In vivo safety of trypan blue use in vitreoretinal surgery

Purpose: The purpose of this study was to evaluate "in vivo" safety of trypan blue (TB) in patients undergoing TB-assisted internal limiting membrane or epiretinal membrane peeling. Methods: Prospective study including 21 patients (21 eyes) with full-thickness macular hole and/or epiretinal membrane undergoing TB-assisted internal limiting membrane/epiretinal membrane peeling. Main outcome measures included distance visual acuity, near visual acuity, amplitude of P50 and N95 of the pattern electroretinogram, and fundus autofluorescence; these were assessed preoperatively, at 6 months (n = 21) and 12 months (n = 10) postoperatively. Results: There was a statistically significant improvement in distance visual acuity, near visual acuity, P50, and N95 amplitude at 6 months and 12 months postoperatively. The mean logarithm of the minimum angle of resolution distance visual acuity and near visual acuity improved from baseline by 0.31 (SD 0.37) and 0.17 (SD 0.31) at 6 months, respectively, and by 0.4 (SD 0.25) and 0.35 (SD 0.28) at 12 months, respectively. The mean P50 and N95 component amplitudes improved by 28% compared with baseline at 6 months (P50 0.4 [SD 0.8]; N95 0.53 [SD 1.07]) and by 63% at 12 months (P50 0.9 [0.85]; N95 1.04 [1.34]). Autofluorescence did not demonstrate damage to the retinal pigment epithelium attributable to TB. Conclusion: No deleterious effects of TB were observed in this study. Copyright © 2011 Lippincott Williams &Wilkins.

Electric currents and lens regeneration in the rat

We studied the process of lens regeneration in the rat following an extracapsular lens extraction preserving the anterior lens capsule and anterior lens epithelium. We assessed clinically the clarity of the newly regenerated lens, evaluated changes in the lens electrical currents following surgery and during the regeneration process and correlated these changes with findings on light microscopy. Protein analysis of the regenerated lens was also undertaken. Experiments were performed in 41 Sprague-Dawley rats, sacrificed at 0, 2, 4 and 8 weeks postoperatively. Our results showed that complete lens regeneration occurred 8 weeks postoperatively only if the anterior epithelium was preserved and the lens capsule was closed surgically. Lens electrical currents, altered following surgery, recovered in parallel with the process of regeneration of the lens. The newly regenerated lens was optically clear and biochemical analysis revealed a pattern of protein expression resembling that observed during lens development. In conclusion, complete lens regeneration occurs in the rat and it is possible that lens electrical signals, together with other cues, may play an important role in this process. © 2009 Elsevier Ltd.

Fundus autofluorescence in patients with age-related macular degeneration and high risk of visual loss

PURPOSE: To describe fundus autofluorescence (AF) patterns and their change over time in patients with age-related macular degeneration (AMD) and high risk of visual loss participating in the drusen laser study (DLS). DESIGN: Randomized clinical trial. METHODS: The study population consisted of 29 patients (35 eyes) participating in the DLS, which is a prospective, randomized, controlled clinical trial of prophylactic laser therapy in patients with AMD and high risk of neovascular complications. The intervention consisted of 16 eyes having prophylactic laser and 19 receiving no treatment. The main outcome measures were changes in the distribution of drusen and AF. Patients were reviewed for a median follow-up or 24 months (range 12-36 months). RESULTS: At baseline, four patterns of fundus AF were recognized: focal increased AF (n = 18), reticular AF (n = 3), combined focal and reticular AF (n = 2), and homogeneous AF (n = 12). At last follow-up, fundus AF remained unchanged in 15 untreated (78%) and in seven treated (43%) eyes. In only one untreated eye, focal areas of increased AF returned to background levels and were no longer detectable at last follow-up, compared with six treated eyes. This difference was statistically significant (P = .03). Only large foveal soft drusen (drusenoid pigment epithelium detachments) consistently corresponded with focal changes in AF, whereas no obvious correspondence was found between small soft drusen located elsewhere and changes in AF. CONCLUSION: The lack of obvious correspondence between the distribution of drusen and of AF found in this study appears to indicate that drusen and AF represent independent measures of aging in the posterior pole. © 2002 Elsevier Science Inc. All rights reserved.

Iris cysts in children:Classification, incidence, and management. The 1998 Torrence A Makley Jr Lecture

Background - Iris cysts in children are uncommon and there is relatively little information on their classification, incidence, and management. Methods - The records of all children under age 20 years who were diagnosed with iris cyst were reviewed and the types and incidence of iris cysts of childhood determined. Based on these observations recommendations were made regarding management of iris cysts in children. Results - Of 57 iris cysts in children, 53 were primary and four were secondary. There were 44 primary cysts of the iris pigment epithelium, 34 of which were of the peripheral or iridociliary type, accounting for 59% of all childhood iris cysts. It was most commonly diagnosed in the teenage years, more common in girls (68%), was not recognised in infancy, remained stationary or regressed, and required no treatment. The five mid-zonal pigment epithelial cysts were diagnosed at a mean age of 14 years, were more common in boys (83%), remained stationary, and required no treatment. The pupillary type of pigment epithelial cyst was generally recognised in infancy and, despite involvement of the pupillary aperture, also required no treatment. There were nine cases of primary iris stromal cysts, accounting for 16% of all childhood iris cysts. This cyst was usually diagnosed in infancy, was generally progressive, and required treatment in eight of the nine cases, usually by aspiration and cryotherapy or surgical resection. Among the secondary iris cysts, two were post-traumatic epithelial ingrowth cysts and two were tumour induced cysts, one arising from an intraocular lacrimal gland choristoma and one adjacent to a peripheral iris naevus. Conclusions - Most iris cysts of childhood are primary pigment epithelial cysts and require no treatment. However, the iris stromal cyst, usually recognised in infancy, is generally an aggressive lesion that requires treatment by aspiration or surgical excision.

Clinicopathological correlation of epiretinal membranes and posterior lens opacification following perfluorohexyloctane tamponade

Background/aims - Epiretinal and retrolental proliferation may occur during prolonged use of the novel tamponade agent perfluorohexyloctane (F H ). This study aims to determine whether there is any histological evidence that F H has a role in the formation of these membranes. Methods - Eight epiretinal membranes and three opaque posterior lens capsules were excised from patients in whom F H had been used as a long term retinal tamponade agent. The membranes and capsules were examined employing light microscopic methods, including immunohistochemistry. Results - The epiretinal membranes showed histological features typical of proliferative vitreoretinopathy (PVR) epiretinal membranes, but they also exhibited a dense macrophagic infiltration. In addition, three of the membranes contained multinucleated cells. Macrophages represented up to 30% of the cells present and appeared to contain large intracytoplasmic vacuoles. Similar cells were seen on the back of the posterior lens capsule in one specimen and all three capsules had posterior migration of lens epithelium. Conclusion - The pathological findings are not simply those of PVR. The macrophage infiltration suggests that there may be a biological reaction to F H which could reflect its surmised propensity to emulsify. Further investigations concerning the cellular response to this promising tamponade agent are warranted.

Age- and Light-Dependent Development of Localised Retinal Atrophy in CCL2(-/-)CX3CR1(GFP/GFP) Mice

Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2(-/-)CX3CR1(GFP/GFP) mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2(-/-)CX3CR1(GFP/GFP) mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17~60% of 12-month, and 30~100% of 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice respectively, but not in wild-type mice. All CCL2(-/-)CX3CR1(GFP/GFP) mice exposed to extra-light (~800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20-25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2(-/-)CX3CR1(GFP/GFP) mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2(-/-)CX3CR1(GFP/GFP) mice. GABA expression was reduced in the inner retina of aged CCL2(-/-)CX3CR1(GFP/GFP) mice. Significantly increased Müller glial and microglial activation was observed in CCL2(-/-)CX3CR1(GFP/GFP) mice compared to age-matched WT mice. Macrophages from CCL2(-/-)CX3CR1(GFP/GFP) mice were less phagocytic, but expressed higher levels of iNOS, IL-1ß, IL-12 and TNF-a under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.

A tale of two chitons: Is habitat specialisation linked to distinct associated bacterial communities?

Although most chitons (Mollusca: Polyplacophora) are shallow-water molluscs, diverse species also occur in deep-sea habitats. We investigated the feeding strategies of two species, Leptochiton boucheti and Nierstraszella lineata, recovered on sunken wood sampled in the western Pacific, close to the Vanuatu Islands. The two species display distinctly different associations with bacterial partners. Leptochiton boucheti harbours Mollicutes in regions of its gut epithelium and has no abundant bacterium associated with its gill. Nierstraszella lineata displays no dense gut-associated bacteria, but harbours bacterial filaments attached to its gill epithelium, related to the Deltaproteobacteria symbionts found in gills of the wood-eating limpet Pectinodonta sp. Stable carbon and nitrogen isotope signatures and an absence of cellulolytic activity give evidence against a direct wood-feeding diet; both species are secondary consumers within the wood food web. We suggest that the distinct associations with bacterial partners are linked to niche specialisations of the two species. Nierstraszella lineata is in a taxonomic family restricted to sunken wood and is possibly adapted to more anoxic conditions thanks to its gill-associated bacteria. Leptochiton boucheti is phylogenetically more proximate to an ancestral form not specialised on wood and may itself be more of a generalist; this observation is congruent with its association with Mollicutes, a bacterial clade comprising gut-associated bacteria occurring in several metazoan phyla.

Autologous limbal transplantation in patients with unilateral corneal stem cell deficiency

Aim - To describe a surgical technique for autologous limbal stem cell transplantation and the outcome of a series of patients with unilateral stem cell deficiency. Methods - A report of six consecutive patients who underwent autologous limbal stem cell transplantation is presented. The primary diagnosis included alkali burn (n = 3), conjunctival intraepithelial neoplasia (CIN) (n = 1), recurrent pterygium (n = 1), and contact lens induced keratopathy (n = 1). The autologous transplanted tissue consisted of peripheral cornea, limbus, and conjunctiva obtained from the contralateral eye. Three of the above patients underwent penetrating keratoplasty in association with autolimbal transplantation. A significant modification to established techniques was the close monitoring of conjunctival epithelial migration in the immediate postoperative period. If conjunctival epithelium threatened to migrate on to the corneal surface, it was mechanically removed at the slit lamp and prevented from crossing the limbus. This was required in three patients. Results - The mean follow up was 18.8 months. The outcome was satisfactory in all cases: a stable corneal surface was restored and there was a substantial improvement in vision and symptoms. One patient had a primary failure of the corneal allograft associated with glaucoma, and 6 months later developed a retinal detachment. No complications were noted in the donor eye with the exception of one patient who developed filamentary keratitis along the edge of the donor site. Conclusion - Autologous limbal transplantation with corneal, limbal, and conjunctival carriers was found to be useful for ocular surface reconstruction, over a mid-term follow up, in patients with unilateral stem cell deficiency. Close monitoring of the migration of conjunctival epithelium in the immediate postoperative period, and preventing it from crossing the limbus, ensured that the corneal surface was re-epithelialised exclusively from epithelial cells derived from the transplanted limbal tissue. This approach should improve the success of this procedure.

Amniotic membrane transplantation for ocular surface reconstruction

Aims - To evaluate the efficacy of amniotic membrane transplantation (AMT) for ocular surface reconstruction. Methods - 10 consecutive patients who underwent AMT were included. The indications were: group A, cases with persistent epithelial defect after corneal abscess (n = 1), radiation (n = 1), or chemical burn (n = 3); group B, cases with epithelial defect and severe stromal thinning and impending or recent perforation, due to chemical burn (two patients, three eyes) or corneal abscess (n = 2); group C, to promote corneal epithelium healing and prevent scarring after symblepharon surgery with extensive corneo-conjunctival adhesion (n = 1). Under sterile conditions amniotic membrane was prepared from a fresh placenta of a seronegative pregnant woman and stored at -70°C. This technique involved the use of amniotic membrane to cover the entire cornea and perilimbal area in groups A and B, and the epithelial defect only in group C. Results - The cornea healed satisfactorily in four of five patients in group A, but the epithelial defect recurred in one of these patients. After AMT three patients underwent limbal transplantation and one penetrating keratoplasty and cataract extraction. In group B amniotic membrane transplantation was not helpful, and all cases underwent an urgent tectonic corneal graft. Surgery successfully released the symblepharon, promoted epithelialisation and prevented adhesions in the case of group C. Conclusion - AMT was effective to promote corneal healing in patients with persistent epithelial defect, and appeared to be helpful after surgery to release corneo-conjunctival adhesion. Most surgery for further surface rehabilitation. Amniotic membrane used as a patch was not effective to prevent tectonic corneal graft in cases with severe stromal thinning and impending or recent perforation.

Chronic IL9 and IL-13 Exposure Leads to an Altered Differentiation of Ciliated Cells in a Well-Differentiated Paediatric Bronchial Epithelial Cell Model

Asthma is a chronic inflammatory disease characterised by airways remodelling. In mouse models IL-9 and IL-13 have been implicated in airways remodelling including mucus hypersecretion and goblet cell hyperplasia. Their role, especially that of IL-9, has been much less studied in authentic human ex vivo models of the bronchial epithelium from normal and asthmatic children. We assessed the effects of IL-9, IL-13 and an IL-9/IL-13 combination, during differentiation of bronchial epithelial cells from normal (n?=?6) and asthmatic (n?=?8) children. Cultures were analysed for morphological markers and factors associated with altered differentiation (MUC5AC, SPDEF and MMP-7). IL-9, IL-9/IL-13 combination and IL-13 stimulated bronchial epithelial cells from normal children had fewer ciliated cells [14.8% (SD 8.9), p?=?0.048, 12.4 (SD 6.1), p?=?0.016 and 7.3% (SD 6.6), p?=?0.031] respectively compared with unstimulated [(21.4% (SD 9.6)]. IL-9 stimulation had no effect on goblet cell number in either group whereas IL-9/IL-13 combination and IL-13 significantly increased goblet cell number [24.8% (SD 8.8), p?=?0.02), 32.9% (SD 8.6), p?=?0.007] compared with unstimulated normal bronchial cells [(18.6% (SD 6.2)]. All stimulations increased MUC5AC mRNA in bronchial epithelial cells from normal children and increased MUC5AC mucin secretion. MMP-7 localisation was dysregulated in normal bronchial epithelium stimulated with Th2 cytokines which resembled the unstimulated bronchial epithelium of asthmatic children. All stimulations resulted in a significant reduction in transepithelial electrical resistance values over time suggesting a role in altered tight junction formation. We conclude that IL-9 does not increase goblet cell numbers in bronchial epithelial cell cultures from normal or asthmatic children. IL-9 and IL-13 alone and in combination, reduce ciliated cell numbers and transepithelial electrical resistance during differentiation of normal epithelium, which clinically could inhibit mucociliary clearance and drive an altered repair mechanism. This suggests an alternative role for IL-9 in airways remodelling and reaffirms IL-9 as a potential therapeutic target.© 2013 Parker et al.

Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?

Aims/hypothesis: Blood–retina barrier leakage in diabetes results in extravasation of plasma lipoproteins. Intra-retinal modified LDLs have been implicated in diabetic retinopathy (DR), but their effects on retinal pigment epithelial (RPE) cells and the added effects of extravasated modified HDLs are unknown.

Methods: In human retinas from individuals with and without diabetes and DR, immunohistochemistry was used to detect ApoB, ApoA1 and endoplasmic reticulum (ER) stress markers. In cell culture, human RPE cells were treated with native LDL (N-LDL) or heavily-oxidised glycated LDL (HOG-LDL) with or without pretreatment with native HDL (N-HDL) or heavilyoxidised glycated HDL (HOG-HDL). Cell viability, oxidative stress, ER stress, apoptosis and autophagy were assessed by Cell Counting Kit-8 assay, dichlorofluorescein assay, western blotting, immunofluorescence and TUNEL assay. In separate
experiments, RPE cells were treated with lipid oxidation products, 7-ketocholesterol (7-KC, 5–40 µmol/l) or 4-hydroxynonenal (4-HNE, 5–80 µmol/l), with or without pretreatment with N-HDL or HOG-HDL.

Results: ApoB, ApoA1 staining and RPE ER stress were increased in the presence of DR. HOG-LDL but not N-LDL significantly decreased RPE cell viability and increased reactive oxygen species generation, ER stress, apoptosis and autophagy. Similarly, 4-HNE and 7-KC decreased viability and induced ER stress. Pretreatment with N-HDL mitigated these effects, whereas HOG-HDL was less effective by most, but not all, measures.

Conclusions/interpretation: In DR, extravascular modified LDL may promote RPE injury through oxidative stress, ER stress, autophagy and apoptosis. N-HDL has protective effects, but HOG-HDL is less effective. Extravasation and modification of HDL may modulate the injurious effects of extravasated modified LDL on the retinal pigment epithelium.

Mucociliary clearance in cystic fibrosis: Physiology and pharmacological treatments

The understanding of how mutations of the cystic fibrosis gene results in recurrent infection and the development of bronchiectasis is now well established. This review examines aspects of lung pathophysiology specifically, abnormal mucociliary clearance, inflammation and infection which are the basis of the daily symptoms encountered by people with cystic fibrosis. Other components of the lung epithelium and their potential contribution to cystic fibrosis disease are discussed. Therapeutic interventions which aim to target abnormal mucociliary clearance are summarized. © 2011 Elsevier Ltd.

AKT in Stromal Fibroblasts Controls Invasion of Epithelial Cells

The tumour microenvironment has an important role in cancer progression and recent reports have proposed that stromal AKT is activated and regulates tumourigenesis and invasion. We have shown, by immuno-fluorescent analysis of oro-pharyngeal cancer biopsies, an increase in AKT activity in tumour associated stromal fibroblasts compared to normal stromal fibroblasts. Using organotypic raft co-cultures, we show that activation of stromal AKT can induce the invasion of keratinocytes expressing the HPV type 16 E6 and E7 proteins, in a Keratinocyte Growth Factor (KGF) dependent manner. By depleting stromal fibroblasts of each of the three AKT isoforms independently, or through using isoform specific inhibitors, we determined that stromal AKT2 is an essential regulator of invasion and show in oro-pharyngeal cancers that AKT2 specific phosphorylation events are also identified in stromal fibroblasts. Depletion of stromal AKT2 inhibits epithelial invasion through activating a protective pathway counteracting KGF mediated invasions. AKT2 depletion in fibroblasts stimulates the cleavage and release of IL1B from stromal fibroblasts resulting in down-regulation of the KGF receptor (fibroblast growth factor receptor 2B (FGFR2B)) expression in the epithelium. We also show that high IL1B is associated with increased overall survival in a cohort of patients with oro-pharyngeal cancers. Our findings demonstrate the importance of stromal derived growth factors and cytokines in regulating the process of tumour cell invasion.

Matrilysin-1 mediates bronchiolization of alveoli, a potential premalignant change in lung cancer

Matrilysin-1 (also called matrix metalloproteinase-7) is expressed in injured lung and in cancer but not in normal epithelia. Bronchiolization of the alveoli (BOA), a potential precursor of lung cancer, is a histologically distinct type of metaplasia that is composed of cells resembling airway epithelium in the alveolar compartment. We demonstrate that there is increased expression of matrilysin-1 in human lesions and BOA in the CC10-human achaete-scute homolog-1 transgenic mouse model. Forced expression of the matrilysin-1 gene in immortalized human normal airway epithelial BEAS-2B and HPLD1 cells, which do not normally express matrilysin-1, promoted cellular migration, suggesting a functional link for BOA formation via bronchiolar cell migration. In addition, matrilysin-1 stimulated proliferation and inhibited Fas-induced apoptosis, while a knockdown by RNA interference decreased cell growth, migration, and increased sensitivity to apoptosis. Western blotting demonstrated increased levels of phospho-p38 and phospho-Erk1/2 kinases after matrilysin-1 expression. Gene expression analysis uncovered several genes that were related to cell growth, migration/movement, and death, which could potentially facilitate bronchiolization. In vivo, the formation of BOA lesions was reduced when CC10-human achaete-scute homolog-1 mice were crossed with matrilysin-1 null mice and was correlated with reduced matrilysin-1 expression in BOA. We conclude that matrilysin-1 may play an important role in the bronchiolization of alveoli by promoting proliferation, migration, and attenuation of apoptosis involving multiple genes in the MAP kinase pathway.

Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells. © The Author 2008. Published by Oxford University Press. All rights reserved.