Matthias Grueb

Muscarinic acetylcholine receptor subtypes in human corneal epithelium and endothelium.

BackgroundMuscarinic acetylcholine receptors are located throughout the body. The demonstration of muscarinic receptors in corneal tissue has been inconsistent. Using freshly fixed human corneal tissue, we show a complete profile of muscarinic receptor subtypes in human corneal epithelium and endothelium.MethodsMuscarinic receptor sites were studied using immunocytochemistry, immunofluorescence and immunoblotting.ResultsAntibodies to M2, M4 and M5 muscarinic receptor subtypes bound in human corneal epithelium and endothelium. No binding was found for antibodies to M1 and M3 muscarinic receptor subtypes.ConclusionsOur studies indicate the presence of M2, M4 and M5 muscarinic acetylcholine receptor subtypes in human corneal epithelium and endothelium. These receptors may play a role in the regulation of corneal homeostasis, other functions, like wound healing, or the pathogenesis of corneal diseases.

Adrenergic Regulation of cAMP/Protein Kinase A Pathway in Corneal Epithelium and Endothelium

Purpose: The G-protein-coupled receptor/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway is one of the most common and versatile signal pathways in eukaryotic cells. The aim of this study was to characterize subtypes of adrenergic G-protein-coupled receptors and their influence on cAMP concentration and PKA activity in bovine corneal epithelial and endothelial cells. Procedures: Adrenergic receptors and PKA were studied using polyclonal antibodies. cAMP concentration was determined with an enzyme immunoassay, and PKA activity was estimated by the kinases consumption of adenosine triphosphate. Results: In bovine corneal epithelial and endothelial cells, immunocytochemistry and Western blot were positive for α1-, α2A-, β1- and β2-adrenergic receptors. Stimulation of corneal epithelial and endothelial β-adrenoceptors with isoprenaline led to a dose-dependent increase in cAMP concentration and activation of PKA. Stimulation of corneal α2A-adrenoceptors with brimonidine resulted in a dose-dependent decrease in cAMP concentration and the inhibition of PKA activity. Conclusions: In corneal epithelial and endothelial cells, β-adrenergic stimulation leads to activation of PKA via stimulation of adenylyl cyclase, and α2A-adrenoceptor stimulation inhibits PKA activity via inhibition of adenylyl cyclase. Stimulation and inhibition of the corneal cAMP-PKA pathway may play a role in important corneal functions such as wound healing or homeostasis. Long-term therapy with α2A-agonists or β-antagonists may influence these functions in a currently unknown way.

Effect of brimonidine on corneal thickness.

PURPOSE Brimonidine, an alpha-2 adrenoceptor agonist, is an effective and safe medication that is widely used in glaucoma treatment. Although it is known that it is quickly taken up by the cornea following topical administration and that the cornea has alpha-2 adrenoceptors, there are only few studies available on the impact brimonidine has on the cornea. METHODS Twenty healthy test persons (12 female and 8 male subjects)-mean age about 33 years (22 to 38 years)-were tested in a double-blind, prospective, randomized study. Intraocular pressure as well as epithelial, stromal, and endothelial thickness was measured before, at 25 days while, and at 5 days after administration of brimonidine 0.1% eye drops twice daily. To check the impact of this medication, placebo (proper solution of preservative) eye drops were administered to the other eye twice daily. RESULTS Administration of brimonidine 0.1% resulted in a reduction of intraocular pressure from an initial value of 14 to 9 mmHg after 5 days (P=0.001) as well as an increase in total corneal thickness from 556 μm from the time of the baseline examination to 578 μm (P=0.001), an increase of epithelial thickness from 58 to 66 μm (P<0.001), and stromal thickness from 488 to 502 μm (P=0.008) after 2 days each. Another 2 days later, total corneal thickness was 559 μm (P=0.276), epithelial thickness 56 μm (P=0.561), and stromal thickness 493 μm (P=0.315), which means that the values had returned more or less toward the initial values measured. In contrast, endothelial thickness did not vary following administration of brimonidine 0.1% (P=0.965). With treatment with brimonidine 0.1%, mean intraocular pressure in thin corneas (<556 μm) was lower than in the thick corneas (>556 μm, P=0.018). CONCLUSIONS Topical administration of brimonidine 0.1% results in a reversible increase in corneal thickness. The question whether this increase is of clinical significance and whether it is the result of epithelial and/or endothelial receptor stimulation cannot be finally answered at the present time.

Bilateral ocular surface squamous neoplasia after wood dust exposure

To the Editor:Ocular surface squamous neoplasia (OSSN) includes conjunctival intraepithelial neoplasia with dysplasia, conjunctival carcinoma in situ and conjunctival squamous cell carcinoma (SCC). The incidence of OSSN is approximately 2 per 100 000 per year [1]. Documented cases of bilateral neoplasia/dysplasia are rare [2]. OSSN has been associated with ultraviolet B irradiation, human papillomavirus type 16 and 18, p53 gene overexpression, herpessimplex-virus type 1, trauma, flour, arsenic, tobacco, vitamin A deficiency, immunodeficiency, atopic keratoconjunctivitis and xeroderma pigmentosum [3 /5]. In sinonasal carcinomas wood dust and wood additives are discussed as carcinogenic factors [6 /10]. We report on a patient with bilateral OSSN associated with a 30 year exposure to wood dust. A 75-year-old man was referred with a one year history of a small tumour of the tarsal conjunctiva of the right lower eyelid and a large neoplasm of the tarsal conjunctiva of the left upper eyelid. Visual acuity was 20/25 on both eyes. He had a history of chronic obstructive pulmonary disease and radical prostatectomy due to prostate carcinoma with no evidence of recrudescence or metastatic disease in recent urologic controls. The patient reported a 30-year exposure to wood dust for professional reasons. A bilateral full thickness biopsy of both bilateral tumours was performed. Histopathology of the tumour of the right eye revealed a completely removed conjunctival squamous cell carcinoma with only slight invasion. Histopathology of the neoplasm of the left eye disclosed an invasive moderate differentiated conjunctival squamous cell carcinoma (Figure 1). On immunohistochemistry, the tumour of the conjunctiva presented with p53-protein overexpression, seen only in the nuclei of the carcinoma, which contrasts to the p53-negative epithelium in the vicinity (Figure 2). The polymerase chain reaction based assay proved negative for HPV-DNA in both of the bilateral tumours. Eight months after complete removal of the tumour the patient presented with an extensive relapse on the left eye, covering parts of the tarsal conjunctiva of the upper eyelid and the entire tarsal conjunctiva of the lower eyelid. He underwent full thickness excision with partly removal of the lower eyelid. Histopathologically, a recurrent moderately differentiated squamous cell carcinoma of the conjunctiva was found, which could be excised completely. After one year follow up no recrudescence occurred. Documented cases of bilateral ocular surface squamous neoplasia (OSSN) are rare [2]. OSSN in general and bilateral cases in particular have mainly been associated with ultraviolet B irradiation, human papillomavirus infection and p53 gene overexpression [3 /5], with the p53 overexpression generally being more indicative of an altered cell cycle control than being an etiopathogenetic cause. We report on a patient with bilateral OSSN with a history of a 30 year exposure to wood dust showing p53 gene overexpression. A large excess risk for sinonasal carcinomas has been documented among workers in wood-related occupations, particularly adenocarcinomas, and workers exposed to wood additives, particularly squamous cell carcinomas [6 /10]. However, the pathophysiologic mechanisms of the carcinogenesis are unknown. In our patient the lack

Amphotericin B in the therapy of Candida glabrata endophthalmitis after penetrating keratoplasty.

Purpose: Candida glabrata is a rare cause of endophthalmitis after penetrating keratoplasty. Adequate therapy is still under discussion. With respect to severe complications and side effects of antifungal therapy, a substantial knowledge of sensitivity and resistance of the organism is necessary. Methods: We report on a 26-year-old man with a hyperacute onset of the infection only 10 hours after surgery. A combined therapy for fluconazole and steroids administered over 3 months had shown no effect on intraocular infection. Results: After topical and intracameral application of amphotericin B in combination with topical prednisolone 3 months after the onset of the endophthalmitis, the infection disappeared within 14 days, and the graft remained clear for 2 months. No toxic effects were noticed. Conclusion: In the case presented here, topical and intracameral application of amphotericin B was sufficient and safe in the therapy for C. glabrata endophthalmitis after penetrating keratoplasty. Although typically the intraocular infection is first noticed within the first 2 weeks, a hyperacute onset has to be considered.

Effect of Timolol on Central Corneal Thickness

Purpose Timolol is an effective and safe medication that is widely used in glaucoma treatment. Although it is known that it is quickly taken up by the cornea following topical administration and that the cornea exhibits β-adrenergic receptors, there are few studies available on the clinical impact of timolol on central corneal thickness (CCT). Methods Twenty healthy subjects were tested in a double-blind, prospective, and randomized study. Intraocular pressure (IOP) and CCT were measured before and during administration of timolol 0.5% eyedrops over 28 days. Results Administration of timolol 0.5% resulted in a reduction of IOP from an initial value of 16 ± 2 mm Hg to 13 ± 0 mm Hg (p<0.001, R2 = 0.7033) as well as an increase in CCT from 555 ± 11 μm from the time of the baseline examination to 567 ± 9 μm (p = 0.005, R2 = 0.8754), an increase of epithelial thickness from 53 ± 2 μm to 59 ± 3 μm (p<0.001, R2 = 0.5063), and an increase of stromal thickness from 494 ± 4 μm to 498 ± 9 μm (p = 0.045, R2 = 0.4352) after 9 days each. From day 10 on, a decrease in CCT (R2 = 0.6164), epithelial thickness (R2 = 0.2216), and stromal thickness (R2 = 0.2092) was observed. At the end, the values had returned toward the initial values measured (CCT 553 ± 8 μm, p = 0.391; epithelial thickness, 50 ± 2 μm, p = 0.214; and stromal thickness, 493 ± 8 μm, p = 0.483). In contrast, endothelial thickness did not vary following administration of timolol 0.5% (p = 0.727, R2 = 0.009). Conclusions Topical administration of timolol 0.5% results in a reversible increase in CCT. These modest changes are unlikely to influence tonometry or clinical decision-making.

Muscarinic cholinoceptor-stimulated phosphatidyl inositol pathway in corneal epithelial and endothelial cells

BackgroundMuscarinic cholinoceptors are distributed widely in both the central and peripheral nervous system. The presence of muscarinic cholinoceptors in corneal tissue is well established. Previous reports have shown that corneal muscarinic cholinoceptors are of the m2 or m4 subtype. However, recent studies have indicated the presence of the m5 muscarinic cholinoceptor subtype in human corneal epithelium and endothelium. The aim of the study was to confirm the presence of the m5 cholinoceptor subtype in bovine corneal epithelium and endothelium and the activation of phosphatidyl inositol pathway by its stimulation.MethodsMuscarinic m5 cholinoceptor sites, phosphatidyl inositol 4,5-biphosphate, inositol 1,4,5-triphosphate and protein kinase C, were studied using immunocytochemistry and immunofluorescence. Activation of protein kinase C after stimulation of the m5 muscarinic cholinoceptor subtype was measured using the HTS protein kinase C assay kit.ResultsImmunocytochemistry/immunofluorescence revealed the presence of the m5 muscarinic cholinoceptor subtype, phosphatidyl inositol 4,5-biphosphate and protein kinase C in bovine corneal epithelial and endothelial cells. In bovine corneal epithelium and endothelium, protein kinase C activity was stimulated by acetylcholine in a dose-dependent manner (P<0.0001).ConclusionsOur findings indicate that acetylcholine-induced stimulation of muscarinic m5 cholinoceptors activates the phosphatidyl inositol pathway in corneal epithelial and endothelial cells, resulting in increased protein kinase C activity. Further work will be needed to clear the physiologic role of this signaling pathway in corneal epithelium and endothelium.

Serotonin (5-HT7) Receptor-Stimulated Activation of cAMP-PKA Pathway in Bovine Corneal Epithelial and Endothelial Cells

Background: 5-Hydroxytryptamine (5-HT; serotonin) is a major neurotransmitter, and its receptors are found throughout the whole body. The 5-HT7 receptor subtype was detected in human corneal epithelial and endothelial cells and found to be functionally active in a corneal epithelial cell line. The aim of the present study was to demonstrate that native bovine corneal epithelial and endothelial cells express a functional 5-HT7 receptor positively coupled to adenylyl cyclase and protein kinase A (PKA) formation. Methods: 5-HT7 receptors were studied using polyclonal antibodies. cAMP concentration after 5-HT7 receptor stimulation with 5-carboxamidotryptamine (5-CT, a 5-HT7 agonist) was tested by enzyme immunoassay, PKA activity was estimated by kinase consumption of ATP. Results: Immunocytochemistry and immunofluorescence revealed the presence of 5-HT7 receptors in corneal epithelial and endothelial cells. Stimulation of corneal 5-HT7 receptors with 5-CT revealed a dose-dependent increase in intracellular cAMP concentration in corneal epithelium (0.01–0.34 pmol/ml) and endothelium (0.01–0.19 pmol/ml) between 10–10 and 10–7 mg/ml 5-CT (p = 0.001) with maximal stimulation from 10–7 to 10–3 mg/ml 5-CT (0.30 ± 0.03 and 0.18 ± 0.01 pmol/ml, respectively). Incubation with 10–6 mg/ml SB269970 (a selective 5-HT7 antagonist) blocked 5-CT-induced cAMP increase in corneal epithelial (0.03 pmol/ml) and endothelial cells (0.02 pmol/ml; p = 0.001). Stimulation of corneal 5-HT7 receptors with 5-CT revealed a dose-dependent increase in PKA activity between 10–10 and 10–8 mg/ml 5-CT in corneal epithelium and endothelium (<1 to >99%; p = 0.013 and p = 0.017, respectively) with maximal stimulation from 10–8 to 10–4 mg/ml (>99%) 5-CT. Conclusions: Our data demonstrate that native corneal epithelial and endothelial cells express a functional 5-HT7 receptor positively coupled to adenylyl cyclase and PKA formation. However, at the present time, the physiological role of 5-HT receptors and the cAMP-PKA pathway in the cornea remains a matter of speculation.

International Uveitis-Expert-Network: a web based platform on the Internet

The management of chronic diseases, e.g. uveitis, often needs a lot of experience, expert knowledge and skill. As diagnosis and therapy of rare, chronic diseases are difficult, communication between experts and updates to the latest treatment options are necessary. The Internet, with its various possibilities, provides the opportunity to establish an international data exchange network in the field of uveitis where a group of experts can discuss difficult cases in a protected forum. By integrating the advantages of the World Wide Web and services such as e-mail and discussion groups, we have created a platform for easy and effective international information exchange in uveitis. This relates to data concerning scientific information as well as the introduction and discussion of difficult cases. The platform is a regular website programmed in Hypertext Markup Graefe’s Arch Clin Exp Ophthalmol (2002) 240:593