Ocular surface immune transcriptome and tear cytokines in corneal infection patients.

Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1ß, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.

Reduction in Ophthalmic Presentations to Australian Emergency Departments During the COVID-19 Period: Are We Seeing the Full Picture?

PURPOSE: To examine changing patterns of ophthalmic presentations to emergency departments (EDs) during the lockdowns associated with the first wave of the COVID-19 pandemic in Australia and the two months immediately following lockdown relaxation. PATIENTS AND METHODS: This was a retrospective audit of triage coding and ICD-10-AM coding for all patient presentations to four Australian EDs from March 29 to May 31 in 2019 and 2020 (the COVID-19 lockdown period and the corresponding period in 2019), and from June 1 to July 31 in 2019 and 2020 (the post-lockdown period and the corresponding period in 2019). Number of ophthalmic presentations triaged per day and number of seven common and/or time-sensitive, vision threatening ophthalmic diagnoses were examined. Differences in mean daily presentation numbers were assessed with non-paired Student's t-test with Bonferroni correction. RESULTS: Total ophthalmic presentations per day during COVID-19 lockdowns fell by 16% compared to the corresponding period in 2019 (13.0 ± 4.0 in 2019 vs 10.8 ± 3.3 in 2020, mean ± standard deviation; p=0.01). There was also a significant decrease in presentations of atraumatic retinal detachment, conjunctivitis, and eye pain. In the two months following easing of lockdown restrictions, total ophthalmic presentations per day returned to the same level as that of the corresponding period in 2019 (12.2 ± 4.3 in 2019 vs 12.3 ± 4.1 in 2020, p=0.97). CONCLUSION: Total ophthalmic presentations and presentations of atraumatic retinal detachment, conjunctivitis and, eye pain to EDs fell during the lockdowns associated with the first wave of COVID-19 in Australia. These may represent delays in patients seeking appropriate medical attention and may have implications on patient morbidity long after the COVID-19 pandemic.

A technique for shotgun proteomic analysis of the precorneal tear film in dogs with naturally occurring primary glaucoma.

OBJECTIVE: To introduce a protocol for the characterization of protein patterns in tears of dogs with primary angle closure glaucoma (PACG) and primary open-angle glaucoma (POAG). ANIMALS: Nineteen dogs (25 eyes). METHODS: Tear samples were collected using a Schirmer tear strip, from dogs with PACG (PACG-affected eyes, n = 8; unaffected eyes predisposed to PACG, n = 7), POAG (n = 4), and healthy controls (n = 6). Protein precipitation and trypsin digestion were performed for analyses via liquid chromatography-tandem mass spectrometry. Proteins were identified using the SwissProt protein sequence database. Relative protein expression in 17 eyes (15 dogs) was evaluated using Proteome Discoverer 2.0. Pathway analyses were performed to investigate molecular mechanisms associated with primary glaucoma. RESULTS: Unique peptides were identified in 505 proteins, with Major allergen Can f 1 and albumin identified with high confidence. Proteins unique to tears from diseased eyes (PACG: n = 7; POAG: n = 14) were identified. Nucleoside diphosphate was unique to tears in PACG eyes naïve to therapy, while retinal binding protein and NSFL1 cofactor p47 were unique to medicated PACG eyes. Relative expression of 34 proteins differed between disease states. Pathway analyses identified that the 'inflammatory response' was among the top disease/disorders in dogs with primary glaucoma (PACG and POAG) but not in healthy controls. CONCLUSION: Tear samples suitable for mass spectrometry were readily obtained from pet dogs without needing specialized equipment. Further studies to validate the findings and explore potential candidate biomarkers for early disease detection and potential therapeutic targets are indicated.

Evaluating the cost and wait-times of a task-sharing model of care for diabetic eye care: a case study from Australia.

OBJECTIVES: To determine whether a collaborative model of care that uses task-sharing for the management of low-risk diabetic retinopathy, Community Eye Care (C-EYE-C), can improve access to care and better use resources, compared with hospital-based care. DESIGN: Retrospective audit of medical and financial records to compare two models of care. SETTING: A large, urban tertiary Australian publicly funded hospital. INTERVENTION: C-EYE-C is a collaborative care model, involving community-based optometrist assessment and 'virtual review' by ophthalmologists to manage low-risk patients. The C-EYE-C model of care was implemented from January to October 2017. PARTICIPANTS: New low-risk patient referrals with diabetes received at a tertiary hospital ophthalmology unit. PRIMARY AND SECONDARY OUTCOMES: Historical standard hospital care was compared with C-EYE-C for attendance, wait-times, outcomes and costs. Clinical concordance between the optometrist and ophthalmologist diagnosis and management was assessed using weighted kappa statistic. RESULTS: There were 133 new low-risk referrals, managed in standard hospital care (n=68) and C-EYE-C (n=65). Attendance rates were similar between the models of care (72.1% hospital vs 67.7% C-EYE-C, p=0.71). C-EYE-C had shorter appointment wait-time (53 vs 118 days, p<0.01). In the C-EYE-C model of care, 68.2% of patients did not require hospital appointments and costs were 43% less than hospital care. There was substantial agreement between optometrists and ophthalmologists for diagnosis (κ=0.64, CI 0.47-0.81) and management (κ=0.66, CI 0.45-0.87). CONCLUSION: This Australian study showed that collaborative eye care resulted in reduced patient waiting times and considerable cost-savings, while maintaining a high standard of patient care compared with traditional hospital-based care in the management of low-risk hospital referrals with diabetic eye disease. The improved access and reduced costs were largely the result of better task allocation through greater utilisation of primary eye care professionals to provide services for low-risk patients. Better resource use may free up further resources for other eye care services.

Multitrait analysis of glaucoma identifies new risk loci and enables polygenic prediction of disease susceptibility and progression.

Glaucoma, a disease characterized by progressive optic nerve degeneration, can be prevented through timely diagnosis and treatment. We characterize optic nerve photographs of 67,040 UK Biobank participants and use a multitrait genetic model to identify risk loci for glaucoma. A glaucoma polygenic risk score (PRS) enables effective risk stratification in unselected glaucoma cases and modifies penetrance of the MYOC variant encoding p.Gln368Ter, the most common glaucoma-associated myocilin variant. In the unselected glaucoma population, individuals in the top PRS decile reach an absolute risk for glaucoma 10 years earlier than the bottom decile and are at 15-fold increased risk of developing advanced glaucoma (top 10% versus remaining 90%, odds ratio = 4.20). The PRS predicts glaucoma progression in prospectively monitored, early manifest glaucoma cases (P = 0.004) and surgical intervention in advanced disease (P = 3.6 × 10-6). This glaucoma PRS will facilitate the development of a personalized approach for earlier treatment of high-risk individuals, with less intensive monitoring and treatment being possible for lower-risk groups.

Improving Patient Access and Reducing Costs for Glaucoma with Integrated Hospital and Community Care: A Case Study from Australia.

INTRODUCTION: Glaucoma, a chronic eye disease requires regular monitoring and treatment to prevent vision-loss. In Australia, most public ophthalmology departments are overburdened. Community Eye Care is a 'collaborative' care model, involving community-based optometrist assessment and 'virtual review' by ophthalmologists to manage low-risk patients. C-EYE-C was implemented at one Australian hospital. This study aims to determine whether C-EYE-C improves access to care and better utilises resources, compared to hospital-based care. METHODS: A clinical and financial audit was conducted to compare access to care and health system costs for hospital care and C-EYE-C. Attendance, wait-time, patient outcomes, and the average cost per encounter were calculated. A weighted kappa assessed agreement between the optometrist and ophthalmologist decisions. RESULTS: There were 503 low-risk referrals, hospital (n = 182) and C-EYE-C (n = 321). C-EYE-C had higher attendance (81.6% vs 68.7%, p = 0.001); and shorter appointment wait-time (89 vs 386 days, p < 0.001). Following C-EYE-C, 57% of patients avoided hospital; with 39% requiring glaucoma management. C-EYE-C costs were 22% less than hospital care. There was substantial agreement between optometrists and ophthalmologist for diagnosis (k = 0.69, CI 0.61-0.76) and management (k = 0.66, CI 0.57-0.74). DISCUSSION: C-EYE-C showed higher attendance, and reduced wait-times and health system costs. CONCLUSIONS: Upscale of the C-EYE-C model should be considered to further improve capacity of public eye services in Australia.

Can the appropriateness of eye care be measured through cross-sectional retrospective patient record review in eye care practices in Australia? The iCareTrack feasibility study.

OBJECTIVES: The CareTrack study found that a wide range of appropriateness of care (ie, care in line with evidence-based or consensus-based guidelines) was delivered across many health conditions in Australia. This study therefore aimed to demonstrate the feasibility of using the CareTrack method (a retrospective onsite record review) to measure the appropriateness of eye care delivery. DESIGN: Cross-sectional feasibility study. SETTING AND PARTICIPANTS: Two hundred and thirteen patient records randomly selected from eight optometry and ophthalmology practices in Australia, selected through a combination of convenience and maximum variation sampling. METHODS: Retrospective record review designed to assess the alignment between eye care delivered and 93 clinical indicators (Delphi method involving 11 experts) extracted from evidence-based clinical practice guidelines. PRIMARY OUTCOME MEASURE: Number of eligible patient records, sampling rates and data collection time. This feasibility study also tested the ability of 93 clinical indicators to measure percentage appropriate eye care for preventative, glaucoma and diabetic eye care. A secondary outcome was the percentage of practitioner-patient encounters at which appropriate eye care was received. RESULTS: A median of 20 records (range 9 to 63) per practice were reviewed. Data collection time ranged from 3 to 5.5 hours (median 3.5). The most effective sampling strategy involved random letter generation followed by sequential sampling. The appropriateness of care was 69% (95% CI 67% to 70%) for preventative eye care, 60% (95% CI 56% to 58%) for glaucoma and 63% (95% CI 57% to 69%) for diabetic eye care. CONCLUSIONS: Appropriateness of eye care can be measured effectively using retrospective record review of eye care practices and consensus-based care indicators.

Myocilin Gene Gln368Ter Variant Penetrance and Association With Glaucoma in Population-Based and Registry-Based Studies.

Importance: The p.Gln368Ter (rs74315329) risk allele in the myocilin gene (MYOC) was initially reported to have high penetrance in glaucoma registry-based studies, but much lower estimates were recently obtained from population-based studies. We investigated this disparity using data from Australia and the United Kingdom. Objectives: To examine the penetrance and effect size of the MYOC p.Gln368Ter variant with glaucoma and ocular hypertension (OHT). Design, Setting, and Participants: This cross-sectional study within the UK Biobank (UKBB) included participants of white British ancestry. Glaucoma cases were defined by International Classification of Diseases, Ninth Revision (ICD-9) and Tenth Revision (ICD-10) diagnoses and self-reported questionnaires. Carriers of the MYOC p.Gln368Ter variant were identified using genotype imputation from arrays. In contrast, 2 Australian registry-based studies, the Australian and New Zealand Registry of Advanced Glaucoma and the Glaucoma Inheritance Study in Tasmania, ascertained glaucoma cases referred by eye care clinicians, with historic control participants recruited from other Australian studies. Samples were either directly sequenced or had genotypes determined by imputation (for the Australian registry and historic control participants). Recruitment to the UKBB occurred between 2006 and 2010, and data analysis occurred from September 2017 to July 2018. Main Outcomes and Measures: The penetrance and odds ratio (OR) were estimated for the MYOC p.Gln368Ter variants in participants with glaucoma and OHT. Results: A total of 411 337 UKBB participants of white British ancestry (mean [SD] age, 56.6 [8.0] years) were included, plus 3071 Australian registry and 6750 historic control participants. In the UKBB, the minor allele frequency of the MYOC p.Gln368Ter variant was 1 in 786 individuals (0.13%). The odds ratio of p.Gln368Ter in patients with primary open-angle glaucoma (POAG) was 6.76 (95% CI, 4.05-11.29); glaucoma (POAG, self-reported glaucoma, and unspecified glaucoma), 4.40 (95% CI, 3.38-5.71); OHT, 3.56 (95% CI, 2.53-4.92); and OHT and glaucoma combined, 4.18 (95% CI, 3.05-5.67). The penetrance of the MYOC p.Gln368Ter variant was 7.6% in patients with glaucoma, 24.3% in patients with OHT, and 30.8% in patients with OHT and glaucoma combined. In the Australian registry studies, the odds of MYOC p.Gln368Ter variant were 12.16 (95% CI, 6.34-24.97) in patients with advanced glaucoma and 3.97 (95% CI, 1.55-9.75) in those with nonadvanced glaucoma; the penetrance of glaucoma was 56.1%, and penetrance in those considered to have glaucoma or be glaucoma suspects was 69.5%. Conclusions and Relevance: The MYOC p.Gln368Ter variant confers a very high-risk effect size for advanced glaucoma; the risk is lower in nonadvanced glaucoma and OHT. In the general population sample, approximately 50% of MYOC p.Gln368Ter carriers 65 years and older had glaucoma or OHT, with higher prevalence in the Australian registry studies.

Genome-wide association study of intraocular pressure uncovers new pathways to glaucoma.

Intraocular pressure (IOP) is currently the sole modifiable risk factor for primary open-angle glaucoma (POAG), one of the leading causes of blindness worldwide1. Both IOP and POAG are highly heritable2. We report a combined analysis of participants from the UK Biobank (n = 103,914) and previously published data from the International Glaucoma Genetic Consortium (n = 29,578)3,4 that identified 101 statistically independent genome-wide-significant SNPs for IOP, 85 of which have not been previously reported4-12. We examined these SNPs in 11,018 glaucoma cases and 126,069 controls, and 53 SNPs showed evidence of association. Gene-based tests implicated an additional 22 independent genes associated with IOP. We derived an allele score based on the IOP loci and loci influencing optic nerve head morphology. In 1,734 people with advanced glaucoma and 2,938 controls, participants in the top decile of the allele score were at increased risk (odds ratio (OR) = 5.6; 95% confidence interval (CI): 4.1-7.6) of glaucoma relative to the bottom decile.

Inhibitory Effects of Angiotensin II Receptor Blockade on Human Tenon Fibroblast Migration and Reactive Oxygen Species Production in Cell Culture.

PURPOSE: We investigate the effect of angiotensin receptor blockade on the migration of human Tenon fibroblasts (HTF), using irbesartan, an angiotensin II receptor type 1 (AT1R) blocker (ARB) as a potential antifibrotic agent in glaucoma filtration surgery. METHODS: Confluent HTF cultures were scratched with a 1 mL pipette tip and treated with either irbesartan (10, 50, and 100 µg/mL) or angiotensin II (2 µg/mL). The extent of HTF migration up to 30 hours, and cell number and morphology at 72 hours was evaluated. To assess the effect on reactive oxygen species (ROS) level, HTF were treated with either irbesartan (10 µg/mL) or angiotensin II (2 µg/mL) for 24 hours after scratching, and then stained with dihydroethidium (DHE) before evaluation by confocal microscopy. RESULTS: Irbesartan inhibited HTF migration by 50% to 70% compared to controls (P < 0.05). Levels of ROS were almost completely attenuated by irbesartan (DHE fluorescence intensity of 5.68E-09) (P < 0.05). Irbesartan reduced cell numbers by 50% and induced morphologic changes with loss of pseudopods (P < 0.05). Conversely, angiotensin II increased cell numbers up to 4-fold while retaining cell viability. CONCLUSIONS: Irbesartan inhibited HTF migration and ROS production. It also reduced cell numbers and altered HTF morphology. Angiotensin II increased cell number without altering morphology. This initial study warrants future investigations for further potential antifibrotic effects of this drug. TRANSLATIONAL RELEVANCE: This in vitro study focused on investigations of irbesartan's effects on HTF migration, ROS production, as well as HTF cell numbers and morphology. It suggests a potential therapeutic strategy worth further exploration with a view towards postoperative wound healing modulation in glaucoma filtration surgery.

Oxidative stress and reactive oxygen species: a review of their role in ocular disease.

For many years, oxidative stress arising from the ubiquitous production of reactive oxygen species (ROS) has been implicated in the pathogenesis of various eye diseases. While emerging research has provided some evidence of the important physiological role of ROS in normal cell function, disease may arise where the concentration of ROS exceeds and overwhelms the body's natural defence against them. Additionally, ROS may induce genomic aberrations which affect cellular homoeostasis and may result in disease. This literature review examines the current evidence for the role of oxidative stress in important ocular diseases with a view to identifying potential therapeutic targets for future study. The need is particularly pressing in developing treatments for conditions which remain notoriously difficult to treat, including glaucoma, diabetic retinopathy and age-related macular degeneration.

The Capacity of Eye Care Services for Patients with Glaucoma in Botswana.

PURPOSE: To evaluate current delivery of glaucoma care in Botswana; in particular, the service infrastructure available and glaucoma-related workload. METHODS: A multi-center cross-sectional study was undertaken comprising government eye care institutions and ophthalmic personnel across Botswana. Data on human resources, equipment types and numbers, diagnostic criteria routinely used, treatments routinely provided, and new and repeat glaucoma consultations were obtained through quantitative and qualitative surveys. RESULTS: In 27 government eye care institutions there were two general ophthalmologists, neither of whom had a subspecialty interest in glaucoma, 64 ophthalmic nurses, two optometrists, one low vision therapist, one refractionist, and two equipment technicians. Only 8.5% of available ophthalmic human resources were taken up with provision of glaucoma care. About 1/3 of hospitals did not have tonometers, most primary hospitals lacked slit lamp biomicroscopes and most hospitals lacked sensitive diagnostic equipment. A diagnosis of glaucoma was made by either an ophthalmic nurse or an ophthalmologist, but only 10% of institutions could meet recommendations for follow-up assessment. Topical glaucoma medications were prescribed by almost all hospital clinics, usually by ophthalmic nurses. Drug choices were largely determined by local availability. Glaucoma surgery accounted for 0.8% of total eye operations. Glaucoma patients took up 8.5% of total clinic visits. The total number of glaucoma visits was highest in the two hospitals with ophthalmologists. New glaucoma cases took up 10.3% of total glaucoma visits. CONCLUSION: This study highlights the challenges faced in caring for glaucoma patients in Botswana; in particular, lack of professional human resources, equipment and availability of effective treatments.

Retinal ganglion cell neuroprotection by an angiotensin II blocker in an ex vivo retinal explant model.

PURPOSE: An ex vivo organotypic retinal explant model was developed to examine retinal survival mechanisms relevant to glaucoma mediated by the renin angiotensin system in the rodent eye. METHODS: Eyes from adult Sprague Dawley rats were enucleated immediately post-mortem and used to make four retinal explants per eye. Explants were treated either with irbesartan (10 µM), vehicle or angiotensin II (2 µM) for four days. Retinal ganglion cell density was estimated by ßIII tubulin immunohistochemistry. Live imaging of superoxide formation with dihydroethidium (DHE) was performed. Protein expression was determined by Western blotting, and mRNA expression was determined by RT-PCR. RESULTS: Irbesartan (10 µM) almost doubled ganglion cell survival after four days. Angiotensin II (2 µM) reduced cell survival by 40%. Sholl analysis suggested that irbesartan improved ganglion cell dendritic arborisation compared to control and angiotensin II reduced it. Angiotensin-treated explants showed an intense DHE fluorescence not seen in irbesartan-treated explants. Analysis of protein and mRNA expression determined that the angiotensin II receptor At1R was implicated in modulation of the NADPH-dependent pathway of superoxide generation. CONCLUSION: Angiotensin II blockers protect retinal ganglion cells in this model and may be worth further investigation as a neuroprotective treatment in models of eye disease.

Severe intraocular pressure response to periocular or intravitreal steroid treatment in Australia and New Zealand: data from the Australian and New Zealand Ophthalmic Surveillance Unit.

BACKGROUND: Increase in intraocular pressure is a recognized complication of corticosteroid treatment via intravitreal or periocular injections for treatment of a range of conditions including macular oedema and retinal neovascularization. DESIGN: This surveillance study was designed to determine the incidence and nature of severe intraocular pressure elevation as a complication of intravitreal or periocular corticosteroid injections in Australia and New Zealand. PARTICIPANTS: Seventeen cases meeting the defined criteria of severe intraocular pressure elevation, above 35 mmHg, following an intravitreal or periocular corticosteroid injection were included in the study. METHODS: Over an 18-month period, ophthalmologists were invited to report cases to the Australian and New Zealand Ophthalmic Surveillance Unit. After reporting, further demographic and clinical information was sought via a follow-up questionnaire. MAIN OUTCOME MEASURES: Intraocular pressure elevation above 35 mmHg. RESULTS: Follow-up questionnaires were received for 20 cases of 34 initially reported to the unit. Seventeen met the defined criteria. Triamcinolone acetonide was used in all 17 cases, with 16 delivered as a 4-mg intravitreal injection. There was an absence of identified underlying risk factors in the majority of cases with personal history of glaucoma in 2 of 17 cases. No cases reported a positive family history of glaucoma. Trabeculectomy was performed in 8 of 17 patients (47%) for intraocular pressure management. CONCLUSIONS: Severe intraocular pressure elevation following intravitreal or periocular corticosteroid injection can occur in the absence of risk factors such as personal and family history of glaucoma. The severe intraocular pressure elevation may ultimately require trabeculectomy.

Association of open-angle glaucoma loci with incident glaucoma in the Blue Mountains Eye Study.

PURPOSE: To determine if open-angle glaucoma (OAG)-associated single nucleotide polymorphisms (SNPs) are associated with incident glaucoma and if such genetic information is useful in OAG risk prediction. DESIGN: Case-control from within a population-based longitudinal study. METHODS: study population: Individuals aged over 49 years of age living in the Blue Mountains region west of Sydney and enrolled in the Blue Mountains Eye Study. observation: Cases for this sub-study (n = 67) developed incident OAG between baseline and 10-year visits, in either eye, while controls (n = 1919) had no evidence for OAG at any visit. All participants had an ocular examination and DNA genotyped for reported OAG risk SNPs. main outcome measure: Incident OAG. RESULTS: Two loci also known to be associated with cup-to-disc ratio as well as OAG (9p21 near CDKN2B-AS1 and SIX1/SIX6) were both significantly associated with incident OAG in the Blue Mountains Eye Study cohort (P = .006 and P = .004, respectively). The TMCO1 locus was nominally associated (P = .012), while the CAV1/CAV2 and 8q22 loci were not associated. Multivariate logistic regression and neural network analysis both indicated that the genetic risk factors contributed positively to the predictive models incorporating traditional risk factors. CONCLUSIONS: This study shows that previously reported genetic variations related to OAG and cup-to-disc ratio are associated with the onset of OAG and thus may become useful in risk prediction algorithms designed to target early treatment to those most at risk of developing glaucoma.

Expression of classical components of the renin-angiotensin system in the human eye.

PURPOSE: The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. METHODS: We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). RESULTS: Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. CONCLUSION: This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions.

Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

BACKGROUND: The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. METHODS: A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. RESULTS: Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. CONCLUSIONS: Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression.