR. Cell viability and proliferation was calculated using a MTS Assay. Cell death was measured by LDH assay and cleaved caspase-3 using Western Blot. Gene expression of collagen subtypes Col1, Col3 and Col14 was analyzed using qPCR. Results: Stimulation with Dex decreased cell viability and LDH levels. Dex also induced a significant reduction of Scx gene expression and a marked loss of fibroblast like cell shape. The mRNA for all examined collagen subtypes was found to be down-regulated. Among non-tendinous genes only Pparg was significantly increased, whereas Acan, Alpl and Sox9 were reduced. Conclusions: These results indicate a Dex induced phenotype drift of the tenocytes by reducing scleraxis expression. Reduction of several collagen subtypes, but not cell death, seems to be a feature of Dex induced tissue degeneration.

Purpose: Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea – such as stimulating epithelial cell proliferation – processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis. Methods: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR). Results: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor. Conclusions: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.

Synopsis Tendinopathy has become the accepted term to describe a spectrum of changes that occur in damaged and/or diseased tendons. Over the past 2 decades, there have been new insights into tendon pathophysiology of relevance to clinicians, including (1) better characterization of the overuse injury process and the resultant structural and functional disruption in chronically painful tendons, (2) improved understanding of the pathomechanics associated with chronic tendon injury, and (3) greater knowledge about the influence of lifestyle factors and drugs on tendon pathology. The implications of these new insights are discussed. J Orthop Sports Phys Ther 2015;45(11):833-841. Epub 21 Sep 2015. doi:10.2519/jospt.2015.5884.

Is it merely a curiosity that fat pads are found adjacent to the area of tendon affected by tendinopathy? We propose that fat pads share an anatomical and functional relationship with their adjacent tendons and may therefore contribute to the pathogenesis of tendinopathy. Fat pads and tendons have a shared blood supply,1 and cytokines produced in the fat pad have only a short distance to travel in order to affect the tendon.2 Fat pads lubricate, insulate, protect and provide structural support for tendons. However, the functional significance of the fat pad is often overlooked. In an early study of fat pad function, the distal tip of Kager's fat pad migrated into the retrocalcaneal bursa during ankle movement in healthy individuals, but not in an individual with a hindfoot disorder.3 This ‘variable plunger’ mechanism minimises pressure changes within the bursa during ankle movement. …

Corneal dystrophies are a clinically and genetically heterogeneous group of inherited disorders that bilaterally affect corneal transparency. They are defined according to the corneal layer affected and by their genetic cause. In this study, we identified a dominantly inherited epithelial recurrent erosion dystrophy (ERED)-like disease that is common in northern Sweden. Whole-exome sequencing resulted in the identification of a novel mutation, c.2816C>T, p.T939I, in the COL17A1 gene, which encodes collagen type XVII alpha 1. The variant segregated with disease in a genealogically expanded pedigree dating back 200 years. We also investigated a unique COL17A1 synonymous variant, c.3156C>T, identified in a previously reported unrelated dominant ERED-like family linked to a locus on chromosome 10q23-q24 encompassing COL17A1. We show that this variant introduces a cryptic donor site resulting in aberrant pre-mRNA splicing and is highly likely to be pathogenic. Bi-allelic COL17A1 mutations have previously been associated with a recessive skin disorder, junctional epidermolysis bullosa, with recurrent corneal erosions being reported in some cases. Our findings implicate presumed gain-of-function COL17A1 mutations causing dominantly inherited ERED and improve understanding of the underlying pathology.

Studies of human patellar and Achilles tendons have shown that primary tendon fibroblasts (tenocytes) not only have the capacity to produce acetylcholine (ACh) but also express muscarinic ACh receptors (mAChRs) through which ACh can exert its effects. In patients with tendinopathy (chronic tendon pain) with tendinosis, the tendon tissue is characterised by hypercellularity and angiogenesis, both of which might be influenced by ACh. In this study, we have tested the hypothesis that ACh increases the proliferation rate of tenocytes through mAChR stimulation and have examined whether this mechanism operates via the extracellular activation of the epidermal growth factor receptor (EGFR), as shown in other fibroblastic cells. By use of primary human tendon cell cultures, we identified cells expressing vimentin, tenomodulin and scleraxis and found that these cells also contained enzymes related to ACh synthesis and release (choline acetyltransferase and vesicular acetylcholine transporter). The cells furthermore expressed mAChRs of several subtypes. Exogenously administered ACh stimulated proliferation and increased the viability of tenocytes in vitro. When the cells were exposed to atropine (an mAChR antagonist) or the EGFR inhibitor AG1478, the proliferative effect of ACh decreased. Western blot revealed increased phosphorylation, after ACh stimulation, for both EGFR and the extracellular-signal-regulated kinases 1 and 2. Given that tenocytes have been shown to produce ACh and express mAChRs, this study provides evidence of a possible autocrine loop that might contribute to the hypercellularity seen in tendinosis tendon tissue.

Tenocytes produce substance P (SP), and its receptor (neurokinin-1 receptor (NK-1R)) is expressed throughout the tendon tissue, especially in patients with tendinopathy and tissue changes (tendinosis) including hypercellularity and vascular proliferation. Considering the known effects of SP, one might ask whether SP contributes to these changes.To test whether development of tendinosis-like changes (hypercellularity and angiogenesis) is accelerated during a 1-week course of exercise with local administration of SP in an established Achilles tendinopathy model.Rabbits were subjected to a protocol of Achilles tendon overuse for 1 week, in conjunction with SP injections in the paratenon. Exercised control animals received NaCl injections or no injections, and unexercised, uninjected controls were also used. Tenocyte number and vascular density, as well as paratendinous inflammation, were evaluated. Immunohistochemistry and in situ hybridisation to detect NK-1R were conducted. Results There was a significant increase in tenocyte number in the SP-injected and NaCl-injected groups compared with both unexercised and exercised, uninjected controls. Tendon blood vessels increased in number in the SP-injected group compared with unexercised controls, a finding not seen in NaCl-injected controls or in uninjected, exercised animals. Paratendinous inflammation was more pronounced in the SP-injected group than in the NaCl controls. NK-1R was detected in blood vessel walls, nerves, inflammatory cells and tenocytes.SP accelerated the development of tendinosis-like changes in the rabbit Achilles tendon, which supports theories of a potential role of SP in tendinosis development; a fact of clinical interest since SP effects can be effectively blocked. The angiogenic response to SP injections seems related to paratendinitis.

It has been hypothesised that substance P (SP) may be produced by primary fibroblastic tendon cells (tenocytes), and that this production, together with the widespread distribution of the neurokinin-1 receptor (NK-1 R) in tendon tissue, could play an important role in the development of tendinopathy, a condition of chronic tendon pain and thickening. The aim of this study was to examine the possibility of endogenous SP production and the expression of NK-1 R by human tenocytes. Because tendinopathy is related to overload, and because the predominant tissue pathology (tendinosis) underlying early tendinopathy is characterized by tenocyte hypercellularity, the production of SP in response to loading/strain and the effects of exogenously administered SP on tenocyte proliferation were also studied. A cell culture model of primary human tendon cells was used. The vast majority of tendon cells were immunopositive for the tenocyte/fibroblast markers tenomodulin and vimentin, and immunocytochemical counterstaining revealed that positive immunoreactions for SP and NK-1 R were seen in a majority of these cells. Gene expression analyses showed that mechanical loading (strain) of tendon cell cultures using the FlexCell (R) technique significantly increased the mRNA levels of SP, whereas the expression of NK-1 R mRNA decreased in loaded as compared to unloaded tendon cells. Reduced NK-1 R protein was also observed, using Western blot, after exogenously administered SP at a concentration of 10(-7) M. SP exposure furthermore resulted in increased cell metabolism, increased cell viability, and increased cell proliferation, all of which were found to be specifically mediated via the NK-1 R; this in turn involving a common mitogenic cell signalling pathway, namely phosphorylation of ERK1/2. This study indicates that SP, produced by tenocytes in response to mechanical loading, may regulate proliferation through an autocrine loop involving the NK-1 R.

Introduction Substance P (SP) which is produced by neuronal and non- neuronal cells has various functions. In addition to its role in pain transmission, this neuropeptide has effects on tissue matrix remodelling, cell growth, angiogenesis and inflammation. 1,2 SP has previously been shown to be correlated to pain levels in tendinopathy. The painful area of rotator cuff tendinosis is correlated with the local tissue level of SP, and also that SP can be endogenously produced by tendon fibroblasts, particularly when these cells are subjected to repetitive mechanical loading. 3 Glucocorticoid injections are frequently used as a treatment for chronic tendinopathy, although clinicians are now increasingly aware of caveats to this approach. 4 Several randomised trials and systematic reviews have demonstrated acute pain-relief in patients with tendinopathy who receive corticosteroid injections, despite the risks of rupture or long-term recurrence of tendon pain 5 . We hypothesise that glucocorticoids reduce the SP production in tendon tissue which subsequently lead to short-term pain relief and decrease in tensile strength of the tendons. Methods Human tendon fibroblasts were isolated from healthy Achilles or hamstrings tendons of male and female recreational athletes aged between 22–40 (n = 6). Cells were cultured in the presence or absence of dexamethasone (1 to 400 nM), an inhibitor of the glucocorticoid receptor (RU486), recombinant TGF-beta (2.5 or 5.0 ng/ml), or an inhibitor of the TGF-beta receptor (A83.01), recombinant human IL-1β and IL-6. Expression levels of the genes encoding for SP (TAC1) and its preferred receptor (NK1R), IL-1α, IL-1β and IL-6 were determined with qPCR, and protein levels of SP were examined by enzyme immunoassay and Western blot. Results Exposure of human tendon cells to dexamethasone resulted in a time-dependent reduction of mRNA for SP in both hamstrings and Achilles tenocytes, whereas NK1R was unaffected. SP protein was also substantially decreased by dexamethasone (Figure 1). The reduction of SP mRNA was dependent on signalling through the glucocorticoid receptor (Figure 2). Dexamethasone also prevented induction of SP by IL-1β and by cyclic mechanical loading (Figure 3). Discussion This study demonstrates that dexamethasone treatment of human tendon fibroblasts reduces the expression of SP through a glucocorticoid receptor-dependent pathway. Given that local fibroblasts contribute to the increased levels of SP in tendinopathy, a reduction of SP by corticosteroids in patients with tendinopathy may contribute pain relief. Drugs interfering with SP signalling could be a future target in the treatment of tendinopathy. References Andersson G, et al . Regul Pept. 2008;150(13):81–87 Coombes BK, et al . Lancet 2010;376(9754):1751–1767 Fong G, et al . J Orthop Res 2013;31(1):91–98 Gotoh M, et al . J Orthop Res. 1998;16(5):618–621 Metcalfe D. Rheumatology (Oxford). 2008;47:Ii183–Ii184

A module of the NESTOR underwater neutrino telescope, was deployed, in March 2003, at a depth of 3800m in order to test the overall detector performance and particularly that of the data acquisition systems. A prolonged period of running under stable operating conditions made it. possible to measure the cosmic ray muon flux, I(0)cos(a)(0). (c) 2006 Elsevier B.V. All rights reserved.

NESTOR is a deep-sea neutrino telescope that is under construction in the Ionian Sea off the coast of Greece at a depth of about 4000 m. This paper briefly reviews the detector structure and deployment techniques before describing in detail the calibration and engineering run of a test detector carried out in 2003. The detector was operated for more than I month and data was continuously transmitted to shore via an electro-optical cable laid on the sea floor. The performance of the detector is discussed and analysis of the data obtained shows that the measured cosmic ray muon flux is in good agreement with previous measurements and with phenomenological cosmic ray models. (c) 2005 Published by Elsevier B.V.