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The mRNA expression of cytokines and their receptors in cultured iris pigment epithelial cells: a comparison with retinal pigment epithelial cells.

It has been suggested that human iris pigment epithelial (IPE) cells isolated from iridectomized tissue could be used as autologous cells for transplantation into the subretinal space in diseases with dysfunctional retinal pigment epithelium (RPE). RPE cells synthesize a number of cytokines and their receptors which are important for its proper function. Nearly nothing is known about the capacity of IPE to synthesize cytokines or responding to them. To compare the mRNA expression of 36 cytokines or their receptors in cultured adult IPE cells and RPE cells we used semi-quantitative reverse transcription polymerase chain reactions (RT-PCR). Included in our assay were cytokines with known expression in RPE to get a broad basis for comparing IPE cells: basic fibroblast growth factor (bFGF or FGF-2), and one of its receptor (FGFR-1), epidermal growth factor (EGF), and its receptor EGF-R, transforming growth factor beta(TGFbeta), and its type III receptor TGFbeta-R3, the platelet-derived growth factors and receptors (PDGF A, PDGF B, PDGF-Ralpha, PDGF-Rbeta), tumor necrosis factor alpha(TNFalpha), and two receptors TNF-R1 and TNF-R2, insulin (INS) with receptor INS-R, insulin-like growth factors (IGF1, IGF2), and receptors (IGF1-R, IGF2-R), vascular endothelial growth factor (VEGF), and two receptors (VEGF-R1 or FLT-1 and VEGF-R2 or FLK-1), the receptor for VEGF-C: VEGF-R3 or FLK-4, interleukin 6 (IL6), and its receptor (IL6-R), nerve growth factor (NGF), interleukin 1alpha(IL1alpha), and a receptor (IL1-R). In addition, cytokines or their receptors not known to be expressed in RPE were included to widen our picture of cytokine gene expression in the eye: stem cell factor (SCF), its receptor (SCF-R), low-affinity nerve growth factor receptor p75 (p75(NGF-R), ciliary neutrothropic factor (CNTF), and its receptor (CNTF-R), glycoprotein 130 interleukin 6 transducer (gp130 (IL6-SD), leukemia inhibitory factor (LIF), and its receptor (LIF-R). Semi-quantitative expression data were obtained using series of fivefold dilutions of each cDNA and a fixed number of PCR cycles. The expression of RPE 65, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta2-microglobulin (B2MG) was used as a control for cellular origin, RNA quality and PCR conditions. With the exception of insulin and tumor necrosis factor alphaall other cytokines analysed and their receptors were expressed in both IPE and RPE cells, even though the levels varied. No qualitative or quantitative difference were observed in the mRNA expression level of 34 (94%) of the cytokines or receptors between IPE and RPE. In contrast, the mRNA expression level of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 [VEGF-RS (FLK-1)] was lower in IPE than in RPE cells. As an increased expression of VEGF in the RPE in maculae with age-related macular disease could be involved in its pathogenesis, a decreased expression of angiogenic growth factors in IPE cells could possibly be beneficial for the therapy of age-related maculopathy if indeed other tasks of non-functional RPE cells could be performed by IPE cells. The similarity of the mRNA expression pattern in 94% of the cytokines analyzed supports the assumption that IPE cells potentially can perform functions of RPE cells in the appropriate environment.

Adult↗

Comparison of tight junction permeability for albumin in iris pigment epithelium and retinal pigment epithelium in vitro.

BACKGROUND: The degenerative retinal diseases are one of the major causes of visual loss in the western world. Although heterologous RPE transplants rescue the photoreceptors in the dystrophic rat model, rejection remains a major limiting factor. Given the common embryonic origin, iris pigment epithelial (IPE) cells might be able to take over the functions of retinal pigment epithelial (RPE) cells, serving as an autologous graft for transplantation and thereby preventing rejection. One of the main functions of RPE cells is the generation of tight junctions which form the outer blood-retinal barrier. In this study we compared the tight junction permeabilities of IPE and RPE cells isolated from Long Evans rats by measuring their albumin clearances. METHODS: IPE and RPE cells were cultured on semipermeable filter supports with and without the addition of 0.02% ethylenediaminetetraacetic acid (EDTA). At selected intervals, the albumin clearances of the IPE and RPE cells were measured spectrophotometrically and compared. The morphology of the cells was compared using electron microscopy and fluorescent labeling. RESULTS: IPE and RPE cells both restricted the passage of albumin in vitro. After the modulation of tight junctions with 0.02% EDTA, the clearance increased in both types of cells in a similar fashion. The morphology of tight junctions was visualized with electron microscopy. CONCLUSION: These results indicate that the functional barrier for macromolecules is similar in IPE and RPE cells in vitro. This raises the possibility that IPE cells would form tight junctions in the subretinal space, thereby substituting for the blood-retinal barrier normally formed by RPE cells.

Actins↗

Investigation of possible metabolism of pigment yellow 17, a 3,3'-dichlorobenzidine-based pigment, after inhalation exposure in rats.

Rats were exposed by inhalation to the technically highest administrable concentration of 230 mg Pigment Yellow 17/m3 air for 4 h. Inhalability of the dust was guaranteed by a mass-median aerodynamic diameter of 1.0-1.1 microns. For 14 days after exposure, urine and serum samples were analysed for 3,3'-dichlorobenzidine, the parent carcinogenic amine of the test compound. No 3,3'-dichlorobenzidine could be detected either in urine or blood, the detection limit being 5 ng/ml for both media. Based on the results of this study there is no evidence for metabolic cleavage of Pigment Yellow 17 to 3,3'-dichlorobenzidine in the rat.

3,3'-Dichlorobenzidine↗

[Correlation between dermoscopy and histopathology in pigmented and non-pigmented skin tumours].

Dermoscopy (dermatoscopy, epiluminescence microscopy, surface microscopy) is a non-invasive method in dermatology. With this method pigmented and non-pigmented skin tumours can be diagnosed with a clear higher sensitivity and specificity compared to clinical examination. Malignant skin tumours are detected earlier and unnecessary excisions of benign skin tumours can be avoided. The knowledge about the correlation between dermoscopy and histopathology is hereby an essential condition. In this continuing medical education article the correlation of both examination techniques is presented for the dermoscopic differential diagnoses.

Basal Cell Carcinoma↗

Human and porcine anterior lens capsule as support for growing and grafting retinal pigment epithelium and iris pigment epithelium.

PURPOSE: To establish a method for transplantation of cultured monolayers of RPE and IPE into the subretinal space, anterior lens capsule was evaluated for its suitability to serve as growth support and carrier for transplantation procedures. MATERIALS AND METHODS: Twenty-four anterior lens capsules were obtained from porcine eyes. The same number of human lens capsules was obtained during cataract surgery. Six lens capsules of each species were stored at -80 degrees C. Subsequently, the capsules were transferred onto type-I collagen. A second set of six lens capsules was treated identically except for the cryo treatment. A third set of six capsules was initially exposed to 0.05% trypsin for 30 min. Suspended porcine RPE and IPE cells (5 x 10(4) cells/well) were seeded on the top of each capsule. The remaining six lens capsules served as controls and were incubated in uncoated 12-well dishes without undergoing experimental treatment. The cultures were maintained in a water-saturated atmosphere at 37 degrees C with 5% CO(2). Six days later, viability, morphology, and cell density were determined. The capsules covered by a confluent monolayer of cells were transferred into uncoated wells and cultivated for another 10 days. At the end of the experiment, light and phase-contrast microscopy was performed on all capsules. RESULTS: Storage at -80 degrees C and exposure to trypsin resulted in significant reduction of cellular contamination. The highest cell density was found after 5 days when capsules which had undergone cryopreservation or trypsin exposure served as support for RPE and IPE. The pigment cell layer was firmly attached to the capsules and permitted a transfer to other culture flasks without significant cell loss. The IPE cell layer remained confluent after transfer to uncoated culture flasks, while the RPE cell layer ceased to proliferate 10 days after transfer. CONCLUSIONS: Lens capsules may be suitable for growing and supporting monolayers of pigment epithelial cells. Especially IPE cells formed stable monolayers on anterior lens capsules which could be transferred to secondary culture flasks without inflicting damage on the cells.

Animals↗

Clinical experience with the PLDL-1 (Pigmented Lesion Dye Laser) in the treatment of pigmented birthmarks: a preliminary report.

Twenty patients aged between 2 years and 17 years (mean 9 years) with pigmented birthmarks, mainly of the head and neck, were treated with the PLDL-1 laser (Pigmented Lesion Dye Laser--Candela Corporation, Wayland, Massachusetts, USA) which emits light with a wavelength of 510 nanometers and a pulse duration of 300 +/- 50 nano-seconds. Nine patients (45%) showed excellent results after a test-patch was performed. Two patients (10%) showed some lightening of colour after initial test-patch. Six patients (30%) showed no improvement and 3 patients (15%) showed some hyperpigmentation at the test-patch sites which had not disappeared at 6 months follow-up. There was no change in the clinical behaviour at 6 months follow-up and no evidence of scarring was encountered.

Adolescent↗

Photoreactivation in pigmented and non-pigmented extreme halophiles.

The sensitivity to ultraviolet radiation (254 nm) and the photoreactivability of four pigmented and three colourless strains of the extremely halophilic bacteria Halobacterium cutirubrum and Halobacterium salinarium have been studied. The results with three pigmented/non-pigmented pairs show that the pigments play an accessory role in photoreactivation at low visible light intensities and confirm that they do not provide passive protection against ultraviolet light. Evidence is presented that photoreactivation plays an unexpected direct role in the resistance of extreme halophiles to ultraviolet radiation and that colourless mutants of H. cutirubrum NRC 34001 only arise in cultures that have been both ultraviolet-irradiated and photoreactivated. None of these extreme halophiles is capable of excision repair of ultraviolet damage to DNA.

DNA Repair↗

Pigmented cutaneous papillomatosis (pigmented epidermal nevus) in three pug dogs; histopathology, electron microscopy and analysis of viral DNA by the polymerase chain reaction.

Canine pigmented epidermal nevus (CPEN) is a skin disorder of some breeds of dog characterized by multiple black plaques of the haired and non-haired skin. Three cases of pigmented cutaneous papillomatosis (previously described also as CPEN) in pug dogs were investigated histopathologically, immunohistochemically and electron microscopically. Additionally, DNA analyses with the polymerase chain reaction (PCR) were performed in two cases. Many nuclei of the stratum granulosa were diffusely immunolabelled for specific structural antigens of bovine papillomavirus (subgroup A), but nuclear inclusion bodies were not detected by retrospective examination of haematoxylin and eosin-stained sections of the affected skin. Aggregates of small numbers of viral particles (ranging from 37 to 43 nm in diameter) with a hexagonal structure were sparsely scattered throughout the nuclei of some of the superficial keratinocytes. PCR amplification targeted for the L1 gene of papillomavirus cloned from a case of CPEN yielded an expected fragment of 194-bp in the two CPEN cases examined but not in a case of canine oral papilloma.

Animals↗

Mechanically stripped pigmented and non-pigmented epithelium of the shark ciliary body: morphology and transepithelial electrical properties.

Sections of intact ciliary epithelium and mechanically stripped non-pigmented (NPE) and pigmented (PE) cell layers of adult sharks (Squalus acanthias) were mounted in Ussing-type chambers (area 0.1 cm2). Addition of 10(-5) M forskolin to the aqueous side of intact epithelium significantly increased short-circuit current (Isc) within 15 min and a maximum of approx. 30 microA cm-2 was reached after 45-60 min. Transepithelial potential difference (V) increased from -0.8 mV (aqueous side negative as compared with blood/stromal side) to -1.5 mV, whereas resistance (R) was unchanged (50 omega cm2). Forskolin was without effect when applied to the blood side. In stripped PE preparations (R 15 omega cm2), 10(-5) M forskolin applied to the apical side induced a qualitatively similar change of Isc and V compared with the intact tissue. The forskolin-induced effects were fully reversed by 10(-4) M bumetanide and were not dependent on pretreatment of the tissue with 10(-3) M BaCl2. In stripped NPE preparations resistance was usually less than 10 omega cm2 and was not stable. This is consistent with the morphologic observation that although tight junctions were still demonstrable in stripped NPE cells, the apical membranes were damaged. In preparations taken for light and electron microscopy the stripped PE layer revealed intact epithelial cells. In particular, the basal thirds of the stripped PE cells were in very close contact with each other. These attachment zones may have the appearance of tight junctions. Thus the PE cells of the shark ciliary epithelium can be successfully isolated for transepithelial transport studies. The adenylate cyclase system is present in PE cells, and transepithelial transport of chloride may be regulated by intracellular cAMP.

Animals↗

Orientational properties of biological pigments in ordered systems studied with polarized light: photosynthetic pigment-protein complexes in membranes.

A discussion is presented of the problems involved in the interpretation of linear dichroism and fluorescence depolarization experiments on macroscopically ordered membrane systems. Particular attention has been paid to ordered membranes containing photosynthetic pigment-protein complexes, but the mathematical treatment can equally well be applied to other systems. The information about the orientational properties of the pigments is obtained by the application of the theories developed for the characterization of the molecular orientational order in liquid-crystalline materials. It is shown that while linear dichroism only yields the order parameter S mu of the absorption transition moment, fluorescence depolarization experiments yield in addition the order parameter Sv of the emission transition moment as well as three orientational correlation functions of the two transition moments. It is argued that in general the latter information can only be obtained on utilizing a number of experimental scattering geometries. In particular, the merits of angle-resolved experiments are illustrated.

Biophysical Phenomena↗

Cone pigment polymorphism in New World monkeys: are all pigments created equal?

Most platyrrhine monkeys have a triallelic M/L opsin gene polymorphism that underlies significant individual variations in color vision. A survey of the frequencies of these polymorphic genes suggests that the three alleles occur with equal frequency among squirrel monkeys (subfamily Cebinae), but are not equally frequent in a number of species from the subfamily Callitrichinae. This departure from equal frequency in the Callitrichids should slightly increase the ratio of dichromats to trichromats in the population and significantly alter the relative representation of the three possible dichromatic and trichromatic phenotypes. A particular feature of the inequality is that it leads to a relative increase in the number of trichromats whose M/L pigments have the largest possible spectral separation. To assess whether these trichromatic phenotypes are equally well equipped to make relevant visual discriminations, psychophysical experiments were run on human observers. A technique involving the functional substitution of photopigments was used to simulate the discrimination between fruits among a background of leaves. The goal of the simulation was to reproduce in the cones of human observers excitations equivalent to those produced in monkey cones as the animals view fruit. Three different viewing conditions were examined involving variations in the relative luminances of fruit and leaves and the spectrum of the illuminant. In all cases, performance was best for simulated trichromacies including M/L pigments with the largest spectral separation. Thus, the inequality of opsin gene frequency in Callitrichid monkeys may reflect adaptive pressures.

Animals↗

Influence of tyrosinase levels on pigment accumulation in the retinal pigment epithelium and on the uncrossed retinal projection.

To study the relationship among tyrosinase activity, melanin production, and the routing of retinal ganglion cell (RGC) axons at the optic chiasm, we analysed mice with varying doses of the tyrosinase gene. These include the dark-eyed albino (Tyrc44H), a radiation-induced hypomorphic allele of tyrosinase; and transgenic mice carrying 1 or 2 alleles of a tyrosinase minigene on both wild-type (Tyr+) and albino (Tyrc) backgrounds. Melanization of the retinal pigment epithelium (RPE) occurred gradually even at <2% wild-type tyrosinase activity and was sensitive to tyrosinase activity up to <35% of wild-type levels, beyond which melanin synthesis appeared to be saturated. Overexpression of tyrosinase led to tyrosinase activity above wild type level, but did not increase melanin production. Although a loss of melanin because of a mutation in tyrosinase is associated with a decrease in the number of uncrossed fibers, elevating tyrosinase levels does not appear to cause an increase in the size of the uncrossed retinal projection. Our results suggest that replacing less than 35% of wild-type tyrosinase activity is sufficient to restore normal pigmentation of the RPE, and potentially, to allay visual defects.

Albinism, Oculocutaneous↗

A reliable radiochromatographic assay technique for hepatic microsomal 16 alpha-hydroxylase activity towards oestrone 3-sulphate. Comparison between pigmented and non-pigmented mature guinea pigs.

A reliable procedure for the assay of liver microsomal 16 alpha-hydroxylation of oestrone 3-sulphate has been developed for the guinea pig. It is based on the rapid, quantitative separation of oestradiol and oestriol by Sephadex LH-20 columns after the chemical reduction and enzymic hydrolysis of the incubation products. Microsomal preparations and incubation conditions that optimized 16 alpha-hydroxylation of oestrone 3-sulphate were employed. Under these circumstances, reduction of the substrate at C-17 and hydrolysis of the sulphate were minimized. Conditions were established that yielded reaction linearity with respect to time and microsomal concentration. This hydroxylation had an absolute requirement for NADPH, which could not be satisfied by NADH. Apparent Km values for oestrone 3-sulphate and NADPH, under the conditions used, were 14 microM and 0.17 mM respectively. 16 alpha-Hydroxylase activity was present in the liver microsomal fraction from heavily pigmented, female English Shorthaired guinea pigs. Much lower activity was detected in mature pigmented males and albino females. No activity could be demonstrated in mature, albino males.

Animals↗

Can we safely diagnose pigmented lesions from stored video images? A diagnostic comparison between clinical examination and stored video images of pigmented lesions removed for histology.

Rapid expansion of communication technology has permitted the clinician to perform a consultation with a patient located at a different site. Assuming adequate diagnostic accuracy, it could theoretically be possible to use telemedical techniques as a triage tool. Images of pigmented lesions sent by the primary care physician could be viewed by the consultant dermatologist, and those with banal lesions spared from attending clinic. Previous studies assessing diagnostic accuracy of images of lesions have used 'face-to-face' diagnoses as the 'gold standard'. We set out to compare diagnostic accuracy of image examination compared with that of clinical examination, using histological examination as the diagnostic benchmark. We found that pigmented lesions may be diagnosed as accurately by stored video image evaluation as by conventional clinical examination. None of the malignant skin tumours was misdiagnosed as benign in either group. Whilst these results are encouraging in terms of the clinical safety of store-and-forward imaging, the inability to examine the whole patient or to palpate the lesions may limit the acceptability of the technique severely. Further evaluation of the cost : benefit ratio of such a system to the health care provider must be undertaken before considering this technique as a potential adjunct to managing outpatient referrals.

Adolescent↗

Tumor-associated retinal pigment epithelial proliferation simulating retinal pigment epithelial tear.

A lesion with both the clinical and fluorescein angiographic appearance of the classical retinal pigment epithelial (RPE) tear was discovered over the dome of an actively growing metastatic choroidal tumor in a patient with a previous history of breast carcinoma. However, our patient exhibited no evidence of pigment epithelial detachment or age-related macular degeneration, the underlying cause of the RPE tear. Although we cannot be sure that a true RPE tear did not exist over our patient's tumor, more likely, we are observing a tumor induced zone of RPE dehiscence accompanied by RPE proliferation at its border, giving a very similar fundus appearance. Possible pathogenic mechanisms for the finding in our patient, and a comparison to those mechanisms responsible for the classical RPE tear, are discussed.

Bone Neoplasms↗

Clinical predictors of malignant pigmented lesions. A comparison of the Glasgow seven-point checklist and the American Cancer Society's ABCDs of pigmented lesions.

Reducing morbidity and mortality from malignant melanoma is the greatest challenge facing dermatology today. We present a comparison of the Glasgow seven-point checklist and the American Cancer Society's ABCDs of pigmented lesions for detecting early melanomas. Logistic regression analysis showed that the Glasgow checklist (N = 205 lesions) contained two significant variables (irregular outline, P = .001, relative odds = 10.9; diameter greater than 1 cm, P = .005, relative odds = 6.7) for differentiating benign from malignant pigmented lesions whereas a three-point version of the ABCDs (N = 192 lesions) had three significant variables (irregular outline, P = .001, relative odds = 9.3; diameter greater than 6 mm, P = .008, relative odds = 5.5; variegation, P = .05, relative odds = 3.7). All six melanomas scored 3 out of 3 on the ABCD scale for a sensitivity of 1.00 and specificity of 0.98 at a threshold of 3 out of 3. Patients rarely sought dermatologic consultation because of these predictors but often sought help for nonsignificant reasons such as inflammation (P = .93), color change (P = .84), itch (P = .72), and increasing size (P = .38). The simpler three-point scale at a threshold of two had equal specificity (0.88 versus 0.94) and better sensitivity (0.73 versus 0.44) than the Glasgow seven-point checklist at the recommended threshold of three out of seven. We therefore recommend that the American public be better educated about the ABCDs to help increase self-referral so that early melanomas can be found and eradicated.

Adolescent↗

Changes in ultrastructure and function of the sheep pigment epithelium and retina induced by sodium iodate. I. The ultrastructure of the normal pigment epithelium of the sheep.

The normal ultrastructure of the sheep pigment epithelial cells is described as a basis for the interpretation of toxic (sodium iodate) effects on these cells dealt with in two following papers. The morphological features of the different cell membranes and cell organelles, particularly the phagosomes and the lipid droplets, are discussed in relation to renewal of the photoreceptor outer segment, pigment epithelial and retinal metabolism, barrier mechanisms and electrical properties.

Animals↗