Search

Introduction The primate retina has evolved regional specialisations for specific visual functions. The macula is specialised towards high acuity vision and is an area that contains an increased density of cone photoreceptors and signal processing neurons. Different regions in the retina display unique susceptibility to pathology, with many retinal diseases primarily affecting the macula. Objectives To better understand the properties of different retinal areas we studied the differential distribution of metabolites across the retina. Methods We conducted an untargeted metabolomics analysis on full-thickness punches from three different regions (macula, temporal peri-macula and periphery) of healthy primate retina. Results Nearly half of all metabolites identified showed differential abundance in at least one comparison between the three regions. Furthermore, mapping metabolomics results from macula-specific eye diseases onto our region-specific metabolite distributions revealed differential abundance defining systemic metabolic dysregulations that were region specific. Conclusions The unique metabolic phenotype of different retinal regions is likely due to the differential distribution of different cell types in these regions reflecting the specific metabolic requirements of each cell type. Our results may help to better understand the pathobiology of retinal diseases with region specificity.

The primate retina has evolved regional specialisations for specific visual functions. The macula is specialised towards high acuity vision and is an area that contains an increased density of cone photoreceptors and signal processing neurons. Different regions in the retina display unique susceptibility to pathology, with many retinal diseases primarily affecting the macula. To better understand the properties of different retinal areas we conducted an untargeted metabolomics analysis on full thickness punches from three different regions (macula, temporal peri-macula and periphery) of primate retina. Half of all metabolites identified showed differential abundance in at least one comparison between the three regions. The unique metabolic phenotype of different retinal regions is likely due to the differential distribution of different cell types in these regions reflecting the specific metabolic requirements of each cell type. Furthermore, mapping metabolomics results from macula-specific eye diseases onto the region-specific distributions of healthy primate retina revealed differential abundance defining systemic metabolic dysregulations that were region specific, highlighting how our results may help to better understand the pathobiology of retinal diseases with region specificity.

Background Detection of homozygous deletion of the p16 gene (CDKN2A) by fluorescence in situ hybridization (FISH) has been investigated as an ancillary technique in the diagnosis of malignant mesothelioma. Method This retrospective study reviewed the results of all p16 FISH tests performed at a regional mesothelioma centre from February 2012 to November 2019 in cases of possible mesothelioma to examine the diagnostic utility of this test as well as patients characteristics and survival in p16 FISH positive mesothelioma versus p16 FISH negative mesothelioma. Results P16 FISH testing was requested in 216 pathological samples in the study period. The test failure rate was 4% (10/216). Median time from request to result was 10 days (IQR 7–13, range 1–30). The sensitivity, specificity, NPV and PPV were 60 %, 100 %, 39 % and 100 % respectively. There were no false positive results and this genetic aberration was only detected in cases of mesothelioma. The prevalence of p16 FISH positive mesothelioma was higher in cytological specimens compared to histological specimens (75 % vs 58 %, p = 0.03) and lower in women compared to men (33 % vs 66 %, p = 0.003). P16 FISH positive mesothelioma was associated with significantly worse survival (median overall survival 285 vs 339 days, p = 0.0018). This remained significant after adjusting for confounding variables (OR 4.4, 95 %CI 1.84–11.14, p = 0.001). Conclusions In this study, 60 % of mesotheliomas harbour a homozygous deletion of CDKN2A and can be accurately, reliably and efficiently identified by p16 FISH testing. This test can be embedded within routine practice in mesothelioma pathways to enhance diagnostic accuracy.

Age-related macular degeneration (AMD) is a leading cause of visual loss. It has a strong genetic basis, and common haplotypes on chromosome (Chr) 1 (CFH Y402H variant) and on Chr10 (near HTRA1/ARMS2) contribute the most risk. Little is known about the early molecular and cellular processes in AMD, and we hypothesized that analyzing submacular tissue from older donors with genetic risk but without clinical features of AMD would provide biological insights. Therefore, we used mass spectrometry–based quantitative proteomics to compare the proteins in human submacular stromal tissue punches from donors who were homozygous for high-risk alleles at either Chr1 or Chr10 with those from donors who had protective haplotypes at these loci, all without clinical features of AMD. Additional comparisons were made with tissue from donors who were homozygous for high-risk Chr1 alleles and had early AMD. The Chr1 and Chr10 risk groups shared common changes compared with the low-risk group, particularly increased levels of mast cell–specific proteases, including tryptase, chymase, and carboxypeptidase A3. Histological analyses of submacular tissue from donors with genetic risk of AMD but without clinical features of AMD and from donors with Chr1 risk and AMD demonstrated increased mast cells, particularly the tryptase-positive/chymase-negative cells variety, along with increased levels of denatured collagen compared with tissue from low–genetic risk donors. We conclude that increased mast cell infiltration of the inner choroid, degranulation, and subsequent extracellular matrix remodeling are early events in AMD pathogenesis and represent a unifying mechanistic link between Chr1- and Chr10-mediated AMD.

Opticin is an extracellular glycoprotein present in the vitreous. Its antiangiogenic properties offer the potential for therapeutic intervention in conditions such as proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the hypothesis that intravitreal administration of recombinant human opticin can safely protect against the development of pathological angiogenesis and promote its regression. We generated and purified recombinant human opticin and investigated its impact on the development and regression of pathological retinal neovascularization following intravitreal administration in murine oxygen-induced retinopathy. We also investigated its effect on normal retinal vascular development and function, following intravitreal injection in neonatal mice, by histological examination and electroretinography. In oxygen-induced retinopathy, intravitreal administration of human recombinant opticin protected against the development of retinal neovascularization to similar extent as aflibercept, which targets VEGF. Opticin also accelerated regression of established retinal neovascularization, though the effect at 18 h was less than that of aflibercept. Intravitreal administration of human recombinant opticin in neonatal mice caused no detectable perturbation of subsequent retinal vascular development or function. In summary we found that intraocular administration of recombinant human opticin protects against the development of pathological angiogenesis in mice and promotes its regression.

Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4× 10-10; FHR-2, p = 6.0× 10-10; FHR-3, p = 1.5× 10-5; FHR-4, p = 1.3× 10-3; FHR-5, p = 1.9× 10-4) and FHL-1 (p = 4.9× 10-4) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.

Purpose To characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients. Design Multicenter case series, in vitro experimentation, and retrospective analysis of previously reported variants. Participants Seven families with apparently autosomal dominant EOMD. Methods Patients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed. Main Outcome Measures Detailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD. Results All affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1G→T het; c.619+1G→A het, c.380G→A, p.(Arg127His) het; c.694C→T p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291T→A, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch’s membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1R127H, FHL-1A415f/s, and FHL-1C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1. Conclusions Early-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities.

Rare variants in the complement factor I (CFI) gene, associated with low serum factor I (FI) levels, are strong risk factors for developing the advanced stages of age-related macular degeneration (AMD). No studies have been undertaken on the prevalence of disease-causing CFI mutations in patients with geographic atrophy (GA) secondary to AMD. A multicenter, cross-sectional, noninterventional study was undertaken to identify the prevalence of pathogenic rare CFI gene variants in an unselected cohort of patients with GA and low FI levels. A genotype-phenotype study was performed. Four hundred and sixty-eight patients with GA secondary to AMD were recruited to the study, and 19.4% (n = 91) demonstrated a low serum FI concentration (below 15.6 μg/ml). CFI gene sequencing on these patients resulted in the detection of rare CFI variants in 4.7% (n = 22) of recruited patients. The prevalence of CFI variants in patients with low serum FI levels and GA was 25%. Of the total patients recruited, 3.2% (n = 15) expressed a CFI variant classified as pathogenic or likely pathogenic. The presence of reticular pseudodrusen was detected in all patients with pathogenic CFI gene variants. Patients with pathogenic CFI gene variants and low serum FI levels might be suitable for FI supplementation in therapeutic trials.

Retinal pigment epithelial (RPE) cells that underlie the neurosensory retina are essential for the maintenance of photoreceptor cells and hence vision. Interactions between the RPE and their basement membrane, i.e. the inner layer of Bruch’s membrane, are essential for RPE cell health and function, but the signals induced by Bruch’s membrane engagement, and their contributions to RPE cell fate determination remain poorly defined. Here, we studied the functional role of the soluble complement regulator and component of Bruch’s membrane, Factor H-like protein 1 (FHL-1). Human primary RPE cells adhered to FHL-1 in a manner that was eliminated by either mutagenesis of the integrin-binding RGD motif in FHL-1 or by using competing antibodies directed against the α5 and β1 integrin subunits. These short-term experiments reveal an immediate protein-integrin interaction that were obtained from primary RPE cells and replicated using the hTERT-RPE1 cell line. Separate, longer term experiments utilising RNAseq analysis of hTERT-RPE1 cells bound to FHL-1, showed an increased expression of the heat-shock protein genes HSPA6, CRYAB, HSPA1A and HSPA1B when compared to cells bound to fibronectin (FN) or laminin (LA). Pathway analysis implicated changes in EIF2 signalling, the unfolded protein response, and mineralocorticoid receptor signalling as putative pathways. Subsequent cell survival assays using H2O2 to induce oxidative stress-induced cell death suggest hTERT-RPE1 cells had significantly greater protection when bound to FHL-1 or LA compared to plastic or FN. These data show a non-canonical role of FHL-1 in protecting RPE cells against oxidative stress and identifies a novel interaction that has implications for ocular diseases such as age-related macular degeneration.

Purpose For oropharynx squamous cell carcinoma (OPSCC) this study aimed to: (i) compare 5-year overall survival (OS) stratification by AJCC/UICC TNM versions 7 (TNMv7) and 8 (TNMv8), (ii) determine whether changes to T and N stage groupings improve prognostication and (iii) develop and validate a model incorporating additional clinical characteristics to improve 5-year OS prediction. Material and Methods All OPSCC treated with curative-intent at our institution between 2011 and 2017 were included. The primary endpoint was 5-year OS. Survival curves were produced for TNMv7 and TNMv8. A three-way interaction between T, N stage and p16 status was evaluated for improved prognostication. Cox proportional hazards modelling was used to derive a new predictive model. Results Of 750 OPSCC cases, 574 (77%) were p16-positive. TNMv8 was more prognostic than TNMv7 (concordance probability estimate [CPE] ± SE = 0.72 ± 0.02 vs 0.53 ± 0.02). For p16-positive disease, TNMv8 discriminated stages II vs I (HR 2.32, 95% CI 1.47–3.67) and III vs II (HR 1.75, 95% CI 1.13–2.72). For p16-negative disease, TNMv7 and TNMv8 demonstrated poor hazard discrimination. Different T, N stage and p16-status combinations did not improve prognostication after adjusting for other factors (CPE = 0.79 vs 0.79, p = 0.998). A model for p16-positive and p16-negative OPSCC including additional clinical characteristics improved 5-year OS prediction beyond TNMv8 (c-index 0.76 ± 0.02). Conclusions TNMv8 is superior to TNMv7 for p16-positive OPSCC, but both performed poorly for p16-negative disease. A novel model incorporating additional clinical characteristics improved 5-year OS prediction for both p16-positive and p16-negative disease.

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family found in vitreous humour and cartilage. It was first identified associated with the surface of vitreous collagen fibrils and several other SLRPs are also known to bind collagen fibrils and it some cases alter fibril morphology. The purpose of this study was to investigate the binding of opticin to the collagen II-containing fibrils found in vitreous and cartilage. Electron microscopic studies using gold labelling demonstrated that opticin binds vitreous and thin cartilage collagen fibrils specifically at a single site in the gap region of the collagen D-period corresponding to the e2 stain band; this is the first demonstration of the binding site of a class III SLRP on collagen fibrils. Opticin did not bind thick cartilage collagen fibrils from cartilage or tactoids formedin vitrofrom collagen II, but shows high specificity for thin, heterotypic collagen fibrils containing collagens II, and XI or V/XI. Vitreous collagen fibrils from opticin null and wild-type mice were compared and no difference in fibril morphology or diameter was observed. Similarly,in vitrofibrillogenesis experiments showed that opticin did not affect fibril formation. We propose that when opticin is bound to collagen fibrils, rather than influencing their morphology it instead hinders the binding of other molecules to the fibril surfaces and/or act as an intermediary bridge linking the collagen fibrils to other non-collagenous molecules.

Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supra-normal concentrations supress preretinal neovascularisation. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/ml. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94 % of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5 h, 24 h, 72 h, 7 days, 14 days and 28 days post-injection (n=6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 ml and the vitreous half-life 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available anti-angiogenic therapeutics. Whilst opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the non-pigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate and the turnover in vitreous is approximately 15% per day.

Age-related macular degeneration (AMD) is a leading cause of blindness. Genetic variants at the chromosome 1q31.3 encompassing the complement factor H (CFH, FH) and CFH related genes (CFHR1-5) are major determinants of AMD susceptibility, but their molecular consequences remain unclear. Here we demonstrate that FHR-4 plays a prominent role in AMD pathogenesis. We show that systemic FHR-4 levels are elevated in AMD (P-value = 7.1 × 10−6), whereas no difference is seen for FH. Furthermore, FHR-4 accumulates in the choriocapillaris, Bruch’s membrane and drusen, and can compete with FH/FHL-1 for C3b binding, preventing FI-mediated C3b cleavage. Critically, the protective allele of the strongest AMD-associated CFH locus variant rs10922109 has the highest association with reduced FHR-4 levels (P-value = 2.2 × 10−56), independently of the AMD-protective CFHR1–3 deletion, and even in those individuals that carry the high-risk allele of rs1061170 (Y402H). Our findings identify FHR-4 as a key molecular player contributing to complement dysregulation in AMD., A locus on chromosome 1 encompassing the CFHR genes is highly associated with AMD risk. Here, Cipriani and colleagues investigate the role of CFHR4, encoding FHR-4, and demonstrate a relationship between AMD risk, circulating FHR-4 levels and genetic variants at this locus.

Introduction Adequate intraoperative lymph node sampling is a fundamental part of lung cancer surgery, but adherence to standards is not well known. This study sought to measure the adequacy of intraoperative lymph node sampling at a regional Thoracic Surgery Centre and a tertiary lung cancer center in the United Kingdom. Methods This retrospective study analyzed the pathological reports from NSCLC resections over the 4-year period 2011–2014. Adequacy of sampling was assessed against International Association for the Study of Lung Cancer recommendations of at least three mediastinal lymph node stations: station 7 in all patients, station 5 or 6 in left upper lobe tumors, and station 9 in lower lobe tumors. The influence of clinical variables (age, tumor T stage, type of surgery, and laterality) on adequacy of sampling and the effect of adequacy on overall survival were also assessed. Results A total of 1301 NSCLC resections were performed from January 11, 2011, to December 31, 2014. Adequate intraoperative lymph node sampling increased significantly from 14% (22 of 160) in 2011 to 53% (206 of 390) in 2014 ( p = 0.001). Secondary analysis of clinical variables also revealed that patients with T1a or T4 tumors, those undergoing sublobar resections, those undergoing video-assisted thoracic surgery resections, and those undergoing left-sided resections have significantly higher rates of inadequate lymph node sampling. Overall, there was no statistically significant difference in survival between patients with adequate versus inadequate intraoperative lymph node sampling or when survival was stratified according to overall stage. There was worse survival in inadequate sampling for patients with pN2 disease than for patients with pN2 disease and adequate sampling. Conclusion This study provides a much-needed benchmark of current thoracic surgical practice in lung cancer in the United Kingdom and important granularity to facilitate changes to improve adequacy of staging.

Metabolomics is the study of endogenous and exogenous metabolites in biological systems, which aims to provide comparative semi-quantitative information about all metabolites in the system. Metabolomics is an emerging and potentially powerful tool in ophthalmology research. It is therefore important for health professionals and researchers involved in the speciality to understand the basic principles of metabolomics experiments. This article provides an overview of the experimental workflow and examples of its use in ophthalmology research from the study of disease metabolism and pathogenesis to identification of biomarkers.

Floaters are a common ocular condition which form as a consequence of aging changes in the vitreous. Although in most patients the symptoms are minimal, they can cause significant impairment in vision-related quality of life in a small population of patients. Recently there has been an increase in awareness of the visual disability caused by floaters, and the evidence-base for treatment of this condition using small-gauge vitrectomy has increased. In this review, we define the term 'floaters' as symptomatic vitreous opacities (SVO). We suggest a classification dependent on the presence or absence of posterior vitreous detachment and discuss their pathogenesis and natural history. We review their impact on patients' quality of life related to visual function. We review the psychological factors that may have a role in some patients who appear to be affected by SVO to the extent that they pursue all options including surgery with all its attendant risks. We summarise the available evidence-base of treatment options available for SVO with special emphasis on the safety and efficacy of vitrectomy for this condition.

Background Age-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation. Methods Epigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes. Results Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10−9), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10−7), and Tenascin X (TNXB) (p = 6.30 × 10−6) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors. Conclusions We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4). Electronic supplementary material The online version of this article (10.1186/s13148-019-0608-2) contains supplementary material, which is available to authorized users.

Aim: It has been reported that a high body mass index (BMI) is associated with better survival in patients with idiopathic pulmonary fibrosis (IPF). We aimed to find out whether body mass index influences survival of patients with idiopathic pulmonary fibrosis. Methods: We examined the relationship between BMI and survival in our cohort of patients with IPF. These patients were first seen in our clinic between May 2008 and January 2018. All had a diagnosis of IPF based on clinical assessment and high resolution CT scan; one third underwent a surgical lung biopsy. Height and body weight were ascertained at the first consultation. Survival analysis was performed using SPSS version 16. Results: We had data on 367 patients. The median age at first consultation was 66 years (range 39 to 97). There were 272 men and 95 women. At presentation, 6 were underweight (BMI 30). On Kaplan-Meier survival analysis, there was no significant difference in survival between these categories (p=0.75). Conclusion: The differences between the groups did not reach clinical significance. The graph suggests that being underweight is an adverse prognostic factor, but the number of such patients was very small. It also suggests that obesity may be marginally beneficial for the first four years but not thereafter.

The SLRP opticin (OPTC) has been demonstrated to be produced and degraded in osteoarthritic (OA) human cartilage. Here, we investigated the in vivo effect of OPTC deficiency in OA cartilage. OA was induced in 10-week-old Optc−/− and Optc+/+ mice. Ten weeks post-surgery, cartilage was processed for histology and immunohistochemistry. SLRP expression was determined in non-operated mouse cartilage. OA Optc−/− demonstrated significant protection against cartilage degradation. Data revealed that in non-operated Optc−/− cartilage, expression of SLRPs lumican and epiphycan was up-regulated at day 3 and in 10-week-olds (p ≤ 0.039), and fibromodulin down-regulated in 10-week-olds (p = 0.001). Immunohistochemistry of OA mice showed a similar pattern. In OA Optc−/− cartilage, markers of degradation and complement factors were all down-regulated (p ≤ 0.038). In OA Optc−/− cartilage, collagen fibers were thinner and better organized (p = 0.038) than in OA Optc+/+ cartilage. The protective effect of OPTC deficiency during OA results from an overexpression of lumican and epiphycan, known to bind and protect collagen fibers, and a decrease in fibromodulin, contributing to a reduction in the complement activation/inflammatory process. This work suggests that the evaluation of the composition of the different SLRPs in OA cartilage could be applied as a new tool for OA prognosis classification.

The recent revolution in age-related macular degeneration (AMD) genetics has demonstrated that genetic alterations affecting the alternative pathway of the complement cascade have a major influence on AMD risk. One of the two most important genetic loci is on chromosome 1 and contains genes encoding complement factor H (FH) and the factor H related proteins (FHR proteins). In macular tissue, especially Bruch’s membrane, relatively high levels of a truncated splice variant of FH called factor H-like protein 1 (FHL-1) are present. Here we discuss how genetic variations may alter the amounts, or by altering their protein sequences, the functions of these proteins. In particular, the common Y402H polymorphism affects the ability of FHL-1 and FH to localize to Bruch’s membrane and the inner choroid because it alters the ability of these complement regulators to bind heparan sulphate (HS) in these structures. In addition, there is an age-related loss of HS from Bruch’s membrane. We hypothesize that a combination of poor binding of the 402H variants of FHL-1 and FH to Bruch’s membrane, combined with a decrease in binding due to age-related HS loss, eventually results in insufficient FHL-1 and FH binding to Bruch’s membrane. This could result in complement activation, inflammation and thereby predispose to AMD.

This study is based on 122 patients with interstitial lung disease (ILD) who underwent right heart catheterisation (RHC) to assess the pulmonary arterial pressure (43 patients with connective tissue disease (CTD-ILD), 52 IPF and 27 other ILDs). 19 were excluded from analysis because they had group 2 pulmonary hypertension (PH) or a pulmonary vascular resistance 35 mmHg in 56 of the 67 with PH by RHC. GAP and CPI did not correlates with PH. Those with CTD-ILD lived significantly longer than those with IPF (mean for CTD 28 months vs 14 months vs other 29 months, p 0.014). The same was found for the subgroup with PH. The development of PH has a significant impact on survival.

Introduction: The aim of this study was to evaluate the prevalence of latent tuberculosis infection (LTBI) in our interstitial lung disease (ILD) patients. Methods: Patients with ILD were tested by using a commercial test QuantiFERON®-TB-Gold In Tube (QFT) at the time of initial diagnosis, before any immunomodulatory drug was initiated. Those patients with a historical or active TB were excluded, as were any with an equivocal QFT result. They were compared with 272 control cases who had a normal chest x-rays or thoracic CT, and no history of TB. Comparisons were classified by age (

Viral gene delivery is showing great promise for treating retinal disease. Although subretinal vector delivery has mainly been used to date, intravitreal delivery has potential advantages if low retinal transduction efficiency can be overcome. To this end, we investigated the effects of co-injection of glycosaminoglycan-degrading enzymes, singly or in combination, with AAV2 as a method of increasing retinal transduction. Experiments using healthy mice demonstrated that these enzymes enhance retinal transduction. We found that heparinase III produced the greatest individual effect, and this was enhanced further by combination with hyaluronan lyase. In addition, this optimized AAV2-enzyme combination led to a marked improvement in transduction in retinas with advanced retinal degeneration compared with AAV2 alone. Safety studies measuring retinal function by flash electroretinography indicated that retinal function was unaffected in the acute period and at least 12 months after enzyme treatment, whereas pupillometry confirmed that retinal ganglion cell activity was unaffected. Retinal morphology was not altered by the enzyme injection. Collectively these data confirm the efficacy and safety of this intravitreal approach in enhancing retinal transduction efficiency by AAV in rodents. Translating this method into other species, such as non-human primates, or for clinical applications will have challenges and require further studies.

Introduction Endoscopic ultrasound (EUS) allows access to the inferior mediastinal lymph node stations (8 and 9) which are beyond the reach of endobronchial ultrasound (EBUS). The addition of EUS to EBUS procedures requires cost and resource investment. This study sought to describe the prevalence of station 8/9 nodal metastases from intra-operative lymph node sampling in a UK region where routine pre-operative EUS is not available. Methods A retrospective review of all lung cancer resections at the University Hospital South Manchester from 2011 to 2014. Surgical variables, pre-operative PET variables and survival outcomes were collected and analysed. Results 1421 surgical resections were performed in the study period. Lymph node stations 8 and/or 9 were sampled in 52% (736/1421) of patients. Overall, there were 34 patients with lymph node metastases at station 8/9. This represents 2.4% of the study populations and 4.6% of patients in whom stations 8/9 were sampled intra-operatively. Of those patients with station 8/9 metastases, 65% (22/34) had multi-station N2 disease and the majority of the additional N2 disease was present in EBUS-accessible areas (lymph node stations 2, 4 and 7). Two percent (16/736) of patients in whom station 8/9 lymph nodes were sampled intra-operatively had N2 disease that was only accessible endoscopically with EUS. There was no significant difference in overall survival in patients with pathological N2 disease stratified according to whether stations 8/9 were involved or not. Conclusions The prevalence of lymph node metastases in stations 8/9 in this UK surgical centre where routine pre-operative EUS is not performed is low at approximately 5%. Given the identification of N2 disease in two-thirds of these patients can potentially be achieved through EBUS alone, this questions whether the resource implications of EUS are justified by the impact on patient management.

Purpose Preliminary assessment of a new prototype ultrasound-based hypersonic vitrector (HV) by qualitatively examining the histopathological changes in the retina and vitreous body after pars plana vitrectomy (PPV) and its ability to fragment vitreous collagen. Methods Fourteen porcine cadaveric eyes, 20 eyes in live swine and six human cadaveric eyes underwent PPV using the HV or a pneumatic guillotine vitrector (GV). An additional 4 porcine crystalline lenses were touched with either the HV or GV for 1 minute. Following PPV, human vitreous was removed and processed for electron microscopy (EM). Eyes and lenses were fixed and sectioned for light microscopy (LM). Results There were no macroscopic retinal or optic nerve defects associated with either HV or GV PPVs. Cadaveric retinal specimens showed separation of the inner limiting membrane (ILM) and vacuolization and fragmentation at the nerve fiber layer (NFL) and the ganglion cell layer (GCL). ILM fragmentation and separation were found after PPV in live swine with both vitrectors. Small disruptions of the posterior capsule or structural lens defects were found after HV touch. The EM analysis revealed more fragmentation of human vitreous collagen fibrils after HV compared to GV PPV. Conclusions LM and EM analysis of retina, vitreous, and crystalline lens after PPV showed similar morphological changes using the HV or the GV. Vitreous fragmentation appeared more effective with the HV. Overall this study suggests that the HV may be a promising new technology. More work is needed to quantitatively assess its safety and efficacy.

The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch’s membrane. Bruch’s membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch’s membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch’s membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies.

Background:Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) enables minimally invasive lymph node sampling during bronchoscopy under conscious sedation. The primary purpose of this study was to investigate the safety profile of EBUS-TBNA in an elderly population. The secondary aim was to assess the efficacy of EBUS-TBNA for nodal staging and pathological diagnosis in elderly patients with primary lung cancer.Methods:This was a prospective cohort study of patients undergoing EBUS-TBNA, between March 2010 and August 2012, at a single U.K. hospital site. Procedure and outcome data including 6-month follow-up were collected prospectively. Patients were divided into less than 70 (

An elderly patient, with a history of depression with psychosis, presented with breathlessness, a right exudative pleural effusion and a peripheral eosinophilia. The pleural fluid was eosinophil-rich (10% of leucocytes). Olanzapine therapy had been commenced 12 months previously. There was a family history of TB and the patient was of African origin. A full diagnostic work-up ensued including computed tomography of the thorax and local anaesthetic thoracoscopy. The pleura was unremarkable on CT and displayed bland smooth thickening at visual inspection during thoracoscopy. Pleural biopsies demonstrated chronic inflammation with eosinophils but no evidence of granulomatous inflammation or malignancy. Pleural tissue culture did not yield mycobacteria. A diagnosis of olanzapine-induced eosinophilic pleuritis was suspected and the pleural disease resolved with withdrawal of olanzapine.Eosinophilic pleural fluid is not a marker of non-malignant aetiology and eosinophilic pleural effusions require a careful and systematic diagnostic work-up. This is the second case report to identify olanzapine as a causative agent in eosinophilic pleural effusion.

To assess repeatability of visual function measures in patients with early, intermediate or late age-related macular degeneration (AMD) without active neovascular disease in the study eye, but active neovascular AMD in the fellow eye. One hundred subjects from an ongoing trial were screened for this study in which their LogMAR acuity, contrast sensitivity and reading performance were assessed using standardised protocols by trained optometrists. The same measures were repeated one month later and repeatability of the visual functions assessed. Data from 83 subjects satisfied inclusion criteria for analysis. Coefficient of repeatability was 14.9 letters for LogMAR visual acuity , 7.2 letters for Pelli Robson contrast sensitivity, 0.72 for LogMAR reading acuity, 110.4 words/ min for reading speed and 0.67 for LogMAR critical print size. Intraclass correlation coefficients allowed comparison between measures and were found to be 0.96 for LogMAR visual acuity, 0.93 for contrast sensitivity, 0.75 for LogMAR reading acuity, 0.79 for reading speed and 0.74 for LogMAR critical print size. Coefficients of variation were 9.4 %, 10.7 %, 48.4 %, 28.4 % and 31.8 % respectively. We found coefficients of repeatability that concurred with previous studies demonstrating variability of visual functions in patients with AMD. In addition, we found intraclass correlation coefficients to be better with visual acuity and contrast sensitivity than with measures of reading performance.

Background— Extracellular matrix expansion is a key element of ventricular remodeling and a potential therapeutic target. Cardiovascular magnetic resonance (CMR) T 1 -mapping techniques are increasingly used to evaluate myocardial extracellular volume (ECV); however, the most widely applied methods are without histological validation. Our aim was to perform comprehensive validation of (1) dynamic-equilibrium CMR (DynEq-CMR), where ECV is quantified using hematocrit-adjusted myocardial and blood T 1 values measured before and after gadolinium bolus; and (2) isolated measurement of myocardial T 1 , used as an ECV surrogate. Methods and Results— Whole-heart histological validation was performed using 96 tissue samples, analyzed for picrosirius red collagen volume fraction, obtained from each of 16 segments of the explanted hearts of 6 patients undergoing heart transplantation who had prospectively undergone CMR before transplantation (median interval between CMR and transplantation, 29 days). DynEq-CMR–derived ECV was calculated from T 1 measurements made using a modified Look-Locker inversion recovery sequence before and 10 and 15 minutes post contrast. In addition, ECV was measured 2 to 20 minutes post contrast in 30 healthy volunteers. There was a strong linear relationship between DynEq-CMR–derived ECV and histological collagen volume fraction ( P r =0.745; P r 2 =0.555 and between-subject: r =0.945; P r 2 =0.893; for ECV calculated using 15-minute postcontrast T 1 ). Correlation was maintained throughout the entire heart. Isolated postcontrast T 1 measurement showed significant within-subject correlation with histological collagen volume fraction ( r =−0.741; P r 2 =0.550 for 15-minute postcontrast T 1 ), but between-subject correlations were not significant. DynEq-CMR–derived ECV varied significantly according to contrast dose, myocardial region, and sex. Conclusions— DynEq-CMR–derived ECV shows a good correlation with histological collagen volume fraction throughout the whole heart. Isolated postcontrast T 1 measurement is insufficient for ECV assessment.

Age-related macular degeneration (AMD) is a common cause of blindness in older individuals. To accelerate the understanding of AMD biology and help design new therapies, we executed a collaborative genome-wide association study, including >17,100 advanced AMD cases and >60,000 controls of European and Asian ancestry. We identified 19 loci associated at P

Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan with a highly diverse range of structures, for which two regions of CFH (CCP6–8 and CCP19–20) have been implicated in HS binding. Mutations/polymorphisms within these glycosaminoglycan-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome. In this study, we demonstrate that CFH has tissue-specific binding properties mediated through its two HS-binding regions. Our data show that the CCP6–8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCP19–20, and that their sulfate specificities are different. Furthermore, the HS binding site in CCP6–8, which is affected by the AMD-associated Y402H polymorphism, plays the principal role in host tissue recognition in the human eye, whereas the CCP19–20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and atypical hemolytic uremic syndrome, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.

Time-dependent post-mortem biochemical changes have been demonstrated in donor cornea and vitreous, but there have been no published studies to date that objectively measure post-mortem changes in the retinal metabolome over time. The aim of the study was firstly, to investigate post-mortem, time-dependent changes in the rat retinal metabolome and secondly, to compare the metabolite composition of healthy rat ocular tissues. To study post-mortem changes in the rat retinal metabolome, globes were enucleated and stored at 4 °C and sampled at 0, 2, 4, 8, 24 and 48 h post-mortem. To study the metabolite composition of rat ocular tissues, eyes were dissected immediately after culling to isolate the cornea, lens, vitreous and retina, prior to storing at -80 °C. Tissue extracts were subjected to Gas Chromatograph Mass Spectrometry (GC-MS) and Ultra High Performance Liquid Chromatography Mass Spectrometry (UHPLC-MS). Generally, the metabolic composition of the retina was stable for 8 h post-mortem when eyes were stored at 4 °C, but showed increasing changes thereafter. However, some more rapid changes were observed such as increases in TCA cycle metabolites after 2 h post-mortem, whereas some metabolites such as fatty acids only showed decreases in concentration from 24 h. A total of 42 metabolites were identified across the ocular tissues by GC-MS (MSI level 1) and 2782 metabolites were annotated by UHPLC-MS (MSI level 2) according to MSI reporting standards. Many of the metabolites detected were common to all of the tissues but some metabolites showed partitioning between different ocular structures with 655, 297, 93 and 13 metabolites being uniquely detected in the retina, lens, cornea and vitreous respectively. Only a small percentage (1.6%) of metabolites found in the vitreous were only detected in the retina and not other tissues. In conclusion, mass spectrometry-based techniques have been used for the first time to compare the metabolic composition of different ocular tissues. The metabolite composition of the retina stored at 4 °C post-mortem is mostly stable for at least 8 h.

The essential trace metals iron, zinc, and copper play important roles both in retinal physiology and disease. They are involved in various retinal functions such as phototransduction, the visual cycle, and the process of neurotransmission, being tightly bound to proteins and other molecules to regulate their structure and/or function or as unbound free metal ions. Elevated levels of "free" or loosely bound metal ions can exert toxic effects, and in order to maintain homeostatic levels to protect retinal cells from their toxicity, appropriate mechanisms exist such as metal transporters, chaperones, and the presence of certain storage molecules that tightly bind metals to form nontoxic products. The pathways to maintain homeostatic levels of metals are closely interlinked, with various metabolic pathways directly and/or indirectly affecting their concentrations, compartmentalization, and oxidation/reduction states. Retinal deficiency or excess of these metals can result from systemic depletion and/or overload or from mutations in genes involved in maintaining retinal metal homeostasis, and this is associated with retinal dysfunction and pathology. Iron accumulation in the retina, a characteristic of aging, may be involved in the pathogenesis of retinal diseases such as age-related macular degeneration (AMD). Zinc deficiency is associated with poor dark adaptation. Zinc levels in the human retina and RPE decrease with age in AMD. Copper deficiency is associated with optic neuropathy, but retinal function is maintained. The changes in iron and zinc homeostasis in AMD have led to the speculation that iron chelation and/or zinc supplements may help in its treatment.

Background Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. Methods We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). Results Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. Conclusions Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.

PURPOSE:To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD).DESIGN:Case series.PARTICIPANTS:A total of 562 patients diagnosed with IRD.METHODS:We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data.MAIN OUTCOME MEASURES:Diagnostic yield of genomic testing.RESULTS:Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield.CONCLUSIONS:We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS.Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Background: Recently, we demonstrated that the glycoprotein opticin is anti-angiogenic. Here, the underpining mechanism is explored. Results: By binding to collagen, opticin competitively inhibits integrin-mediated endothelial cell adhesion. Conclusion: Opticin inhibits angiogenesis by weakening endothelial cell adhesion to the surrounding extracellular matrix. Significance: Elucidating the regulatory mechanisms of angiogenesis is important for understanding pathology and drug discovery., Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and MatrigelTM. We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α1β1 and α2β1 integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.