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.

Pan-European survey of the topical ocular use of cyclosporine A.

OBJECTIVE: To assess medical practices surrounding the use of topical ocular cyclosporine A across European Union nations. METHODS: Key stakeholders (ophthalmologists, hospital pharmacists, regulatory health authorities) from European Union member states were interviewed by telephone using a semi-structured, open-ended questionnaire. Ophthalmologists responded to questions about practice patterns of cyclosporine A use (prescription frequency, indication, dosage), pharmacists about cyclosporine A formulations (composition, manufacturing process, quality control, distribution), and the regulatory authorities about market authorization and pharmacovigilance for various cyclosporine A products. RESULTS: Over the years, cyclosporine A use for ophthalmic indications has increased across all European Union nations. Prevalence of cyclosporine A use was heterogeneous, with Belgium, France, Germany, Italy, Portugal, Spain and the United Kingdom reporting the highest frequency. Compounded cyclosporine A formulations and other cyclosporine A products were prescribed through temporary authorization on a compassionate use or named-patient basis. Cyclosporine A was prescribed for dry eye disease, atopic and vernal keratoconjunctivitis, corneal graft rejection, and other autoimmune and inflammatory diseases. Concentrations of prescribed topical cyclosporine A ranged between 0.05-2% and formulations were instilled 1-6 times daily. Interviewed stakeholders expressed concern regarding, (1) paucity of product information, (2) lack of standardized manufacturing processes and quality control of cyclosporine A formulations, and (3) poor regulation and pharmacovigilance of ocular cyclosporine A-based products. CONCLUSIONS: Medical practice surrounding ocular cyclosporine A use in European Union nations differs based on variations in concentration, dosage, prescription indication, formulation, availability and distribution, manufacturing, quality, and regulatory monitoring.

Antiangiogenic activity in tears: presence of pigment-epithelium-derived factor. New insights and preliminary results.

BACKGROUND/PURPOSE: Pigment-epithelium-derived factor (PEDF) is of major importance to prevent neovascularization of the retina. Recently found to occur in the cornea and conjunctival tissue, its presence in tears has this far not been reported. Initial results, based on the analysis of diluted tear fluid samples, even indicated the absence of this factor in human tears. Considering the clinical importance of PEDF as regulator of corneal vascularization, we investigated undiluted human tears. METHODS: Samples of 18 healthy individuals were collected and analyzed using a commercial ELISA. Samples were also collected from 5 patients with pterygium and as a positive control 2 samples of subretinal fluid from patients following retinal detachment surgery. RESULTS: PEDF concentrations were below the detection limit (i.e. <0.1 ng/ml) in the majority of samples from the healthy individuals. However, PEDF was discovered in 3 of the samples taken, with significant concentrations of 2, 32 and 53 ng/ml. In the group of 5 patients with pterygium, there were no detectable concentrations of PEDF. CONCLUSION: PEDF may be found in measurable amounts in human tear fluid of healthy individuals and may therefore play a role in the effects and regulation of PEDF at the ocular surface.

The triple classification of dry eye for practical clinical use.

PURPOSE: "Dry Eye is a condition produced by the inadequate interrelation between lacrimal film and ocular surface epithelium, and is caused by quantitative and qualitative deficits in one or both of them. It can be produced by one or combined etiologic causes, affecting one or several of the secretions of the glands serving the ocular surface, and producing secondary manifestations of different grades of severity". Clinicians need a practical classification to face diagnosis, prognosis and treatment. Dry eyes have many etiologies and pathogenesis, different affectation of the various dacryoglands and ocular surface epithelium, and diverse grades of severity. The specialists in xero-dacryology must know these three parameters to evaluate any case of dry eye, and to establish an adequate treatment. METHODS: To facilitate this, an open session in the 8th congress of the International Society of Dacryology and Dry Eye (Madrid, April, 2005) proposed modifying the Triple Classification of dry eye approved in the XIV congress of the European Society of Ophthalmology (Madrid, June, 2003). There was consensus of all conclusions. CONCLUSIONS: The following classification has been established: First, a classification of the etio-pathogenesis, distributed in ten groups: age-related, hormonal, pharmacologic, immunopathic, hyponutritional, dysgenic, infectious/inflammatory, traumatic, neurologic and tantalic. Second, a classification of the affected glands and tissues, which under the acronym of ALMEN includes the Aqueo-serousdeficient, Lipodeficient, Mucindeficient and Epitheliopatic dry eyes, and the Non dacryological affected exocrine glands (saliva, nasal secretion, tracheo-pharyngeal secretion, etc). And thirdly, a classification of severity, in three grades: Grade 1 or mild (symptoms without slitlamp signs), grade 2 or moderate (symptoms with reversible signs), and grade 3 or severe (symptoms with permanent signs).

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.

Cellular proliferation and leukocyte infiltration in the rabbit cornea after photorefractive keratectomy.

PURPOSE: To map the proliferative activity of corneal cells during wound healing following photorefractive keratectomy (PRK) and to compare two markers for proliferation. METHODS: PRK, 5- mm in diameter with a -6 D setting, was performed in one eye of 28 New Zealand White Rabbits. The rabbits were sacrificed at time points between 12 hours and three months after surgery. The treated and fellow corneas were fixed in 10% formaldehyde, paraffin embedded, and immunohistochemically stained for proliferate cell nuclear antigen (PCNA) and at one time point, 1 week, also for Ki-67. RESULTS: Following initial sliding of the epithelial cells, the proliferative activity in the wound area starts in the leading edge (24 hours) and is spread towards the periphery. The proliferative activity peaks after one week and subsides during the following two weeks. Early (24 hours) proliferative activity is also seen in the limbal epithelium which peaks after three days. The keratocytes express PCNA in the peripheral stroma 48 hours after injury. They then also migrate to repopulate the stroma under the wound area. The expression period lasts 1 week and subsides the following week. Leukocytes are found in the wound as early as 12 hours after injury. The cells disappear around the time of epithelial wound closure, i.e. after 3 days. The two proliferative markers PCNA and KI 67 show a similar distribution after surgery. CONCLUSION: Epithelial proliferative activity starts earlier after injury, and is preceded by leukocyte presence in the wound. The PCNA expression starts later in the keratocytes but lasts somewhat longer (3 weeks). PCNA expression appears more efficient than Ki-67 to show proliferative activity of slow cycling cells in the cornea

Anterior lenticonus: histological evaluation and approach for cataract surgery.

PURPOSE: To present 2 cases of anterior lenticonus in patients without Alport's syndrome, a surgical technique of cataract extraction in eyes with anterior lenticonus, and histological results of lenticonus specimens obtained intraoperatively. SETTING: From St. Eriks Eye Clinic, Karolinska Institute, Stockholm, Sweden. METHODS: Two otherwise healthy patients presented with anterior lenticonus but no history of Alport's or other pathology. Best corrected visual acuity (BCVA) was decreased. Both patients had cataract extraction by phacoemulsification with intraocular lens implantation under topical anesthesia. Two continuous curvilinear capsulorhexes (CCCs) were created. The entire lenticonus was embedded in formaldehyde buffer 4% for histological analysis. RESULTS: In the first patient, BCVA did not improve postoperatively because of amblyopia. The patient subjectively reported a substantial improvement in visual field clarity. The surgical and postoperative course in the other patient was uneventful. The sections were positive for collagen types IV and VI, and the arrangement of the collagen fibers was highly irregular. CONCLUSION: Anterior lenticonus was detected in patients without Alport's syndrome who were otherwise healthy. A modified 2-step CCC technique can be used to make cataract surgery in such eyes safe and relatively easy.

Basic fibroblast growth factor in nasal polyps immunohistochemical and quantitative findings.

Basic fibroblast growth factor (bFGF) is a mediator with potent mitogenic properties. Increased amounts of this mediator have been demonstrated in damaged lung tissue, and it has been suggested to increase the healing of gastro-duodenal ulcers. In order to quantify the amounts and document the localization of bFGF in nasal polyps, polyp tissue from 12 patients undergoing polypectomy was analyzed by ELISA and immunohistochemistry. Mucosa from the inferior turbinate was analyzed in the same manner for comparison. The amount of bFGF detected in polyp tissue was significantly higher than that in turbinate mucosa. The amount of bFGF was also significantly higher in the group of patients with high degree of inflammation. The immunohistochemical findings demonstrated abundant bFGF activity mainly in the glandular acini, in the epithelium, in infiltrating inflammatory cells and in the vessel walls. We propose that bFGF may contribute in a significant way to the formation of nasal polyps.

Enhanced short-term plasmid transfection of filtration surgery tissues.

PURPOSE: To quantify and localize plasmid transfection of filtration surgery tissues using two delivery techniques. METHODS: Full-thickness filtering procedures were performed on eyes of New Zealand albino rabbits. In 10 eyes, naked plasmid DNA in saline was either injected beneath Tenon's capsule at the filtration site or absorbed into a collagen shield that was then placed external to the sclerostomy and under the Tenon's capsule. Forty-eight hours after surgery, levels of the reporter gene, chloramphenicol acetyltransferase (CAT) were measured in samples of ocular tissues. In two additional eyes, the ss-galactosidase (ss-GAL:) reporter gene expression was localized histologically. RESULTS: Injection of plasmid DNA in saline vehicle into the filtration bleb produced readily detectable CAT activity in bleb tissue (conjunctiva, Tenon's capsule, and sclera) whereas CAT activity was nearly undetectable in samples of the cornea, iris-ciliary body, and tissues located opposite the bleb site. Delivery of the plasmid DNA into the bleb through a collagen shield increased CAT activity 30-fold over injection of plasmid in saline (2711 +/- 567 mU/mg versus 92 +/- 38 mU/mg). ss-Gal activity was imaged only in the region of the bleb, and microscopic examination showed ss-Gal activity localized to Tenon's capsule fibroblasts, with minimal ss-Gal activity observed in inflammatory cells or scleral fibroblasts. CONCLUSIONS: Transfection of filtration tissues is enhanced by absorption of naked DNA into a collagen shield. Furthermore, transfection is localized to the fibroblasts and inflammatory cells of the filtration bleb site. Gene therapy using naked plasmid DNA and a simple collagen shield delivery vehicle may be useful for regulating wound healing after glaucoma surgery.

Basic fibroblast growth factor (bFGF) in saliva--physiological and clinical implications.

Basic fibroblast growth factor (bFGF) has significant properties in wound healing, tissue regeneration and ulcer repair of the upper digestive tract. The purpose of the present study was to identify and analyse factors affecting the concentration of bFGF in saliva from healthy human individuals. A commercially available enzyme-linked immunosolvent assay kit (ELISA) was used for the analyses of bFGF in saliva. In total, 56 samples were collected from 28 healthy subjects, 15 male and 13 female. Determination of bFGF was performed by spectrophotometer (wavelength 490 nm). bFGF was detected in all samples. Mean bFGF concentration was 0.87 pg/ml (SD 0.49) and the concentration ranged from 0.3 to 1.9 pg/ml. In subjects aged 22 to 49 years, no age-dependent variation in bFGF was present, females did not differ from males, and no difference was evident between European and Asian subjects. Smokers had significantly higher saliva concentrations of bFGF than non-smokers. Since bFGF, together with other growth factors, is involved in wound healing and tissue repair, we suggest that bFGF in saliva is involved in the reparative processes of mucous membranes.

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.