Cytomegalovirus detected by qPCR in iris and ciliary body of immunocompetent corneal donors.

BACKGROUND: Cytomegalovirus (CMV) can cause a wide panel of ocular infections. The involvement of CMV as a cause of anterior uveitis in the immunocompetent patient is recent and remains poorly understood. OBJECTIVE: To investigate the presence of CMV in anterior uveal tissues of immunocompetent corneal donors. STUDY DESIGN: We collected aqueous humor, iris, and ciliary body from both eyes of 25 donors died at the Limoges University Hospital between January 2020 and July 2021. CMV serology was determined for all patients from post-mortem blood sample. Ocular tissues were split in 2 fragments for qPCR and 2 for histological analysis. CMV genomes copies were quantified by Multiplex qPCR after DNA extraction. RESULTS: 16 of 25 patients (64%) displayed positive CMV serology, with a median age of 67 years. Viremia was positive in 3 of 16 (19%) CMV-positive patients. No CMV DNA copies were found from the aqueous humor samples. CMV DNA was detected in iris and ciliary body of 28 of 32 eyes of seropositive donors, and 5 of 18 eyes of seronegative donors. The median viral copy number [IQR] was 2.41 × 102 [8.91 × 101 - 1.01 × 103] copies/1 × 106 cells in the CMV-positive group and 0.00 [0.00 - 3.54 × 102] copies/1 × 106 cells in the CMV-negative group (p<0.001). Histology and immunohistochemistry did not reveal any CMV lesions from any sample. CONCLUSION: CMV DNA was found in iris and ciliary body of immunocompetent seropositive patients, but also, although less frequently, from seronegative donors. These results highlight mechanisms of infection, latency and reactivation of CMV in ocular tissues.

[Mesectodermal leiomyoma. Unusual tumor of the ciliary body]. / Leiomioma mesoectodérmico. Un tumor poco frecuente del cuerpo ciliar.

BACKGROUND: Mesectodermal leiomyoma is a benign tumor of smooth muscle of the ciliary body, which is derived from the neural crest. CLINICAL CASE: We report the case of a 35-year-old Mexican woman with visually impaired and blurred vision of the right eye of 2 months duration. The clinical and imaging presuntional diagnosis was adenoma of the non pigmented epithelium of the ciliary body and it was surgically resected. Microscopically, the tumor was composed of cells with round nuclei and scant cytoplasm without atypia or mitosis, arranged in a fibrillary background. The immunohistochemical markers for vimentin, muscle specific actin, smooth muscle actin and calponin were strongly positive in the cytoplasm of the neoplastic cells, while for glial fibrillary acidic protein and S-100 protein were negative in the same cellular population. CONCLUSIONS: Mesectodermal leiomyoma of the ciliary body is benign tumor of smooth muscle extremely rare in this location. Until now, there are just 25 previous reported cases in the literature and, the main differential diagnosis is uveal malignant melanoma, therefore some eyes were enucleated. The ultrabiomicroscopy, A and B-scan imaging studies are useful in the evaluation, however, is mandatory the microsocpic examination with routine and histochemical stains as well as the use of immunohistochemical markers such as vimentin, specific muscle actin, smooth muscle actin andcalponin to stablish the smooth muscle origin of this neoplasm, and rule out other malignant neoplams such as malignant melanoma.

Antecedentes: el leiomioma mesoectodérmico es un tumor benigno excepcional que se origina en el músculo liso del cuerpo ciliar y deriva de la cresta neural. Caso clínico: se comunica el caso de una mujer de 35 años, con disminución de la agudeza visual y visión borrosa de 2 meses de evolución en el ojo derecho. El diagnóstico presuncional clínico e imagenológico fue: adenoma del epitelio no pigmentado del cuerpo ciliar, por lo que se resecó quirúrgicamente. Microscópicamente, el tumor estaba formado por células de núcleos redondos de escaso citoplasma sin atipia ni mitosis, dispuestas en una matriz fibrilar. Los inmunomarcadores para vimentina, actina músculo específica, actina de músculo liso y calponina fueron todos positivos en el citoplasma de las células neoplásicas, excepto de los inmunomarcadores para la proteína ácida gliofibrilar y la proteína S-100 que resultaron negativos en la misma población celular. Conclusiones: el leiomioma mesoectodérmico del cuerpo ciliar es un tumor benigno de músculo liso extremadamente raro en esta localización. Hasta el momento, sólo hay 25 casos informados en la bibliografía médica y su principal diagnóstico diferencial es melanoma uveal, motivo por el que algunos ojos se enuclearon. Los estudios de ultrabiomicroscopia y ecografía modos A y B son útiles en la evaluación; sin embargo, es obligado el estudio microscópico con tinciones de rutina, y el uso de marcadores inmunohistoquímicos, como los utilizados en este caso para establecer la naturaleza del músculo liso de esta neoplasia y descartar algunas otras, como el melanoma.

Development and validation of a liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of latanoprost free acid in rabbit aqueous humor and ciliary body.

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (< 15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost.

Comparison of tight junction protein expression in the ciliary epithelia of mouse, rabbit, cat and human eyes.

Tight junctions in the nonpigmented epithelium (NPE) of the ciliary processes and the iris vascular endothelium form the ocular blood aqueous barrier that prevents leakage of proteins, immune cells and non-immune cells of blood into the anterior chamber. We attempted to determine whether ultrastructural differences in tight junctions reported in earlier studies are reflected in the expression pattern of tight junction proteins (TJP) and whether the TJP in mice, rabbits and cats resemble those of humans. For immunohistochemistry, 10 µm thick cryosections were rehydrated in PBS and fixed in 50 mM ammonium chloride at room temperature. After rinses in PBS, the sections were incubated twice in 0.1% Triton X-100, 10% goat serum, specific primary antibody or in PBS. After rinses in PBS, the sections were incubated in FITC-conjugated secondary antibody. After rinses in PBS, the sections were mounted with Vectashield mounting medium with propidium iodide, examined and photographed using a confocal microscope. The expression patterns of TJP in ocular ciliary epithelium of human, rabbit, cat and mouse were similar. Occludin immunoreactivity was observed as a sharp line along the junction between pigmented epithelium (PE) and NPE, and along the apico-lateral surfaces of NPE. Very light staining of the ciliary stroma was observed in cat and mouse. Claudin-1 was expressed along the entire boundaries of NPE and was more distinct between PE and NPE in rabbit. The ciliary stroma showed faint staining in cat and mouse. ZO-1 showed staining between PE and NPE, and at the adjacent membrane. Moderate staining was seen in PE in cat and mouse, which suggests that claudin-1, occludin and ZO-1 are expressed along the junction between PE and NPE, and the apico-lateral border of NPE. Lack of major difference in the expression patterns among the different species is important for validating the use of rabbit, mouse and cat in studies of intraocular inflammation in humans.

Simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor and ciliary body by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

A rapid, sensitive and selective method for the simultaneous quantification of carteolol and dorzolamide in rabbit aqueous humor (AH) and ciliary body (CB) has been developed and validated using reversed phase-high performance liquid chromatography (RP-HPLC) with isocratic elution coupled with atmospheric pressure chemical ionization mass spectrometry/mass spectrometry (APCI-MS/MS). The analytes and nadolol (used as internal standard, IS) were purified from AH by protein precipitation. The sample preparation from CB was based on a two steps extraction procedure at different pH, utilizing a liquid-liquid extraction with a mixture of ethyl acetate, toluene and isopropanol 50:40:10 (v/v) at pH 8, followed by a second extraction with ethyl acetate at pH 11. The combined organic extracts were then back extracted into 0.1% aqueous trifluoroacetic acid (TFA). The accuracy and precision values, calculated from three different sets of quality control samples analyzed in sestuplicate on three different days, were within the generally accepted criteria for analytical methods (<15%). The assay proved to be accurate and precise when applied to the in vivo study of carteolol and dorzolamide in rabbit AH and CB after single administration of an eye drops containing both drugs.

Is the positive c-kit immunostaining associated with the presence of cells analogous to the intersticial cells of Cajal in the ciliary muscle?

PURPOSE: Interstitial cells of Cajal were identified in the gastrointestinal tract of several species, with close relation to the enteric nervous system. Since it was recognized that interstitial cells of Cajal express the gene product of c-kit, we performed immunohistochemistry for c-kit protein in ciliary muscle specimens of monkeys' eyes. METHODS: Eight eyes from four adult male new world monkeys (Cebus apella) were studied. After blocking endogenous peroxidase activity and nonspecific protein binding, 1:100 dilution of mouse monoclonal antibody against c-kit human oncoprotein was applied to tissues. Antigen-antibody reaction was visualized using the avidin-biotinylated horseradish peroxidase complex in each slide. RESULTS: We observed some groups of fusiform c-kit expressing cells located amongst muscle bundles of the ciliary muscle. Other pigment cells and mast cells were also observed. CONCLUSION: C-kit expressing cells observed in the ciliary muscle of Cebus apella, showed no similarity to melanocytes or mast cells and they could be associated with their gastrointestinal interstitial cells of Cajal counterpart.

Adenoma of the non-pigmented ciliary epithelium: a rare intraocular tumor with unusual immunohistochemical findings.

A case of adenoma of the non-pigmented ciliary epithelium with smooth muscle differentiation is reported. This uncommon ocular tumor affected a 36-year-old woman, and had caused decreased visual acuity and a total cataract. Ultrasound biomicroscopy disclosed an associated persistent hyperplasic primary vitreous (PHPV). Sectoral cyclectomy with removal of the mass and intracapsular cataract extraction were performed. The tumor was diffusely positive for vimentin, smooth muscle actin, NSE, and S-100, focally for CD68 and Melan-A, and was negative for desmin, EMA, HMB-45, and CD99. Occasional cells reacted for cytokeratin. The proliferation index, as assessed by Ki-67, was below 10%. The overlying non-neoplastic ciliary epithelium was positive for vimentin, NSE, and S-100. Myofilaments are not totally unexpected in ciliary adenomas; however, such a diffuse and strong positivity for smooth muscle actin, as in the present case, has only been observed in one case before, but should be considered in the differential diagnosis of these neoplasms.

Is the positive c-kit immunostaining associated with the presence of cells analogous to the intersticial cells of Cajal in the ciliary muscle? / A imunomarcação positiva para c-kit está associada com a presença de células análogas às intersticiais de Cajal no músculo ciliar?

PURPOSE: Interstitial cells of Cajal were identified in the gastrointestinal tract of several species, with close relation to the enteric nervous system. Since it was recognized that interstitial cells of Cajal express the gene product of c-kit, we performed immunohistochemistry for c-kit protein in ciliary muscle specimens of monkeys' eyes. METHODS: Eight eyes from four adult male new world monkeys (Cebus apella) were studied. After blocking endogenous peroxidase activity and nonspecific protein binding, 1:100 dilution of mouse monoclonal antibody against c-kit human oncoprotein was applied to tissues. Antigen-antibody reaction was visualized using the avidin-biotinylated horseradish peroxidase complex in each slide. RESULTS: We observed some groups of fusiform c-kit expressing cells located amongst muscle bundles of the ciliary muscle. Other pigment cells and mast cells were also observed. CONCLUSION: C-kit expressing cells observed in the ciliary muscle of Cebus apella, showed no similarity to melanocytes or mast cells and they could be associated with their gastrointestinal interstitial cells of Cajal counterpart.

OBJETIVO: As células intersticiais de Cajal estão presentes no trato gastrintestinal de diversas espécies animais, em íntima relação com o sistema nervoso entérico. Uma vez que as células intersticiais de Cajal expressam o produto do gene c-kit, realizou-se um ensaio imuno-histoquímico a fim de se verificar a marcação da proteína c-kit no músculo ciliar de amostras de olhos de macacos. MÉTODOS: Oito olhos de quatro macacos do novo mundo (Cebus apella) foram estudados. Após bloqueio da peroxidase endógena e de ligação protéica não específica, os tecidos receberam aplicação de anticorpos de camundongos antioncoproteína c-kit humana (1:100). A reação antígeno-anticorpo foi verificada através da aplicação do complexo avidina-biotinilada-peroxidase em cada lâmina. RESULTADOS: Foram observados grupos de células que expressam c-kit, localizadas entre as fibras do músculo ciliar. Mastócitos e outras células pigmentadas também foram observadas. CONCLUSÃO: Algumas células que expressam c-kit, observadas no músculo ciliar de Cebus apella, não mostraram similaridade com mastócitos ou melanócitos e podem ser classificadas como análogas das células intersticiais de Cajal gastrintestinais.

A rapid and sensitive LC/MS/MS assay for the quantitation of brimonidine in ocular fluids and tissues.

We report here the development and validation of an LC/MS/MS method for the rapid and accurate quantitation of brimonidine in ocular tissues and fluids using brimonidine-d(4) as an internal standard (IS). Brimonidine was extracted from retina, iris/ciliary body, and vitreous humor samples with an acetonitrile:water (1:1) solution followed by sonication and vortexing. Aliquots of aqueous humor, iris/ciliary body, retina, and vitreous humor samples were diluted with acetonitrile containing IS and were separated on a reverse-phase HPLC column under isocratic conditions. Brimonidine (m/z transition: 292-->212) and the internal standard (m/z transition: 296-->216) were analyzed via multiple-reaction monitoring (MRM) in the positive electrospray mode on a 4000 Q TRAP instrument. The total analysis time for each sample was less than 2.0 min. The calibration curves for brimonidine (1-1000 ng/mL) were constructed using a linear regression with 1/x(2) weighing. The lower limit of quantitation for brimonidine was 1.0 ng/mL for aqueous humor, 10 ng/g for iris/ciliary body, 12.5 ng/g for retina, and 1.6 ng/g for vitreous humor. Intra-day and inter-day estimates of accuracy and precision were within 15% of their nominal values indicating that the method is reliable for quantitation of brimonidine in ocular tissues and fluids.

Unusual primary ocular neoplasm in a child: leiomyosarcoma of the ciliary body.

Primary uveal-tract neoplasms are extremely rare in childhood; the most common lesions found are melanocytic. We report here the case of a 7-year-old girl who underwent enucleation of the right eye with clinical suspicion of choroid melanoma as a result of a ciliary body mass that extended to the posterior chamber. Histologically, the neoplasm featured spindle cell morphology, atypia, and mitoses. The tumor expressed smooth muscle alpha actin, pan-actin HHF-35, and desmin, whereas immunohistochemistry for melanocytic markers, such as S-100, Melan-A, and HMB-45, was negative. Based on these features, the diagnosis of leiomyosarcoma of the ciliary body was firmly established. Although several leiomyomas have been reported in the literature, there are only 2 previously reported cases of primary leiomyosarcoma of the uveal tract. Immunohistochemical expression of muscle proteins allowed distinction from the most common melanocytic tumors arising in this location.

Medulloepithelioma masquerading as chronic anterior granulomatous uveitis.

CASE REPORT: We report a rare clinical case of unilateral ciliary body teratoid medulloepithelioma presented first with infantile cataract, subsequently masquerading as chronic granulomatous anterior uveitis, followed by appearance of a tumour over the iris surface. COMMENTS: Diagnosis of the tumour in the early stages allows proper management and avoids enucleation.

The differing embryonic origins of retinal and uveal (iris/ciliary body and choroid) melanosomes are mirrored by their phospholipid composition.

The phospholipids present in uveal (iris/ciliary body and choroid) and retinal bovine ocular melanosomes were identified using mass spectrometry. Similar phospholipid content is found for the two types of uveal melanosome, with sphingomyelin being the major species. Significant differences are found between the uveal and retinal melanosome. Glycerophosphoethanolamine (GPEtn) is the major species in the retinal pigment epithelium (RPE); 93% of the GPEtn contain polyunsaturated fatty acids, notably docosahexanoic acid and arachidonic acid, in the sn-2 position. RPE melanosomes also contain detectable quantities of glycerophosphoserine and glycerophosphate; these species were not detected in the uveal samples. While the structural and functional roles of melanosomal lipids largely remain to be determined, these different lipid compositions reported herein offer new insights into the roles of melanosomes in the different ocular tissues.

Dietary omega 3 fatty acids decrease intraocular pressure with age by increasing aqueous outflow.

PURPOSE: To determine whether there is an association between dietary omega-3 (omega-3) fatty acid intake, age, and intraocular pressure (IOP) caused by altered aqueous outflow. METHODS: Sprague-Dawley rats were fed either omega-3-sufficient (omega-3(+)) or omega-3-deficient (omega-3(-)) diets from conception. The diets had 7% lipid content. The omega-3(+) diet contained safflower, flaxseed, and tuna oils (5.5:1.0:0.5), and the omega-3(-) diet contained safflower oil only. Intraocular pressure was measured at 5 to 40 weeks of age under light anesthesia (omega-3(+), n = 39; omega-3(-), n = 48). Aqueous outflow was determined at 45 weeks in a subgroup of animals (omega-3(+), n = 15; omega-3(-), n = 22) using pulsed infusion. Ciliary body tissues (n = 6 per group) were assayed for fatty acid content by thin-layer and gas-liquid chromatography in both diet groups. RESULTS: Animals raised on omega-3(+) diets had a 13% decrease in IOP at 40 weeks of age (13.48 +/- 0.32 mm Hg vs. 15.46 +/- 0.29 mm Hg; P < 0.01). When considered as a change in IOP relative to 5 weeks of age, the omega-3(+) group showed a 23% decrease (P < 0.001). This lower IOP in the omega-3(+) diet group was associated with a significant increase (+56%; P < 0.001) in outflow facility and a decrease in ocular rigidity (-59%; P < 0.001). The omega-3(+) group showed a 3.3 times increase in ciliary body docosahexaenoic acid (P < 0.001). CONCLUSIONS: Increasing dietary omega-3 reduces IOP with age because of increased outflow facility, likely resulting from an increase in docosanoids. This indicates that dietary manipulation may provide a modifiable factor for IOP regulation. However, further studies are needed to consider whether this can modify the risk for glaucoma and can play a role in treatment of the disease.

Agonist and antagonist sensitivity of non-selective cation channel currents evoked by muscarinic receptor stimulation in bovine ciliary muscle cells.

1 In the bovine ciliary muscle, stimulation of muscarinic receptors with carbachol (CCh) opens two types of non-selective cation channels (NSCCS and NSCCL) with widely different unitary conductances (100 fS and 35 pS). Here we examined the dependence of the activity of NSCCS on the agonist (CCh) concentration by whole-cell voltage clamp in freshly isolated bovine ciliary muscle cells. We also examined the sensitivity of CCh-evoked NSCCS currents to several muscarinic receptor antagonists. 2 The voltage clamp experiments were carried out using Ba2+ as the charge carrier, as this divalent cation is the most permeant for NSCCS of the alkali and alkaline earth metal ions hitherto examined, whereas it is relatively impermeant to NSCCL. For the dose-activation relationship obtained, the apparent dissociation constant K was estimated to be 0.5 +/- 0.2 microm (n = 31), a value of an order of magnitude smaller than the one reported for CCh-evoked NSCCL currents in our previous experiments. 3 In the dose-inhibition experiments we observed that the CCh-evoked NSCCS currents were inhibited by the muscarinic antagonists with the following potency sequence: atropine approximately 4-DAMP >> pirenzepine > AF-DX116, indicating that the activation of NSCCS by CCh is mediated by an M3 muscarinic receptor. 4 We have previously shown by reverse transcriptase-polymerase chain reaction that the bovine ciliary muscle contains mRNAs for several transient receptor potential channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6) which are attracting attention as molecular candidates for receptor-operated NSCCs. In the present experiments, we succeeded in visually identifying these TRPCs in the plasma membrane of cultured bovine ciliary muscle cells by immunofluorescence microscopy.

Caveolin-1 is up-regulated in transdifferentiated lens epithelial cells but minimal in normal human and murine lenses.

Caveolae are flask-shaped invaginations of the plasma membrane found in many cell types. Caveolae play a role in lipid transport, endocytosis, signal transduction, and cell transformation. Expression of caveolin-1, the principal component of caveolae and a regulator of caveolae-dependent signaling and endocytosis, was investigated in lens epithelial cells and lens fiber cells in wild-type (wt) and SPARC-null mice and normal human donors in vivo and in vitro. RT-PCR, immunofluorescence and immunoblot analyses of human and murine ocular tissues revealed that caveolin-1 was expressed in the corneal epithelium, corneal endothelial cells, and blood vessels of iris, ciliary body and retina, but minimal in the normal lens epithelia or fiber cells of murine and human lens. In contrast, caveolin-1 was significantly up-regulated in mesenchymal-transdifferentiated lens epithelia in SPARC-null cataract lenses. In addition, lens epithelial cells from primary culture or from cultures of immortalized lens epithelial cell lines expressed significant amounts of caveolin-1. The lens epithelial cells expressed epidermal growth factor (EGF) receptor and were responsive to EGF-mediated cell proliferation, but they did not show EGF-dependent caveolin-1 tyrosine phosphorylation. Caveolin-1 might have a role in the process of epithelial-mesenchymal transdifferentiation (EMT) in the lens, the most common cause of vision loss in human secondary cataracts.

A topical nitric oxide-releasing dexamethasone derivative: effects on intraocular pressure and ocular haemodynamics in a rabbit glaucoma model.

BACKGROUND: Topical nitric oxide-releasing dexamethasone (NCX1021) may avoid the negative effects of dexamethasone phosphate. AIMS: To obtain more information on the role of nitric oxide in glaucoma and to compare a nitric oxide-releasing dexamethasone with dexamethasone phosphate with regard to intraocular pressure (IOP) and ocular haemodynamics in an experimental rabbit model. METHODS: Six rabbits were treated with dexamethasone phosphate 0.1% in the right eye and with NCX1021 in the left eye for 5 weeks. The parameters considered were IOP, nitric oxide marker levels in aqueous humour, ocular haemodynamics of ophthalmic artery (by means of colour Doppler imaging), expression of endothelial nitric oxide synthase (eNOS)in ciliary processes and histology of ciliary bodies. RESULTS: Dexamethasone increased IOP levels, NCX1021 did not. Nitrite and cyclic guanosine monophosphate levels in aqueous humour were lowered by dexamethasone and increased by NCX1021. Resistivity index of the ophthalmic artery was increased, eNOS expression was reduced and ciliary bodies showed histological lesions in dexamethasone-treated eyes, not in NCX1021-treated ones. CONCLUSIONS: NCX1021 may avoid the IOP increase, impairment of ocular blood flow and the morphological changes in the ciliary bodies possibly induced by corticosteroid treatment.

Characterisation of the prereceptor regulation of glucocorticoids in the anterior segment of the rabbit eye.

The prereceptor regulation of glucocorticoids (GCs) by 11beta-hydroxysteroid dehydrogenase type-1 (11beta-HSD1), a bidirectional isozyme that interconverts active (cortisol) and inactive (cortisone) GCs, is an established determinant of GC function in tissues such as liver, adipose and bone. Although the therapeutic use of GCs is abundant in ophthalmic practice, where GC interactions with nuclear receptors modulate gene transcription, the prereceptor regulation of endogenous cortisol is not well described in ocular tissues. Recent descriptive studies have localised 11beta-HSD1 to the human corneal epithelium and non-pigmented epithelium (NPE) of the ciliary body, indicating a link to corneal epithelial physiology and aqueous humour production. In this study, we characterise the functional aspects of the autocrine regulation of GCs in the anterior segment of the rabbit eye. Using our in-house generated primary antibody to human 11beta-HSD1, immunohistochemical analyses were performed on paraffin-embedded sections of whole New Zealand white albino rabbits, (NZWAR) eyes. As in human studies, 11beta-HSD1 was localised to the corneal epithelium and the NPE. No staining was seen in the albino 'pigmented' ciliary epithelium. Specific enzyme assays for oxo-reductase (cortisone-->cortisol) and dehydrogenase (cortisol-->cortisone) activity indicated predominant 11beta-HSD1 oxo-reductase activity from both the intact ciliary body tissue (n=12, median 2.1 pmol/mg per h and range 1.25-2.8 pmol/mg per h; P=0.006) and primary cultures of corneal epithelial cells (n=12, median 3.0 pmol/mg per h and range 1.0-7.4 pmol/mg per h, P=0.008) compared with dehydrogenase activity (median 1.0 pmol/mg per h and range 0.5-2.0 pmol/mg per h; median 0.5 pmol/mg per h and range 0.25-1.9 pmol/mg per h respectively). These findings were supported by expression of 11beta-HSD1 protein as visualised by Western blotting of ciliary body tissue and immunocytochemistry of corneal epithelial cells. Reduction of corneal epithelial cell proliferation was seen after primary cultures were co-incubated with cortisol and cortisone. 11beta-HSD1 activity was not demonstrated in naïve conjunctival fibroblasts or corneal stromal keratocytes. Our results indicate that the distribution of 11beta-HSD1 in the rabbit resembles that of the human eye and activates cortisone to cortisol in both corneal and uveal tissues. The NZWAR provides a suitable in vivo model for the further evaluation of 11beta-HSD1 activity in the eye, especially its role in corneal epithelial and ciliary body physiology.

Fas-ligand is stored in secretory lysosomes of ocular barrier epithelia and released with microvesicles.

Previously we described the release of hr44 from the ciliary epithelium to coincide with the loss of the late endosomal/lysosomal marker protein CD63 in mildly inflamed rat eyes. We showed that both proteins are released with microvesicles into the supernatant of cultured retinal pigmented epithelial cells (ARPE-19). Here we wish to determine whether there is a concomitant loss of fas-ligand (FasL) in vivo and whether ocular epithelial cells have secretory lysosomes similar to T cells, from where FasL and hr44 could derive. FasL plays an important role in immunity, immune cell homeostasis and in the maintenance of immune privilege in the eye. However the mode of release of FasL from ocular epithelial cells or its activity in the eye is not fully understood. In normal rat eyes, FasL was detected in the epithelia of the iris and ciliary body and in the anterior region of the retinal pigmented epithelium. FasL is expressed constitutively and is associated with vesicular structures in the normal ciliary epithelium but is not detectable in the ciliary epithelium of inflamed eyes. In contrast, the posterior RPE, which under normal conditions is negative for FasL and hr44 showed strong staining for both molecules in areas adjacent to sub-retinal inflammatory infiltrates. Immunofluorescence and Western blot analysis indicated that cultured ARPE-19 cells express both the soluble and membrane form of FasL. The intracellular concentration of FasL was significantly increased in cells grown in presence of interferon (INF)-gamma. The microvesicles released by cultured ARPE-19 cells and previously shown to be positive for hr44 and CD63 are also positive for membrane FasL. Expression of a recombinant fluorescent construct of FasL together with immuno-staining for CD63 demonstrated that FasL localises to the endocytic compartment of ARPE-19 cells and of melanoma cells (positive control). In cells with lysosomes devoid of specialised secretory functions (e g. HeLa cells) recombinant FasL localised to the cell membrane, demonstrating that RPE cells have secretory lysosomes. We suggest that ocular epithelial cells release soluble FasL and the membrane form of FasL with vesicles. Both forms may contribute in different ways to the effectiveness of the ocular immune response and immune privilege.

Immunolocalization of CYP1B1 in normal, human, fetal and adult eyes.

CYP1B1 is a cytochrome P450 enzyme implicated in autosomal recessive primary congenital glaucoma (PCG). The mechanism and function of CYP1B1 in the development of the PCG phenotype is unknown. Previously, investigators have reported detection of Cyp1b1 mRNA in the ciliary body and epithelium and neuroepithelium in the developing mouse eye, employing in situ hybridization techniques. Similarly, additional investigators have detected CYP1B1 mRNA in the iris, ciliary body, non-pigmented ciliary epithelial line, cornea, retinal-pigment epithelium, and retina in the human adult eye, using Northern blotting. This study was designed to immunolocalize CYP1B1 protein in the various ocular structures of normal, human fetal and adult eyes. Normal fetal and adult eyes were immunolabeled with a polyclonal antibody against human CYP1B1 using indirect immunofluorescence, and then compared with appropriate controls. The intensity of immunolabeling of the various ocular structures was assessed by qualitative and semi-quantitative techniques. In the anterior segment anti-CYP1B1 immunoreactivity (IR) was detected early in fetal development in the primitive ciliary epithelium. As well, the most intense CYP1B1 IR was in the non-pigmented ciliary epithelium. In addition, CYP1B1 IR was also present in the corneal epithelium and keratocytes, both layers of the iris pigmented epithelium, and retina. However, CYP1B1 IR was absent in the trabecular meshwork in all of the samples. In general, CYP1B1 immunolabeling in the human fetal eyes was more intense when compared to adult eyes. CYP1B1 IR was primarily immunolocalized to the non-pigmented ciliary epithelium and early in fetal development. In addition, CYP1B1 IR was not detected in the trabecular meshwork. These findings suggest that the abnormalities in the development of the trabecular meshwork in PCG may result from diminished or absent metabolism of important endogenous substrates in the ciliary epithelium due to non-functional CYP1B1 enzyme.