Expression of GPR-68 in Human Corneal and Conjunctival Epithelium. Possible indicator and mediator of attrition associated inflammation at the ocular surface.

INTRODUCTION: Attrition and osmotic stress have been identified as major forces in the pathophysiology of dry eye. Impaired tolerance to mechano-transduction in the presence of insufficient lubrication has been associated with disturbances of ocular surface homeostasis and encouragement of inflammatory reactions, challenging the usual regulatory coping mechanisms. In spite of the probable link between enhanced attrition and secondary inflammation, the key mediators driving the vicious cycle of severe dry eye disease have not yet been identified. The goal of this study was therefore to investigate human corneal and conjunctival epithelium for the presence of the G protein-coupled receptor GPR-68. This protein had most recently been shown to be not only chemically activated but also mechanically, possibly through attrition. METHODS: De-identified sections of human cornea and conjunctiva were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemical methods. Results Specific staining for G-protein-coupled receptor 68 (GPR68) was observed in all samples of the cornea throughout the epithelial layers of the corneal epithelium, most prominently in the area of the wing cells and the basement membrane. Even in the conjunctiva, specific staining for GPR-68 was found. DISCUSSION: The detection of G protein-coupled receptor GPR-68 in human corneal and conjunctival epithelium raises the question of its function and purpose. The mechanical activation of GPR68 in situations with enhanced friction and attrition could modify various cellular functions and possibly jeopardize normal inflammatory homeostasis at the ocular surface. Accordingly, decreased lubrication in dry eye disease could result in activation of GPR-68. This could lead to secondary inflammation, initially in the epithelium and surrounding stroma. Continuous mechanical stress could result in chronic inflammation, also reaching deeper structures of the cornea, possibly making GPR-68 an important actor in the vicious cycle of dry eye disease. CONCLUSION: G protein-coupled receptor GPR-68, sensitive to flow and mechanic stimulation, is present in the human corneal epithelium and conjunctiva. Decreased lubrication and increased attrition, accompanied by sensations typical for dry eye, might lead to local inflammation. It is possible that subtle signs of conjunctival, and later corneal, surface damage in the context of these sensations could be a better indicator of the need for and success of therapy than the clinical signs of dry eye disease alone, at least in the early stages of the disease. Inhibition of G protein-coupled receptor GPR-68 could represent a new strategy in the treatment of dry eye disease.

GPR-68 in human lacrimal gland. Detection and possible role in the pathogenesis of dry eye disease.

INTRODUCTION: In parallel to ocular surface disease in dry eye there is often a dysfunctionality of the lacrimal gland apparatus. The functionality of the lacrimal gland is of major importance for maintenance of ocular surface integrity and health, even in conditions of enhanced stimulation and secretion requirements. Such enhanced secretion demands can push the lacrimal gland to its limits, with maximized tear fluid secretion and increased flow through the lacrimal ducts. The goal of this study was to investigate whether G protein-coupled receptor GPR-68 is present in the lacrimal gland, as this protein has recently been shown to be sensitive to flow rate and osmolarity. METHODS: For this purpose, de-identified sections of human lacrimal gland tissue were stained for the presence of G protein-coupled receptor 68 with specific antibodies using immunohistochemistry. RESULTS: Specific staining was detected in the acini and ducts of human lacrimal gland. In the ducts, the specific staining was found around the lumen of the ducts. In the acini, the specific staining was observed more towards the lumen but also intercellularly between the acinar cells. DISCUSSION: The detection of G protein-coupled receptor GPR-68 in the lacrimal gland, especially around the lumen of the ducts, raises the question about its function and purpose. Activation of GPR68 leads to modification of various cell functions and is associated with regulation of inflammation. Accordingly, enhanced, secretion-induced, augmentation of flow might exert fluid flow stress on the ducts and acini. This might lead to transient, localized activation of GPR-68 and secondary inflammation within the gland. Depending on the intensity, continuity or repetitive nature of the stimuli, exhaustion of the lacrimal gland secretion capacity might follow, and chronicity of the inflammation in the parenchyma as well as around the ducts might be a consequence. CONCLUSION: G protein-coupled receptor GPR-68, sensitive to flow, is present in the human lacrimal gland. Increased flow, triggered by sensations such as are typical for dry eye, might lead to local inflammation. It is possible that these sensations might serve as a better indicator for the need and success of therapy than the clinical signs of dry eye disease, at least in the early stages of the disease.

Detection of TSG-6-like protein in human corneal epithelium. Simultaneous presence with CD44 and hyaluronic acid.

INTRODUCTION: Tumor necrosis factor-inducible gene 6 protein (TSG-6) is member of the hyaluronan-binding protein family (hyaladherins) to which CD44 also belongs. Inflammatory mediators such as tumor necrosis factor α (TNF-α) and interleukin-1 (IL-1) stimulate TSG-6 production. Recently, however, externally applied TSG-6 has been shown to be effective in the treatment of inflammatory dry eye. On the other hand, it is still unknown whether TSG-6 is naturally present in human corneal epithelium. MATERIAL AND METHODS: Corneal sections of 15 eyes enucleated for posterior segment uveal melanoma were immunohistochemically stained for hyaluronic acid (HA), CD44, and TSG-6. RESULTS: Throughout the corneal epithelium of all sections, CD44 and hyaluronic acid were detected most intensely in the basal epithelial layer. Whereas the presence of HA was intense even in the cytoplasm of the cells, CD44 was located predominantly at the cell membranes. The intensity of the specific staining decreased towards the surface, where CD44 was barely detectable. Hyaluronic acid was, on the other hand, detectable in the extracellular matrix and cells, even at the surface. TSG-6 like immunoreactivity was detected in all sections in a pattern similar to CD44 but much more distinct and intense, with a marked localization in the cell membranes and intercellular spaces, i.e., extracellular matrix. TSG-6 like immunoreactivity was clearly detectable through all cell layers of the corneal epithelium. All control sections were negative. DISCUSSION: Tumor necrosis factor-inducible gene 6 (TSG-6)- like protein is present in human corneal epithelium. It might be a natural component of this tissue which is constantly exposed and mechanically traumatized, and displays localization with similarities to that of CD44. The immunohistological detection of HA as major component of the ECM and epithelial tissue only confirms the results of earlier studies. However, the simultaneous presence and colocalization of CD44 and TSG-6, both HA-binding proteins, requires further investigation of the individual role, regulation and interaction of this system. CONCLUSION: The detection of TSG-6 in human corneal epithelium in the absence of inflammation underlines the importance of normal mechanical forces on the gene expression and regulation of this protein in ocular surface tissues. Given the relationship between inflammation and the protein, TSG-6 may be a major unknown and underestimated player in the regulation of the inflammation encountered in the presence of ocular surface desiccation and dry eye disease.

Osmokinetics: A new dynamic concept in dry eye disease.

INTRODUCTION: Tear fluid osmolarity has been increasingly accepted as an accessible parameter in the diagnosis of ocular surface and dry eye disease. After having been proposed as the gold standard, recent results have put this into question. However, the most recent guidelines for dry eye disease identify specific values of osmolarity as thresholds to help to differentiate between various stages of severity of ocular surface disease. The limits of this approach were investigated to propose a new concept, that of osmokinetics. MATERIALS AND METHODS: Available data on tear fluid osmolarity in normal and diseased eyes were compared. The possibility of normo-osmolar dry eye was investigated and repeated measurements of osmolarity performed. RESULTS: The currently applied static model of a threshold value of osmolarity for diagnosing dry eye disease is apparently insufficient. Not only does it not take into account normo-osmolar dry eye, but it also applies too much significance to a single parameter. Instead, it was found that there is a daily variation in osmolarity (DVO), which appears to be higher in eyes with tear film deficiencies than in healthy eyes. DISCUSSION: Tear film osmolarity does vary considerably throughout the day. Its value should be considered in a kinetic model taking into account the dynamics of osmolarity changes moreso than the current static model. The terms of osmotic stress and diurnal variation of osmolarity were found to offer a more physiological understanding of osmolarity. CONCLUSION: A more dynamic model for osmolarity is presented in which not the value itself but the daily variation of osmolarity is identified. It is suggested that the amplitude of change in osmolarity over the course of a day or even shorter time periods could play a decisive role as a stress factor for the surface cells. The varying osmolar stress could be one of the key mechanisms leading to the cell death, inflammation, apoptosis, and goblet cell disappearance as observed in dry eye disease. Perhaps it is the mean osmolarity level at which these changes occur together with the magnitude of DVO which could identify the level of severity of dry eye disease.

Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis.

OBJECTIVE: Levels of tear film matrix metalloproteinases (MMPs) activity are significantly elevated in horses with ulcerative keratitis and contribute to the excessive breakdown of stromal collagen. Changes in the amount of proteolytic activity in horse tear film during corneal healing and stromal remodeling have not yet been reported, but we hypothesize they should decrease. In the present study we analyzed serial tear fluid from horses with ulcerative keratitis to identify any changes in MMP activity during corneal healing and stromal remodeling. PROCEDURES: Samples of tear fluid were obtained from both eyes of 10 horses with ulcerative keratitis on the day of admission (day 1) at the hospital and then at various time points until complete healing of the cornea. Tear film MMP2 and MMP9 activity was determined by quantitative gelatin zymography. In all cases medical treatment included topical applications of equine serum, antibiotics, atropine and systemic administration of anti-inflammatory drugs. Surgical procedures were performed in several cases on day 2 in addition to the medical treatment. RESULTS: The mean total MMP activity (+/- SD) measured in relative standard units (RSU) in the tear fluid of the ulcerated eye (2.44 +/- 1.44) of the 10 horses was significantly higher than the mean in the contralateral eye (0.81 +/- 0.68) (P = 0.006), on the day of admission at the VMTH. The mean MMP activity in these ulcerated eyes significantly decreased (-82.4%) between the first day of admission and the day when the ulcer had completely healed (P = 0.0002). The activity level in the healed eye (0.43 +/- 0.17) was not significantly different to the one in the contralateral eye (0.36 +/- 0.18) on the day of complete corneal healing (P = 0.374). The level of MMP activity in the contralateral eye also decreased from 0.81 +/- 0.68-0.36 +/- 0.18 but this decrease (56%) was not significant (P = 0.069). CONCLUSIONS: Ulcerative keratitis in horses is associated with initially high levels of tear film proteolytic activity that decrease as the ulcers heal. The success of medical and surgical treatment of the corneal ulcers is reflected by the enzyme activity in tears. In horses successful treatment does lead to a rapid reduction in tear film proteolytic activity that corresponded with the improvement in the clinical signs of corneal ulceration. Measurement of MMP activity in the tear film might represent a way to monitor the progression of corneal healing in horses with ulcerative keratitis.

Connective tissue growth factor in tear film of the horse: detection, identification and origin.

BACKGROUND: Healing of corneal ulcers in horses is often associated with profound corneal stromal fibrosis and scar formation resulting in visual impairment. Connective tissue growth factor (CTGF) is a fibrogenic cytokine involved in wound healing and scarring. The purpose of this study was to determine whether CTGF was present in the tear fluid of normal horse eyes and the eyes of horses with corneal ulcers in order to evaluate the role of CTGF in corneal wound healing and corneal scar formation. METHODS: Tear fluid samples were collected from 65 eyes of 44 horses; 32 samples from normal eyes, 21 samples from eyes with corneal ulceration, and 12 samples from the unaffected contralateral eyes of horses with ulcers. CTGF levels in the tears were determined by enzyme immunoassay using goat IgG against human CTGF. Antigenetic similarity of human and horse CTGF was established in a bio-equivalence assay. The identity of horse CTGF was confirmed by western blot. Lacrimal and nictitating membrane glands were investigated by immunohistochemistry in the attempt to clarify the origin of tear fluid CTGF. RESULTS: CTGF was detected in tear film of 23 normal unaffected eyes (72%) and 8 normal contralateral eyes (67%), with the mean CTGF levels (+/- SEM) being 51.5+/-19.2 and 13.4+/-3.9 ng/ml respectively. CTGF was found in 8 eyes with corneal ulcers (38%) with the mean CTGF concentration of 26.3+/-14.8 ng/ml. Western blot identified the protein detected as CTGF. The identification of CTGF in lacrimal glands suggests a major role of these glands in the presence of CTGF in tears. CONCLUSIONS: CTGF is present in horse tear fluid and derives, at least partly, from the lacrimal gland. Equine CTGF has strong antigenic similarity with human CTGF. Corneal disease leads to a decrease of CTGF concentrations in tears. The possible role of CTGF in the healing process of ocular surface requires further investigation.

Detection of connective tissue growth factor (CTGF) in human tear fluid: preliminary results.

PURPOSE: Connective tissue growth factor (CTGF) is one of the main regulators of fibrosis. We aimed to evaluate its presence in the human tear fluid of healthy individuals. METHODS: A total of 70 tear fluid samples were collected from eight volunteers prior to and after stimulation of reflex tears with onion vapour. Specific ELISA analysis was performed with goat IgG against human CTGF. RESULTS: Connective tissue growth factor was detected in seven samples (10%), with maximum levels of 17 ng/mL in basal tears. Induction of reflex tearing resulted in a fast and significant decrease of CTGF concentrations (r = - 0.95). No CTGF was detected in 90% of the samples. CONCLUSION: Connective tissue growth factor may occur in tear fluid in healthy human eyes. This indicates a possible role for tear fluid CTGF in ocular surface fibrosis and wound healing.

Detection of connective tissue growth factor in human aqueous humor.

PURPOSE: Connective tissue growth factor (CTGF) has been shown to be substantially involved in various processes of fibrosis. We investigated if CTGF is present in aqueous humor (AH). METHODS: Samples from AH were collected from 10 volunteers during cataract surgery. Specific ELISA analysis was performed with goat IGG against human CTGF. RESULTS: CTGF was above the detection limit of the assay in 80% of the samples. The concentration of CTGF in the anterior chamber fluid was 1.24 +/- (SD) 0.26 ng/ml. CONCLUSION: CTGF is present in a majority of AH samples, possibly representing a constant component in this fluid. The origin and physiological importance of CTGF is yet unclear. The involvement of CTGF in processes of intraocular fibrosis and wound healing is possible.

Levels of transforming growth factor alpha (TGF-alpha) in human cerebrospinal fluid.

In this study, we investigated cerebrospinal fluid of patients with various neurological symptoms for the presence of transforming growth factor alpha (TGF-alpha). 41 samples of cerebrospinal fluid were collected by lumbar puncture performed routinely due to the clinical suspicion of neurological disease from 22 females (age 15-80 years, median 42 years) and from 19 males (age 18-82 years, median 48 years). A highly sensitive and specific radioimmunoassay was used to determine the concentration of TGF-alpha in the samples. The detection limit of the assay was about 200 pg TGF-alpha. There was no cross-reactivity to human EGF. We showed CSF indeed does contain TGFalpha. As TGF-alpha was detected in all 41 samples investigated, this growth factor appears to be a constant component of CSF. The mean concentration was 5.5 ng TGF-alpha (S.D. +/- 2.7 pg/ml, range 1.1 to 13.9 pg/ml). There was no significant correlation between TGF-alpha concentration in CSF and age (r = -0.006) and there was no significant difference between females (mean 5.8+/-3.10 pg/ml) and males (mean 5.2+/-1.96 pg/ml). No diagnosis was over represented in patients with TGF-alpha concentrations above or below 1 S.D. off the mean. However, highest concentrations of TGF-alpha were found in the group of patients with peripheral neurological sensory dysfunctions and polyneuropathy. We conclude that TGF-alpha is not only a constant component of human cerebrospinal fluid in adults but could also be significantly involved in the pathophysiology of various neurological diseases. The earlier hypothesis that TGF-alpha could mainly have a role in brain development needs hence to be re-evaluated.

Dry eye syndrome, tear film, pharmacology of eye drops, and toxicity.

Over the past years, the main parameters used for the diagnosis of dry eye have been outlined: there is now substantial effort to bring them into correlation with each other, resulting in easier diagnosis of the diseases that appear to cause dry eye. Although increasing awareness of the plethora of dry eye diseases makes the differential diagnosis and the treatment difficult, the increased availability of optimized artificial tears and tear substitutes, especially viscoelastics, does allow easier relief of the most serious symptoms of dry eye disease. Improved topical treatment, in turn, allows various treatments, such as excimer laser treatment, to be performed, even in dry eyes. This, together with the identification of tear substitutes as a major research target and a better understanding for the psychological tasks of chronic dry eye diseases, offers a good basis toward further improvements.

Comparison of corneal epithelial wound healing after photorefractive keratectomy in the rabbit with two types of excimer lasers.

PURPOSE: To evaluate the differences in epithelial wound healing following photorefractive keratectomy when performed with the Summit UV 200 LA and the VISX 20/20 excimer lasers. METHODS: Sixteen New Zealand rabbits were divided into two groups. One group was treated with the Summit laser and the other group treated with the VISX 20/20 laser. The treatment consisted of a -6.00 diopter photorefractive keratectomy with a 5-mm diameter treatment zone. Epithelial wound healing was followed by photography at 4 hour intervals for 64 hours. The length of the wound edge and the size, shape, and closure time of the wound were measured. RESULTS: The median wound edge length at 4 hours was 18.3 mm for the Summit laser and 16.7 mm for the VISX laser. The median wound size at 4 hours was 22.0 mm2 for the Summit and 21.2 mm2 for the VISX. The median wound closure time was 53.4 hours for the Summit laser and 54.0 hours for the VISX laser. CONCLUSION: There was no statistically significant difference in the epithelial healing of rabbit corneal wounds created by photorefractive keratectomy when performed with two current ophthalmic excimer lasers, the Summit UV 200 LA and the VISX 20/20.

Vascular endothelial growth factor (VEGF) in normal human corneal epithelium: detection and physiological importance.

PURPOSE: Corneal neovascularization is a major challenge following chemical burns and corneal inflammation. The factors triggering corneal neovascularization involve various growth factors. In the release and control of these factors the regenerating tissue plays a decisive role. Only recently has vascular endothelial growth factor been shown to be involved in the basic events of retinal neovascularization. From the cornea current knowledge allows only for the statement that corneal epithelium could be able to produce vascular endothelial growth factor. The present study was designed to show the presence of vascular endothelial growth factor-like substances in the corneal epithelium of the normal eye in vivo. METHODS: Using specific antibodies to the N-terminus of human vascular endothelial growth factor indirect immuno-histochemistry was applied to sections of normal human corneal epithelium and to five sections of enucleated human eyes with a morphologically normal anterior segment. RESULTS: In all sections specific staining for vascular endothelial growth factor was found over the entire epithelium. Staining was most intense in the basal layer of epithelial cells. Only a weak staining was detectable in the cell layers closer to the surface. Here, however, the samples of corneal epithelium having been subject to traumatic erosion showed a slightly more intense staining than the sections from the undisturbed corneal tissue of whole globe sections. CONCLUSION: It was shown that vascular endothelial growth factor-like protein is present in the human corneal epithelium. Observed differences in the staining pattern could suggest that the expression of vascular endothelial growth factor might be enhanced due to ocular surface trauma. It is suggested that corneal epithel vascular endothelial growth factor might be an important factor in the cascade leading to the onset of corneal neovascularization.

Beta-amyloid protein protein precursor expression in lacrimal glands and tear fluid.

PURPOSE: Proteases in tear fluid play important roles in the regulation of corneal wound healing. Inhibitors of proteolytic activity are major modulators of the associated events. Although it is known that various enzyme inhibitors exist in tear fluid, it is not known whether certain isoforms of the beta-amyloid protein precursor (beta-APP), a potent inhibitor of serine proteases, are present in tear fluid. The purpose of this study was to investigate whether beta-APP can be detected in human tear fluid and, if so, to determine the isoform composition and cellular origin. METHODS: Tear fluid was collected from healthy volunteers. The beta-APP was identified and characterized by immunoblotting using antibodies specific for domains of the beta-APP. The protein was characterized further by ion exchange chromatography. Expression of the beta-APP gene was studied using in situ hybridization and RNA-RNA solution hybridization assay. RESULTS: beta-APP with protease inhibitory properties was identified in all samples of human tear fluid. Immunologic analysis revealed that it had been processed proteolytically before secretion. Gene expression studies showed that the beta-APP gene was expressed in lacrimal glands, particularly in acinar cells. The gene transcript almost exclusively corresponded to beta-APP containing the protease inhibitor insert. CONCLUSIONS: beta-APP is expressed in lacrimal glands and subsequently is secreted into tear fluid. Because the bulk of the beta-APP contained the protease inhibitor insert, the authors propose that beta-APP is an important regulator of proteolysis in tear fluid and that possibly it plays a role in the events associated with corneal wound healing. This suggests a novel physiological function of beta-APP in addition to those previously described-regulation of blood coagulation and cell growth.

The clinical use of contact lenses and collagen shields.

Contact lens wear before and after photorefractive or phototherapeutic keratectomy with excimer lasers has become an increasingly interesting subject. No negative side effects of contact lens wear prior to photorefractive keratectomy have yet been shown and refractive errors postoperatively do not seem to constitute a major practical problem. Normal side effects include the risk of bacterial infection and keratitis. The major pathogen in this context is still Pseudomonas aeruginosa, although increased insight into the mechanisms of bacterial adhesion and improvements in cleaning procedures now allow more effective preventive care of contact lenses themselves and of contact lens cases. Tear fluid research using leucotriene C4 as a possible indicator for subclinical inflammation has led to interesting results, and the importance of the arachidonic pathway and its metabolites has become more evident. These advances contribute considerably to the safe use of contact lenses in clinical ophthalmology, although major innovations such as the multifocal contact lenses have not reached the stage of perfection.

Basic fibroblast growth factor in human tear fluid: detection of another growth factor.

BACKGROUND: Topical application of various growth factors have shown promising experimental data in speeding up corneal wound healing. Similar effects have been reported for another group of proteins, fibroblast growth factors (FGFs). One of them, basic FGF (bFGF), stimulates the proliferation of cells of mesodermal as well as of neuroectodermal origin and is highly angiogenic. The aim of the present study was to investigate the presence of bFGF in human tear fluid. METHODS: After establishing an advanced enzyme-linked immunosorbent assay technique, tear fluid was collected from 15 healthy individuals before and after stimulation of reflex tearing. RESULTS: In none of the tear fluid samples collected prior to induction of reflex tearing could FGF be detected. Of the 15 samples collected during reflex tearing, six (40%) contained measurable amounts of bFGF. The amount of bFGF appeared to decrease with increasing tear fluid flow, suggesting a dilution effect. CONCLUSION: This study gives strong evidence that bFGF, as previously shown for epidermal growth factor and transforming growth factor-alpha, may occur in human tear fluid of healthy individuals after induction of reflex tearing and, in very low concentrations, under normal conditions. Whether bFGF can to be considered a constant component of tear fluid, however, remains to be investigated.

Detection of transforming growth factor-alpha mRNA and protein in rat lacrimal glands and characterization of transforming growth factor-alpha in human tears.

PURPOSE: To assess whether the lacrimal gland is a possible site of synthesis of transforming growth factor-alpha (TGF-alpha) and to characterize TGF-alpha biochemically in human tears. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) amplification was used to analyze rat lacrimal glands for the presence of TGF-alpha mRNA. Specific monoclonal antibodies were used to localize TGF-alpha immunohistochemically in lacrimal gland tissue of rats. Human tears were analyzed for immunoreactive TGF-alpha protein using a specific radioimmunoassay, and the molecular weight of TGF-alpha in tears was characterized by Western blot analysis. RESULTS: RT-PCR amplification of rat lacrimal gland RNA generated a band of the predicted 492 base pairs for TGF-alpha mRNA. Immunohistochemical staining of rat lacrimal gland localized TGF-alpha protein to lacrymocytes constituting acini but not to interacinar and intraacinar ducts of lacrimal glands. Western blot analysis of human tears detected a single band at MWt 16,000. Logit transformation of radioimmunoassay data for tears and TGF-alpha standard generated parallel displacement lines, indicating the presence of immunoreactive TGF-alpha levels in human tears with an average concentration of 100 +/- 20 pg/ml (mean +/- SEM). CONCLUSIONS: Rat lacrymocytes synthesize TGF-alpha mRNA and protein, and human tears contain immunoreactive TGF-alpha, suggesting that the lacrimal gland may be an exocrine source for TGF-alpha in tears. The single MWt 16,000 form of TGF-alpha in human tears appears to be generated by an unusual proteolytically processing of the pro-TGF-alpha transmembrane precursor protein.

Inhibition of pseudomonal ulceration in rabbit corneas by a synthetic matrix metalloproteinase inhibitor.

PURPOSE: To evaluate the effect of the synthetic matrix metalloproteinase inhibitor, Galardin, on proteases produced by Pseudomonas aeruginosa (PA) and on a rabbit model of Pseudomonas keratitis. METHODS: Protease activities of culture broths from Pseudomonas strains PA-28 and W-186 were characterized in vitro by gelatin zymography and by digestion of Azocasein in the presence and absence of Galardin and the serine protease inhibitor, aprotinin. In a noninfectious in vivo experiment, sterile PA culture broth from W-186 was injected intrastromally into rabbit corneas that were treated topically with Galardin or vehicle, then evaluated clinically and histologically. In an infectious in vivo experiment, rabbit corneas were injected with washed PA-28, then treated topically with Galardin or vehicle and clinically scored. RESULTS: Gelatin zymography of culture broth from W-186 and PA-28 detected two proteases that were both inhibited by Galardin. Galardin reduced the digestion of Azocasein by both PA culture broths by 99%, whereas aprotinin did not significantly reduce the protease activity of PA-28 conditioned broth. Intrastromal injection of sterile W-186 culture broth caused rapid corneal destruction that was prevented by topical treatment with Galardin. Intrastromal injection of washed PA-28 bacteria resulted in progressive corneal melting that was significantly (P < 0.005) delayed, but ultimately not prevented, by topical treatment with Galardin. CONCLUSIONS: Pseudomonal protease activity in culture broth consisted predominantly of metalloproteinases and were effectively inhibited by Galardin in vitro. Topical treatment with Galardin prevented destruction of rabbit corneas by bacterial products present in culture broth, and it delayed corneal destruction after injection of PA bacteria. Galardin may be a useful adjuvant when corneal destruction proceeds despite prompt antibiotic treatment.

Growth factors and their receptors in the anterior chamber. Absence of epidermal growth factor and transforming growth factor alpha in human aqueous humor.

The involvement of growth factors such as epidermal growth factor (EGF) in regenerative processes of corneal endothelium and lens epithelium has recently been suggested. However, knowledge on the presence of growth factors in anterior chamber fluid (ACF) is still very restricted. Although we have previously shown that EGF is undetectable in the ACF of normal eyes undergoing cataract surgery even by the use of high-sensitivity assays, this does not exclude the possible presence of other, EGF-like proteins in ACF such as transforming growth factor alpha (TGF-alpha). In the present study, we have hence determined in ACF samples of 70 human eyes the concentrations of both EGF and TGF-alpha. As assays served ELISA techniques and RIA. In none of all the samples investigated could detectable amounts of EGF, i.e. above 0.2 pg/ml (detection limit of the assay), be found, confirming earlier results. Interestingly, however, also no TGF-alpha could be detected in ACF. If present at all, the level of any TGF-alpha concentration in ACF was hence below the detection limit, i.e. less than 20 pg/ml. Based on the results of this study, it seems therefore that under physiological conditions there is no measurable presence of free EGF or TGF-alpha in human ACF. Existing receptors in the structures of the anterior segment must hence have ample binding capacity which could explain the effect of externally applied growth factors. The physiological and clinical importance of this result is briefly outlined.

Basic fibroblast growth factor in human saliva: detection and physiological implications.

After the detection of epidermal growth factor and transforming growth factor alpha in various body fluids and human saliva the current study aimed to investigate the presence of basic fibroblast growth factor (bFGF) in human saliva. Basic FGF is stimulating the proliferation of cells of mesodermal and neuroectodermal origin and is highly angiogenetic. After ELISA technique was established, saliva was collected from eight healthy individuals. Run in duplicate, 14 (87.5%) of the 16 samples investigated contained measurable amounts of bFGF. In the samples containing bFGF the concentration varied between 0.1 pg/mL and 8.4 pg/mL (mean concentration, 3.8 pg/mL; SD, 3.5). There was no correlation between age and sex and bFGF concentrations. It is therefore concluded that bFGF is present in human saliva and may even constitute a constant component. The physiological importance of this finding is discussed.