A Sentinel Population: The public health benefits of monitoring enhanced body builders

There is heightened recognition of the public health implications of anabolic androgenic steroids (AAS) for the use of image and performance enhancement; with increasing evidence of their long-term negative health impacts, the hazards associated with their administration (often via injection), and the variability and unpredictability of their contents. In order to optimise the effects of these drugs, together with strict dietary and training regimes, AAS users typically supplement their use with an expansive and continually evolving range of ancillary drugs. The discovery and subsequent adoption of these drugs by the broader AAS user population is largely dependent upon a minority of social influencers within the bodybuilding community. Pioneering enhanced bodybuilders who self-experiment with a diverse range of image and performance enhancing drugs (IPEDs) and ancillary drugs have been the forerunners in the development of an underground user-led literature, online discussion forums, and were early adopters of internet-facilitated drug markets. Yet the impact of their self-experimentations extends well beyond the enhanced bodybuilding community, particularly in their use of ancillary drugs. Most significantly has been their role in the diffusion of various enhancement and psychoactive drugs to the wider population. Using the theoretical framework of the 'diffusion of innovation' we consider the role that pioneering enhanced bodybuilders have played in the diffusion of various enhancement and psychoactive drugs to the wider population through a focus on three substances –dinitrophenol (DNP), melanotan II and gamma-hydroxybtyrate (GHB). With an increasing range of drugs used by bodybuilders, coupled with an expansion in the use of online forums and online platforms to purchase pharmacological and new psychoactive drugs, we anticipate this trend of diffusion amongst the wider population will continue to flourish. Therefore, we highlight the need for policy makers to monitor emergent trends, not only in the general AAS population but particularly amongst enhanced bodybuilders

Developmental changes in patterns of distribution of fibronectin and tenascin-C in the chicken cornea: evidence for distinct and independent functions during corneal development and morphogenesis

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior–posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman’s layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles

Cell-independent matrix configuration in early corneal development

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3

Observations on nascent matrix structures in embryonic cornea: Important in cell interactions, or merely vestiges of the lens surface?

Here we present some new observations on early stages in chick corneal development obtained by remining of datasets obtained via serial block face scanning electron microscopy. We focus on matrix cords, proteoglycan-rich structures of apparent ectodermal origin, emerging from the epithelial basal lamina, which extend proximally into the growing collagenous matrix destined to become the corneal stroma. Cords have no known function. In their earliest manifestation, we describe how they appear to run continuously from epithelium to the lens, in contact with both tissues and may therefore be simply vestigial structures, remaining from the earlier detachment of the lens from its parent ectoderm. However, neural crest cells migrating to form the corneal endothelial monolayer appear to form close associations with cords via elaborate pseudopodial extensions. Presumptive endothelium and keratocytes, in the subsequent wave of neural crest cell influx, may conceivably utilise cords, as well as utilising collagenous fibrils of the interstitial matrix, as substrate cues in cell guidance, attachment and migration. The possibility also exists that cords fulfil a functional role in corneal morphogenesis via mechanotransduction through cell matrix interactions

Actin and Type I Collagen Propeptide Distribution in the Developing Chick Cornea

PURPOSE. To determine the organization of actin filaments and distribution of type I procollagen during the development of the chick corneal stroma. METHODS. Embryonic chicken corneas of ages 6 to 18 days and 18 days posthatch were cryosectioned and fluorescently labeled for filamentous actin with phalloidin and for the N-and C-terminal propeptides of type I procollagen with specific monoclonal antibodies. Tissue sections were examined by fluorescence and confocal microscopy. RESULTS. Prominent actin filament bundles were present at all embryonic stages, arranged in orthogonal arrays. Type I collagen propeptides were also present, with the C-propeptide visible as small foci, often associated with the actin label. The N-propeptide was also detected in the stromal matrix, especially in Bowman's layer. Actin filaments were also prominent in the corneal epithelium, along with collagen propeptide labeling, up to embryonic day14. CONCLUSIONS. Actin filament bundles are abundant in the stroma, presumably in the keratocytes of the developing chick cornea, and are arranged in an orthogonal manner suggesting a possible role in cell and matrix organization in this tissue. Filament bundles appear to be closely associated with the foci of type I procollagen label, suggesting a possible association between the actin cytoskeleton and the trafficking of collagen. The presence of the N-propeptide of type I collagen in the extracellular matrix and the restricted distribution of the Cpropeptide suggest differential processing of these molecules after secretion. The persistence of the N-propeptide implies a role in development, possibly in association with control of collagen fibril diameter and spacing. (Invest Ophthalmol Vis Sci

Actin and Type I Collagen Propeptide Distribution in the Developing Chick Cornea

PURPOSE. To determine the organization of actin filaments and distribution of type I procollagen during the development of the chick corneal stroma. METHODS. Embryonic chicken corneas of ages 6 to 18 days and 18 days posthatch were cryosectioned and fluorescently labeled for filamentous actin with phalloidin and for the N-and C-terminal propeptides of type I procollagen with specific monoclonal antibodies. Tissue sections were examined by fluorescence and confocal microscopy. RESULTS. Prominent actin filament bundles were present at all embryonic stages, arranged in orthogonal arrays. Type I collagen propeptides were also present, with the C-propeptide visible as small foci, often associated with the actin label. The N-propeptide was also detected in the stromal matrix, especially in Bowman's layer. Actin filaments were also prominent in the corneal epithelium, along with collagen propeptide labeling, up to embryonic day14. CONCLUSIONS. Actin filament bundles are abundant in the stroma, presumably in the keratocytes of the developing chick cornea, and are arranged in an orthogonal manner suggesting a possible role in cell and matrix organization in this tissue. Filament bundles appear to be closely associated with the foci of type I procollagen label, suggesting a possible association between the actin cytoskeleton and the trafficking of collagen. The presence of the N-propeptide of type I collagen in the extracellular matrix and the restricted distribution of the Cpropeptide suggest differential processing of these molecules after secretion. The persistence of the N-propeptide implies a role in development, possibly in association with control of collagen fibril diameter and spacing. (Invest Ophthalmol Vis Sci

Chondroitin sulfate as a potential modulator of the stem cell niche in cornea

Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells

The response of foetal annulus fibrosus cells to growth factors: modulation of matrix synthesis by TGF-β1 and IGF-1

The annulus fibrosus of the intervertebral disc is a complex radial-ply tissue that derives initially from segmental condensations of axial mesenchyme surrounding the notochord. These mesenchymal condensations differentiate into the early annulus fibrosus during foetal development—their outer part becoming fibrous, containing collagen type I; and their inner part cartilaginous, containing type II collagen and aggrecan. With post-natal growth and ageing, there is a switch from type I to type II collagen and an increase in proteoglycan synthesis in the outer annulus. This fibrocartilaginous metaplasia appears to occur in response to compressive loading of the tissue as occurs in tendons that wrap around bony pulleys, and driven by growth factors, such as TGF-β. In this study, using high-density micromass cultures, we have assessed the response of foetal outer annulus cells to growth factor stimulation with TGF-β1 and IGF-1, growth factors known to occur within the developing disc. We qualitatively and quantitatively describe the stimulatory effects of these growth factors, both alone and in combination, on the synthesis of sulphated glycosaminoglycan, and collagen types I and II by annulus cells. We show a potential role for TGF-β1 in pushing cells towards a fibrocartilaginous phenotype, with possible complementary effects of IGF-1