and +Department of Medicine Labs., Bristol Royal Infirmary, Bristol BS2 8HW UK Hyaluronan (HA) is commonly found in connective tissue as a component of the extracellular aggrecan complex, and in many sites in the eye, notably the vitreous. It has been used in artificial tears to alleviate dry eye disorders [i] HA has also been reported at the surface of conjunctival and corneal cells, but its site of synthesis arid its metabolic fate are not clear [2,3].The present study was carried out to determine whether HA is present in normal preocular fluid and also if it could be detected as a secretory product in primary cultures of human conjunctiva.Tear samples were collected from 12 subjects with no known eye pathologies using lOpl or 20pl glass capillaries placed at the eyelid margin. In some cases 200-300pl sterile saline were instilled to assist collection. Samples were collected from one individual over 24h.Pooled tears from several subjects were diluted to give a solution of 4M-guanidine hydrochloride in phosphate buffered saline pH 7.0, and the density adjusted to 1.45 with CsCI. The sample was centrifuged at 150,000 g at 10% for 24h and fractions of 0.5ml removed from the top of the tube by aspiration. All samples were stored at -20% until tested for HA.Human conjunctival epithelium was separated from stroma under a dissection microscope. Small explants were plated out in RPMl 1640 medium supplemented with 10% foetal calf serum. Epithelial cultures were established by removing explants after 5-7 days, which gave optimal epithelial migration before fibroblastic outgrowth. After 7 days the culture contained fibroblasts whose proportion increased with time . Mouse 3T3 fibroblasts were also cultured under the same conditions for comparison. Culture supernatant was collected from each type of culture starting at 40% confluence and from equivalent cultures daily until 80-90% confluence (5-7 days) without medium change. Cells were counted after trypsinization. HA was measured using a radiometric assay kit with (1251]-labelled link protein (Pharmacia, UK).The concentration of HA in tear fluid varied between 12-1900 ng/ml (figure 1). No significant diurnal variation was found in one individual. These results show that HA is present in soluble form in tear fluid and washings. It is not yet clear whether this is due to release from surface located HA [2,3] or as a secretory product. The basis of variation in preocular HA levels remains to be explained.Fractionation of pooled human tears on a CsCl density gradient yielded two peaks: at 1.42-1.43 glml and at 1.35-1.40 g/ml. These densities are lower than expected for connective tissue hyaluronan, usually approx. 1.5glml under conditions used here. Factors which could account for this result include the size of HA oligosaccharides and interaction of HA with other components of preocular fluid, such as mucins and tear specific proteins.The level of secreted HA in epithelial and fibroblast cultures increased during rapid proliferation (figure 2). The levels of HA were an order ...

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