The association between pterygium and conjunctival ultraviolet autofluorescence : The Norfolk Island Eye Study

Purpose: To investigate the association between conjunctival ultraviolet autofluorescence (UVAF), a biomarker of ocular ultraviolet radiation (UVR) exposure, and prevalent pterygium. Methods: We conducted a cross-sectional study on Norfolk Island, South Pacific. All permanent residents aged ‡15 were invited to participate. Participants completed a sun exposure questionnaire and underwent autorefraction and slit lamp biomicroscope examination. Area of conjunctival UVAF (sum of temporal ⁄ nasal area in right and left eyes) was determined using computerized methods. Multivariate logistic and linear regression models were used to estimate the associations with pterygia and UVAF, respectively. Results: Of 641 participants, 70 people (10.9%) had pterygium in one or both eyes, and prevalence was higher in males (15.0% versus 7.7%, p = 0.003). Significant independent associations with pterygium in any eye were UVAF (per 10 mm2) [odds ratio (OR) 1.16, 95% confidence interval (CI) 1.16–1.28, p = 0.002], tanning skin phenotype (OR 2.17,1.20–3.92, p = 0.010) and spending more than three-quarters of the day outside (OR 2.22, 1.20–4.09, p = 0.011). Increasing quartile of UVAF was associated with increased risk of pterygium following adjustment of age, sex and time outdoors (pTrend = 0.002). Independent associations with increasing UVAF (per 10 mm2) were decreasing age, time outdoors, skin type and male gender (all p < 0.001). UVAF area correlated well with the duration of outdoor activity (pTrend < 0.001). Conclusion: Pterygium occurs in approximately one-tenth of Norfolk Islanders. Increasing conjunctival UVAF is associated with prevalent pterygia, confirming earlier epidemiological, laboratory and ray-tracing studies that pterygia are associated with UVR. Protection from the sun should be encouraged to reduce the prevalence of pterygium in the community.

Reliability and validity of conjunctival ultraviolet autofluorescence measurement

BACKGROUND: Conjunctival ultraviolet autofluorescence (UVAF) photography was developed to detect and characterise pre-clinical sunlight-induced UV damage. The reliability of this measurement and its relationship to outdoor activity are currently unknown. METHODS: 599 people aged 16-85 years in the cross-sectional Norfolk Island Eye Study were included in the validation study. 196 UVAF individual photographs (49 people) and 60 UVAF photographs (15 people) of Norfolk Island Eye Study participants were used for intra- and inter-observer reliability assessment, respectively. Conjunctival UVAF was measured using UV photography. UVAF area was calculated using computerised methods by one grader on two occasions (intra-observer analysis) or two graders (inter-observer analysis). Outdoor activity category, during summer and winter separately, was determined with a UV questionnaire. Total UVAF equalled the area measured in four conjunctival areas (nasal/temporal conjunctiva of right and left eyes). RESULTS: Intra-observer (ρ_c=0.988, 95% CI 0.967 to 0.996, p<0.001), and inter-observer concordance correlation coefficients (ρ_c=0.924, 95% CI 0.870 to 0.956, p<0.001) of total UVAF exceeded 0.900. When grouped according to 10 mm(2) total UVAF increments, intra- and inter-observer reliability was very good (κ=0.81) and good (κ=0.71), respectively. Increasing time outdoors was strongly with increasing total UVAF in summer and winter (p(trend) <0.001). CONCLUSION: Intra- and inter-observer reliability of conjunctival UVAF is high. In this population, UVAF correlates strongly with the authors' survey-based assessment of time spent outdoors.

Distribution of conjunctival ultraviolet autoflourescence in a population-based study : the Norfolk Island Eye Study

OBJECTIVE: The objective of this study was to describe the distribution of conjunctival ultraviolet autofluorescence (UVAF) in an adult population. METHODS: We conducted a cross-sectional, population-based study in the genetic isolate of Norfolk Island, South Pacific Ocean. In all, 641 people, aged 15 to 89 years, were recruited. UVAF and standard (control) photographs were taken of the nasal and temporal interpalpebral regions bilaterally. Differences between the groups for non-normally distributed continuous variables were assessed using the Wilcoxon-Mann-Whitney ranksum test. Trends across categories were assessed using Cuzick's non-parametric test for trend or Kendall's rank correlation τ. RESULTS: Conjunctival UVAF is a non-parametric trait with a positively skewed distribution. Median amount of conjunctival UVAF per person (sum of four measurements; right nasal/temporal and left nasal/temporal) was 28.2 mm(2) (interquartile range 14.5-48.2). There was an inverse, linear relationship between UVAF and advancing age (P<0.001). Males had a higher sum of UVAF compared with females (34.4 mm(2) vs 23.2 mm(2), P<0.0001). There were no statistically significant differences in area of UVAF between right and left eyes or between nasal and temporal regions. CONCLUSION: We have provided the first quantifiable estimates of conjunctival UVAF in an adult population. Further data are required to provide information about the natural history of UVAF and to characterise other potential disease associations with UVAF. UVR protective strategies should be emphasised at an early age to prevent the long-term adverse effects on health associated with excess UVR.

Assessment of conjunctival goblet cell density using laser scanning confocal microscopy versus impression cytology

Purpose To determine the association between conjunctival goblet cell density (GCD) assessed using in vivo laser scanning confocal microscopy and conjunctival impression cytology in a healthy population. Methods Ninety (90) healthy participants undertook a validated 5-item dry eye questionnaire, non-invasive tear film break-up time measurement, ocular surface fluorescein staining and phenol red thread test. These tests where undertaken to diagnose and exclude participants with dry eye. The nasal bulbar conjunctiva was imaged using laser scanning confocal microscopy (LSCM). Conjunctival impression cytology (CIC) was performed in the same region a few minutes later. Conjunctival goblet cell density was calculated as cells/mm2. Results There was a strong positive correlation of conjunctival GCD between LSCM and CIC (ρ = 0.66). Conjunctival goblet cell density was 475 ± 41 cells/mm2 and 466 ± 51 cells/mm2 measured by LSCM and CIC, respectively. Conclusions The strong association between in vivo and in vitro cellular analysis for measuring conjunctival GCD suggests that the more invasive CIC can be replaced by the less invasive LSCM in research and clinical practice.

Diurnal variation of anterior scleral and conjunctival thickness

Purpose To examine whether anterior scleral and conjunctival thickness undergoes significant diurnal variation over a 24-hour period. Methods Nineteen healthy young adults (mean age 22 ± 2 years) with minimal refractive error (mean spherical equivalent refraction -0.08 ± 0.39 D), had measures of anterior scleral and conjunctival thickness collected using anterior segment optical coherence tomography (AS-OCT) at seven measurement sessions over a 24-hour period. The thickness of the temporal anterior sclera and conjunctiva were determined at 6 locations (each separated by 0.5 mm) at varying distances from the scleral spur for each subject at each measurement session. Results Both the anterior sclera and conjunctiva were found to undergo significant diurnal variations in thickness over a 24-hour period (both p <0.01). The sclera and conjunctiva exhibited a similar pattern of diurnal change, with a small magnitude thinning observed close to midday, and a larger magnitude thickening observed in the early morning immediately after waking. The amplitude of diurnal thickness change was larger in the conjunctiva (mean amplitude 69 ± 29 μm) compared to the sclera (21 ± 8 μm). The conjunctiva exhibited its smallest magnitude of change at the scleral spur location (mean amplitude 56 ± 17 μm) whereas the sclera exhibited its largest magnitude of change at this location (52 ± 21 μm). Conclusions This study provides the first evidence of diurnal variations occurring in the thickness of the anterior sclera and conjunctiva. Studies requiring precise measures of these anatomical layers should therefore take time of day into consideration. The majority of the observed changes occurred in the early morning immediately after waking and were of larger magnitude in the conjunctiva compared to the sclera. Thickness changes at other times of the day were of smaller magnitude and generally not statistically significant.

Composite electrospun scaffolds for engineering tubular bone grafts

In this study, poly (e-caprolactone) [PCL] and its collagen composite blend (PCL=Col) were fabricated to scaffolds using electrospinning method. Incorporated collagen was present on the surface of the fibers, and it modulated the attachment and proliferation of pig bone marrow mesenchymal cells (pBMMCs). Osteogenic differentiation markers were more pronounced when these cells were cultured on PCL=Col fibrous meshes, as determined by immunohistochemistry for collagen type I, osteopontin, and osteocalcin. Matrix mineralization was observed only on osteogenically induced PCL=Col constructs. Long bone analogs were created by wrapping osteogenic cell sheets around the PCL=Col meshes to form hollow cylindrical cell-scaffold constructs. Culturing these constructs under dynamic conditions enhanced bone-like tissue formation and mechanical strength.We conclude that electrospun PCL=Col mesh is a promising material for bone engineering applications. Its combination with osteogenic cell sheets offers a novel and promising strategy for engineering of tubular bone analogs.

In vivo confocal microscopy of the palpebral conjunctiva and tarsal plate

Purpose The aim of this work is to develop a more complete understanding of the in vivo histology of the human palpebral conjunctiva and tarsal plate. Methods. The upper eyelids of 11 healthy human volunteer subjects were everted, and laser scanning confocal microscopy was used to examine the various tissue layers of the palpebral conjunctiva and tarsal plate. Results The superficial and basal epithelial layers are composed of cells with gray cytoplasm and thick, light gray borders.Nuclei can not be seen. The stroma has a varied appearance; fibrous tissue is sometimes observed, interspersed with dark,amorphous lacunae, and crevases. Numerous single white or gray cells populate this tissue, and fine blood vessels are seen traversing the field. Occasional conjunctival microcysts and Langerhans cells are observed. The tarsal plate is dark and amorphous, and meibomian gland acini with convoluted borders are clearly observed. Acini are composed of an outer lining of large cuboidal cells, and differentiated secretory cells can be seen within the acini lumen. Conclusions Laser scanning confocal microscopy is capable of studying the human palpebral conjunctiva, tarsal plate, and acini of meibomian glands in vivo. The observations presented here may provide useful supplementary anatomical information relating to the morphology of this tissue.

In vivo confocal microscopy of the bulbar conjunctiva

Background: The aim of this work is to develop a more complete qualitative and quantitative understanding of the in vivo histology of the human bulbar conjunctiva. Methods: Laser scanning confocal microscopy (LSCM) was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 healthy human volunteer subjects. Results: The superficial epithelial layer of the bulbar conjunctiva is seen as a mass of small cell nuclei. Cell borders are sometimes visible. The light grey borders of basal epithelial cells are clearly visible, but nuclei can not be seen. The conjunctival stroma is comprised of a dense meshwork of white fibres, through which traverse blood vessels containing cellular elements. Orifices at the epithelial surface may represent goblet cells that have opened and expelled their contents. Goblet cells are also observed in the deeper epithelial layers, as well as conjunctival microcysts and mature forms of Langerhans cells. The bulbar conjunctiva has a mean thickness of 32.9 1.1 mm, and a superficial and basal epithelial cell density of 2212 782 and 2368 741 cells/ mm2, respectively. Overall goblet and mature Langerhans cell densities are 111 58 and 23 25 cells/mm2, respectively. Conclusions: LSCM is a powerful technique for studying the human bulbar conjunctiva in vivo and quantifying key aspects of cell morphology. The observations presented here may serve as a useful marker against which changes in conjunctival morphology due to disease, surgery, drug therapy or contact lens wear can be assessed.

Expression of chondromodulin-1 in the temporomandibular joint condylar cartilage and disc

BACKGROUND: The temporomandibular joint (TMJ) cartilage consists of condylar cartilage and disc and undergoes continuous remodeling throughout post-natal life. To maintain the integrity of the TMJ cartilage, anti-angiogenic factors play an important role during the remodeling process. In this study, we investigated the expression of the anti-angiogenic factor, chondromodulin- 1 (ChM-1), in TMJ cartilage and evaluate its potential role in TMJ remodeling. METHODS: Eight TMJ specimens were collected from six 4-month-old Japanese white rabbits. Safranin-O staining was performed to determine proteoglycan content. ChM-1 expression in TMJ condylar cartilage and disc was determined by immunohistochemistry. Three human perforated disc tissue samples were collected for investigation of ChM-1 and vascular endothelial growth factor (VEGF) distribution in perforated TMJ disc. RESULTS: Safranin-O stained weakly in TMJ compared with tibial articular and epiphyseal cartilage. In TMJ, ChM-1 was expressed in the proliferative and hypertrophic zone of condylar cartilage and chondrocyte-like cells in the disc. No expression of ChM-1 was observed in osteoblasts and subchondral bone. ChM-1 and VEGF were both similarly expressed in perforated disc tissues. CONCLUSIONS: ChM-1 may play a role in the regulation of TMJ remodeling by preventing blood vessel invasion of the cartilage, thereby maintaining condylar cartilage and disc integrity.

The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.