Exploring patients' expectations and preferences of glaucoma surgery outcomes to facilitate healthcare delivery and inform future glaucoma research

© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ. Introduction: Glaucoma is a lifelong condition often requiring surgical intervention. To allow us to inform patients' expectations of surgery effectively, it is important to understand patients' preferences and concerns regarding outcomes from glaucoma treatments including surgery. Aims: To explore what clinical and social outcomes of glaucoma surgery are important to patients. Methods: Forty-five glaucoma patients undergoing medical glaucoma treatments or surgery were recruited for focus group interviews to determine their opinions regarding the outcomes of glaucoma treatments. Thematic analysis was performed with NVivo software. Results: Themes identified were understanding glaucoma, understanding surgery treatments and understanding treatment outcomes. The most important outcomes of the glaucoma surgery reported by the patients were social factors. Patients felt that being able to maintain their driving licence is a strong indicator of successful glaucoma treatment/surgery. Other important outcomes were independent living, ability to care for their family and having a good-quality social life. When considering novel surgical treatments, most patients felt that certainty of successful outcome and proven longevity of the effect are the primary motivators for choosing these treatments. Conclusions: Patients understood that clinical measures were surrogates for maintaining visual function, but ability to maintain independent living was the most important outcome from their treatment. For newer treatments patients wished to know more about long-term outcomes when considering this option

Comparative transcriptional profiling of the limbal epithelial crypt demonstrates its putative stem cell niche characteristics

<p>Abstract</p> <p>Background</p> <p>The Limbal epithelial crypt (LEC) is a solid cord of cells, approximately 120 microns long. It arises from the undersurface of interpalisade rete ridges of the limbal palisades of Vogt and extends deeper into the limbal stroma parallel or perpendicular to the palisade. There are up to 6 or 7 such LEC, variably distributed along the limbus in each human eye.</p> <p>Morphological and immunohistochemical studies on the limbal epithelial crypt (LEC) have demonstrated the presence of limbal stem cells in this region. The purpose of this microarray study was to characterise the transcriptional profile of the LEC and compare with other ocular surface epithelial regions to support our hypothesis that LEC preferentially harbours stem cells (SC).</p> <p>Results</p> <p>LEC was found to be enriched for SC related Gene Ontology (GO) terms including those identified in quiescent adult SC, however similar to cornea, limbus had significant GO terms related to proliferating SC, transient amplifying cells (TAC) and differentiated cells (DC). LEC and limbus were metabolically dormant with low protein synthesis and downregulated cell cycling. Cornea had upregulated genes for cell cycling and self renewal such as <it>FZD7, BTG1, CCNG</it>, and <it>STAT3 </it>which were identified from other SC populations. Upregulated gene expression for growth factors, cytokines, WNT, Notch, TGF-Beta pathways involved in cell proliferation and differentiation were noted in cornea. LEC had highest number of expressed sequence tags (ESTs), downregulated and unknown genes, compared to other regions. Genes expressed in LEC such as <it>CDH1, SERPINF1, LEF1, FRZB1</it>, <it>KRT19, SOD2, EGR1 </it>are known to be involved in SC maintenance. Genes of interest, in LEC belonging to the category of cell adhesion molecules, WNT and Notch signalling pathway were validated with real-time PCR and immunofluorescence.</p> <p>Conclusions</p> <p>Our transcriptional profiling study identifies the LEC as a preferential site for limbal SC with some characteristics suggesting that it could function as a 'SC niche' supporting quiescent SC. It also strengthens the evidence for the presence of "transient cells" in the corneal epithelium. These cells are immediate progeny of SC with self-renewal capacity and could be responsible for maintaining epithelial turn over in normal healthy conditions of the ocular surface (OS). The limbus has mixed population of differentiated and undifferentiated cells.</p

Profiling ocular surface responses to preserved and non‐preserved topical glaucoma medications: a two‐year randomised evaluation study

BackgroundUse of topical glaucoma medications has been reported to cause ocular surface (OS) discomfort and inflammation. This study explores the profile of inflammatory cytokines and OS symptoms induced in response to preserved and non‐preserved drops.MethodsProspective, randomized evaluation on thirty‐six treatment‐naïve patients over 24 months of three differently preserved glaucoma drop preparations: Preservative‐free (PF), Polyquad (PQ), and Benzalkonium chloride (BAK). Study participants were evaluated at baseline and then at 1, 3, 6, 12 and 24 months whilst on medication. At each visit, participants completed the Ocular Surface disease index (OSDI) questionnaire, had basal tear sampling and impression cytology (IC) of the conjunctival epithelium. Quantitative polymerase chain reaction was performed to measure the gene expression of inflammatory cytokines (Interleukin (IL)‐6, IL‐8, IL‐10, IL‐12A, IL‐12B, IL‐17A, IL‐1β, and tumour necrosis factor (TNF)‐α) in the IC samples. Corresponding protein expression of cytokines in tear samples was assessed by the Becton‐Dickinson cytometric bead arrays.ResultsCompared to PF and PQ groups, mRNA and protein expression of IL‐6, IL‐8, and IL‐1β increased in samples from the BAK group in a time‐dependent fashion, whereas all other cytokines showed a non‐significant increase. In the BAK group, there was a strong correlation between OSDI and the levels of IC/IL‐1β (r=0.832, R squared=0.692 and p=0.040 ); IC/IL‐10 (r=0.925, R squared=0.856 and p=0.008) and tear/IL‐1β (r=0.899, R squared=0.808 and p=0.014 ). ConclusionBAK‐preserved topical drops stimulate a sterile inflammatory response on the OS within 3‐months which is maintained thereafter. Whereas PF‐drops and PQ‐preserved drops showed no significant OS inflammation

Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms

Clinical Characteristics and Outcomes of Fungal Keratitis in the United Kingdom 2011–2020: A 10-Year Study

Fungal keratitis (FK) is a serious ocular infection that often poses significant diagnostic and therapeutic dilemmas. This study aimed to examine the causes, clinical characteristics, outcomes, and prognostic factors of FK in the UK. All culture-positive and culture-negative presumed FK (with complete data) that presented to Queen’s Medical Centre, Nottingham, and the Queen Victoria Hospital, East Grinstead, between 2011 and 2020 were included. We included 117 patients (n = 117 eyes) with FK in this study. The mean age was 59.0 ± 19.6 years (range, 4–92 years) and 51.3% of patients were female. Fifty-three fungal isolates were identified from 52 (44.4%) culture-positive cases, with Candida spp. (33, 62.3%), Fusarium spp. (9, 17.0%), and Aspergillus spp. (5, 9.4%) being the most common organisms. Ocular surface disease (60, 51.3%), prior corneal surgery (44, 37.6%), and systemic immunosuppression (42, 35.9%) were the three most common risk factors. Hospitalisation for intensive treatment was required for 95 (81.2%) patients, with a duration of 18.9 ± 16.3 days. Sixty-six (56.4%) patients required additional surgical interventions for eradicating the infection. Emergency therapeutic/tectonic keratoplasty was performed in 29 (24.8%) cases, though 13 (44.8%) of them failed at final follow-up. The final corrected-distance-visual-acuity (CDVA) was 1.67 ± 1.08 logMAR. Multivariable logistic regression analyses demonstrated increased age, large infiltrate size (>3 mm), and poor presenting CDVA (60 days of healing time or occurrence of corneal perforation requiring emergency keratoplasty; all p < 0.05). In conclusion, FK represents a difficult-to-treat ocular infection that often results in poor visual outcomes, with a high need for surgical interventions. Innovative treatment strategies are urgently required to tackle this unmet need

Validation of Endogenous Control Genes for Gene Expression Studies on Human Ocular Surface Epithelium

PURPOSE: To evaluate a panel of ten known endogenous control genes (ECG) with quantitative reverse transcription PCR (qPCR), for identification of stably expressed endogenous control genes in the ocular surface (OS) epithelial regions including cornea, limbus, limbal epithelial crypt and conjunctiva to normalise the quantitative reverse transcription PCR data of genes of interest expressed in above-mentioned regions. METHOD: The lasermicrodissected (LMD) OS epithelial regions of cryosectioned corneoscleral buttons from the cadaver eyes were processed for RNA extraction and cDNA synthesis to detect genes of interest with qPCR. Gene expression of 10 known ECG--glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta actin (ACTB), peptidylprolyl isomerase (PPIA), TATA-box binding protein (TBP1), hypoxanthine guanine phosphoribosyl transferase (HPRT1), beta glucuronidase (GUSB), Eucaryotic 18S ribosomal RNA (18S), phosphoglycerate kinase (PGK1), beta-2-microglobulin (B2M), ribosomal protein, large, P0 (RPLP0)--was measured in the OS epithelial regions by qPCR method and the data collected was further analysed using geNorm software. RESULTS: The expression stability of ecgs in the os epithelial regions in increasing order as determined with genorm software is as follows: ACTB<18S<TBP<B2M<PGK1<HPRT1<GUSB<GAPDH<PPIA-RPLP0. In this study, geNorm analysis has shown the following ECGs pairs to be most stably expressed in individual OS epithelial regions: HPRT1-TBP in cornea, GUSB-PPIA in limbus, B2M-PPIA and RPLP0-TBP in LEC and conjunctiva respectively. However, across the entire ocular surface including all the regions mentioned above, PPIA-RPLP0 pair was shown to be most stable. CONCLUSION: This study has identified stably expressed ECGs on the OS epithelial regions for effective qPCR results in genes of interest. The results from this study are broadly applicable to quantitative reverse transcription PCR studies on human OS epithelium and provide evidence for the use of PPIA-RPLP0 ECGs pair in quantitative reverse transcription PCR across the OS epithelium

Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1

APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals

Comparative transcriptional profiling of the human limbal epithelial crypt

Limbal stem cell deficiency (LSCD) is one of the most prominent causes of cornea related blindness in the world with an incidence of approximately 240 new cases per year in the UK alone. Symptoms of LSCD include reduced vision, pain, watering, redness, photophobia and blepharospasm due to chronic inflammation and breakdown of the corneal epithelium. LSCD mainly results in loss of barrier between the corneal and conjunctival epithelium with overgrowth of conjunctival epithelium over the cornea resulting in corneal vascularisation and scarring. Therefore, LSCD not only causes debilitating blindness depending on the severity of the condition but also leads to long term morbidity. Despite the availability of advanced treatment options for LSCD such as limbal tissue grafting and ex-vivo corneal epithelium transplantation, basic research questions such as the identification of limbal stem cell markers (LSC) for effective isolation of a pure population of LSCs from the corneal epithelium remains unanswered. Anatomical studies on the ocular surface have identified a new structure at the limbus termed Limbal Epithelial Crypt (LEC). It is a solid cord of cells arising from the undersurface of palisades of Vogt and extends into the subepithelial stroma of the limbus. Immunohistochemical and electron microscopic studies have demonstrated the presence of SCs in this region. The primary aim of this research is to confirm the hypothesis that LEC is a corneal SC niche (SCN) and to identify any SC related genes of interest in the LEe. The transcriptional profiling of the ocular surface (OS) epithelial regions was performed with the in-house manufactured spotted oligonucleotide and the Gene 1.0 ST arrays platform. Comparison of the results of these two microarray studies facilitated validation of the results and also contributed to the gene database of the as epithelial regions. Methods Frozen tissue blocks of corneoscleral buttons were cryosectioned to obtain serial tissue sections. Good quality tissue sections comprising of as epithelial regions were transferred onto PALM slides for laser microdissection. Extracted RNA samples were amplified & hybridised to the in-house manufactured 30,000k spotted human oligonucleotide microarray chips and Gene LOST arrays. Primary analysis of raw data of both the microarrays studies was performed to filter and normalise the microarray data for removal of the noise and to smoothen the raw data . Secondary analysis of the data involved statistical methods such as ANOVA, principal component analysis unpaired t-test and Significance Analysis of Microarrays (SAM). A differentially expressed gene list for each as epithelial region was generated with these statistical analysis methods. The gene lists in both the microarray studies were further analysed at tertiary level for pathway analysis with Ingenuity Pathway Analysis and geneontology with Database for Annotation, Visualisation, and Integrated Discovery (DAVID) software respectively. The genes of interest in the microarray studies were validated with quantitative gene expression analysis (qPCR) and immunohistochemistry.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Distribution of cp values of ECGs in different OS epithelial regions.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022301#pone-0022301-g001" target="_blank">Figure 1A</a>, the scatter graph of the Cp values of all the ECGs shows all the ECGs had cp value≤35. The order of samples 1–12 were as follows: Samples 1, 2, 3 were cornea 1, cornea 2, cornea 3, samples 4, 5, 6 were limbus 1, limbus 2, limbus 3, samples 7, 8, 9 were LEC 1, LEC 2, LEC 3 and samples 10, 11, 12 were conjunctiva 1, conjunctiva 2, conjunctiva 3. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022301#pone-0022301-g001" target="_blank">Figure 1B</a> is a box plot of q PCR cp values of the ECGs. Each box plot is mean Cp value of all OS epithelial regions. The median Cp value is represented as black line within the box plot, and it divides the Cp values into lower and upper quartile ranges. The whiskers represent the upper and lower data range for the samples tested. The box plot of PPIA demonstrates tight distribution of the samples around the median value. Similarly box plot of RPLP0 shows uniform distribution of Cp values around the median with tight whisker distribution.</p

Endogenous control genes analysed with geNorm software in this study.

<p>Endogenous control genes analysed with geNorm software in this study.</p