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Objectives Methods Results Conclusions Patients with polypoidal choroidal vasculopathy (PCV) exhibit variability in response to anti‐VEGF therapy. This study aimed to analyse the aqueous humour proteomic profiles of PCV patients and provide preliminary insights for the identification of biomarkers associated with anti‐VEGF drug responsiveness.PCV patients who were treatment‐naïve or untreated for more than 3 months were prospectively recruited from two hospitals in Beijing and Tianjin. Based on the relative changes in central macular thickness (ΔCMT/baseline‐CMT) before and after anti‐VEGF treatment, the PCV patients were divided into a good response (GR) group (≤−25%) and a poor response (PR) group (>−25%). Aqueous humour proteomics was performed by the Data‐independent Acquisition‐Mass Spectrometry (DIA‐MS) method, and differentially expressed proteins (DEPs) analysis between the different PCV groups and the control group was conducted. Key DEPs were selected for preliminary validation in the aqueous humour using the Luminex method retrospectively.A total of 31 PCV patients (31 eyes) were included, 13 in the GR group and 18 in the PR group. A total of 414 DEPs were identified, including 36 significantly upregulated proteins, such as G protein regulatory factor 10 (RGS10), podocin (PODN) and epidermal growth factor (EGF), and 32 downregulated proteins, including RAB11FIP4 (Rab11 family‐interacting protein 4), α‐synuclein (SNCA), haemoglobin subunit δ (HBD) and interleukin 6 (IL6). Compared to the cataract control group (10 eyes), 134 proteins were significantly upregulated, and 72 were downregulated. KEGG pathway enrichment analysis revealed that the GR and PR groups differ in terms of cell communication, and cell signal transduction. Protein–protein interaction analysis revealed interactions between EGF and various DEPs. Validation of aqueous humour proteins using the Luminex method revealed that changes in the levels of EGF were associated with the anti‐VEGF treatment response in PCV patients.PCV patients with good or poor anti‐VEGF responses exhibit distinct aqueous humour proteomic profiles. Aqueous EGF may serve as a biomarker for the ‘precise treatment’ of PCV. [ABSTRACT FROM AUTHOR]

ABSTRACTTo explore the role of lncRNA m6A methylation modification in aqueous humour (AH) of patients with pseudoexfoliation glaucoma (PXG). Patients with open-angle PXG under surgery from June 2021 to December 2021 were selected. Age- and gender-matched patients with age-related cataract (ARC) were chosen as control. Patients underwent detailed ophthalmic examinations. 0.05–0.1 ml AH were extracted during surgery for MeRIP-Seq and RNA-Seq. Joint analysis was used to screen lncRNAs with differential m6A methylation modification and expression. Online software tools were used to draw lncRNA-miRNA-mRNA network (ceRNA). Expression of lncRNAs and mRNAs was confirmed using quantitative real-time PCR. A total of 4151 lncRNAs and 4386 associated m6A methylation modified peaks were identified in the PXG group. Similarly, 2490 lncRNAs and 2595 associated m6A methylation modified peaks were detected in the control. Compared to the ARC group, the PXG group had 234 hypermethylated and 402 hypomethylated m6A peaks, with statistically significant differences (| Fold Change (FC) |≥2, p

Simple Summary: Identifying the genetic cause of a retinoblastoma tumour can help doctors determine any future cancer risk for the patient or their family. Somatic testing has historically used DNA from the tumour, after the eye has been removed. We and others have previously shown cell-free DNA (cfDNA) from the tumour is present in the eye fluid of retinoblastoma patients. In this study we tested eye fluid from 68 patients, collected at different points in their treatment. Measurable levels of cfDNA were found in all 11 samples of eye fluid taken from patients who had received less than three cycles of chemotherapy, and 95% (21/22) of causal genetic variants were identified. Eye fluid collected later in the treatment had 150 times less cfDNA and only 46% of genetic variants (25/54) could be detected. Eye fluid sampling early in treatment is therefore likely to be required for successful somatic testing in retinoblastoma patients undergoing eye conservation treatment. The identification of somatic RB1 variation is crucial to confirm the heritability of retinoblastoma. We and others have previously shown that, when tumour DNA is unavailable, cell-free DNA (cfDNA) derived from aqueous humour (AH) can be used to identify somatic RB1 pathogenic variation. Here we report RB1 pathogenic variant detection, as well as cfDNA concentration in an extended cohort of 75 AH samples from 68 patients. We show cfDNA concentration is highly variable and significantly correlated with the collection point of the AH. Cell-free DNA concentrations above 5 pg/µL enabled the detection of 93% of known or expected RB1 pathogenic variants. In AH samples collected during intravitreal chemotherapy treatment (Tx), the yield of cfDNA above 5 pg/µL and subsequent variant detection was low (≤46%). However, AH collected by an anterior chamber tap after one to three cycles of primary chemotherapy (Dx1+) enabled the detection of 75% of expected pathogenic variants. Further limiting our analysis to Dx1+ samples taken after ≤2 cycles (Dx ≤ 2) provided measurable levels of cfDNA in all cases, and a subsequent variant detection rate of 95%. Early AH sampling is therefore likely to be important in maximising cfDNA concentration and the subsequent detection of somatic RB1 pathogenic variants in retinoblastoma patients undergoing conservative treatment. [ABSTRACT FROM AUTHOR]

This paper aimed at devising an intelligence-based method to select compounds that can distinguish between open-angle glaucoma patients, type 2 diabetes patients, and healthy controls. Taking the concentration of 188 compounds measured in the aqueous humour (AH) of patients and controls, linear discriminant analysis (LDA) was used to identify the right combination of compounds that could lead to accurate diagnosis. All possibilities, using the leave-one-out approach, were considered through ad hoc programming and in silico massive data production and statistical analysis. Our proof of concept led to the selection of four molecules: acetyl-ornithine (Ac-Orn), C3 acyl-carnitine (C3), diacyl C42:6 phosphatidylcholine (PC aa C42:6), and C3-DC (C4-OH) acyl-carnitine (C3-DC (C4-OH)) that, taken in combination, would lead to a 95% discriminative success. 100% success was obtained with a non-linear combination of the concentration of three of these four compounds. By discarding younger controls to adjust by age, results were similar although one control was misclassified as a diabetes patient. Methods based on the consideration of individual clinical chemical parameters have limitations in the ability to make a reliable diagnosis, stratify patients, and assess disease progression. Leveraging human AH metabolomic data, we developed a procedure that selects a minimal number of metabolites (3–5) and designs algorithms that maximize the overall accuracy evaluating both positive predictive (PPV) and negative predictive (NPV) values. Our approach of simultaneously considering the levels of a few metabolites can be extended to any other body fluid and has potential to advance precision medicine. Artificial intelligence is expected to use algorithms that use the concentration of three to five molecules to correctly diagnose diseases, also allowing stratification of patients and evaluation of disease progression. In addition, this significant advance shifts focus from a single-molecule biomarker approach to that of an appropriate combination of metabolites. [ABSTRACT FROM AUTHOR]

BackgroundThe iridocorneal angle (ICA) is the major pathway of aqueous humour outflow from the anterior chamber of the eye. Ultrasound biomicroscopy (UBM) has been utilised to characterise the morphology of this drainage pathway in numerous species. UBM may allow for early recognition of aqueous humour outflow obstructions in horses, allowing for earlier recognition of risk for glaucoma, a vision-threatening and painful disease. UBM morphology of the normal equine ICA has yet to be described.ObjectivesTo determine the ultrasonographic morphology of the equine ICA by UBM in standing sedated horses.Study designIn vivo experimental study.MethodsThirty healthy adult horses underwent UBM of the ICA at four locations (superior, temporal, inferior, nasal) of each eye utilising standing sedation, topical anaesthesia and auriculopalpebral perineural anaesthesia. Anatomic structures were defined on ultrasound images through comparison to published histologic photomicrographs of the equine ICA.ResultsUltrasound imaging of the ICA at all four locations was easily performed in standing, sedated horses. High-resolution images of the ICA allowed for identification of the pectinate ligament, corneoscleral trabecular meshwork (TM), uveal TM and supraciliary TM.Main limitationsPupil size was midrange in all eyes, but was not strictly controlled. Lighting conditions not controlled. Various breeds included.ConclusionIn vivo UBM of the equine ICA is feasible and provides high-resolution images of the structures of the aqueous humour outflow pathway.

Primary angle‐closure glaucoma (PACG) is the leading cause of irreversible blindness in the world. Angle closure induced by pupil block and secondary iris synechia is the fundamental pathology of the PACG. The molecular mechanisms of angle closure have not yet been clearly illustrated. This study was designed to investigate the protein difference in the aqueous humour and explore new biomarker of the PACG. Aqueous humour (AH) was collected from patients with acute primary angle closure (APAC) and cataract (n = 10 in APAC group) and patients with cataract only (n = 10 in control group). Samples were pooled and measured using label‐free proteome technology. Then, the differentially expressed proteins (DEPs) were verified by ELISA using independent AH samples (n = 20 each group). More than 400 proteins were revealed in both groups through proteomics. Comparing the two groups, there were 91DEPs. These proteins participate in biological activities such as inflammation, fibrosis, nerve growth and degeneration and metabolism. We found that the expression of transforming growth factor‐β2 and matrilin2 was downregulated in the APAC group. The two proteins are related to inflammation and extracellular matrix formation, which might be involved in angle closure. This study characterized DEPs in AH of the APAC and found a downregulated protein matrilin2. [ABSTRACT FROM AUTHOR]

Abstract Background Viral nucleic acid testing of intraocular fluid using polymerase chain reaction (PCR) is a major laboratory examination in the diagnosis of acute retinal necrosis (ARN). Importantly, false negative PCR results may occur in several special situations. We reported a case of ARN with a negative PCR result in the aqueous humour in the very early stages of disease. Case presentation A female patient presented to the ophthalmologist with complaints of blurred vision and redness in her left eye. Her medical history included ARN in her right eye 10 years prior. Although the result of the aqueous viral analysis by PCR in her left eye was negative the first time (one day after the appearance of ocular symptoms), ARN in her left eye was presumed based on the clinical signs. With timely antiviral and anti-inflammatory treatments, the retinal lesions diminished. The viral load of herpes simplex virus (HSV) turned positive (7.25 × 103 copies/mL) one week later, increased to 2.49 × 105 copies/mL after three weeks, and finally turned negative about five weeks after the onset of disease. The initial HSV-IgG level in the aqueous humour was 0.01 U/mL and increased to 222.64 U/mL in the final sampling. Conclusions The results of PCR analysis can be negative in the very early stages of ARN. Diagnosis of ARN should be made based on the clinical features, and antiviral treatments should not be delayed. Repeated PCR analysis of the aqueous humour is necessary to confirm the diagnosis and monitor the disease process.

Abstract Background Neovascular age-related macular degeneration (nAMD) is a major cause of blindness in the elderly. The disease is due to the growth of abnormal blood vessels into the macula, leading to the loss of central vision. Intravitreal injection of vascular endothelial growth factor (VEGF) inhibitors (e.g., anti-VEGF) is the standard of care for nAMD. However, nearly 50% of patients do not respond or respond poorly to the therapy. More importantly, up to 70% of nAMD patients develop macular fibrosis after 10 years of anti-VEGF therapy. The underlying mechanism of nAMD-mediated macular fibrosis is unknown although inflammation is known to play an important role in the development of abnormal macular blood vessels and its progression to fibro-vascular membrane. In this study, we measured the intraocular levels of adhesion molecule VCAM-1, ICAM-1, CD44, CD62L, and CD62P in nAMD patients with and without macular fibrosis and investigated the link between the levels of adhesion molecule and clinical features (e.g., visual improvement, retinal thickness, etc.). We further investigated the effect of VCAM-1 in macrophage function in vitro and the development of subretinal fibrosis in vivo using a two-stage laser-induced protocol. Results The aqueous levels of ICAM-1, VCAM-1, CD44, and CD62L were significantly higher in nAMD patients compared to cataract controls. The aqueous level of VCAM-1 (but not other adhesion molecules) was significantly higher in patients with macular fibrosis than those without and the level correlated positively with the retinal thickness. VCAM-1 was highly expressed at the lesion site in the mouse model of subretinal fibrosis. Blocking VCAM-1 or its receptor VLA-4 significantly prevented macrophage infiltration and reduced subretinal fibrosis in vivo. VCAM-1 induced macrophage migration and upregulated the expression of Arg-1, Mmp12 and Il6 but down-regulated the expression of iNOS and Il1b in macrophages. Conclusions VCAM-1 may contribute to the development of macular fibrosis in nAMD patients by modulating macrophage functions, including migration and profibrotic polarization.

Abstract Background The aim was to study aqueous humour inflammatory mediators’ levels in a cohort of Egyptian patients with diabetic macular oedema (DMO). Methods This was a case-control prospective study conducted on 2 groups: 25 eyes of 22 (11 females) patients seeking treatment for DMO as patients group, and 10 eyes of 10 (4 females) cataract patients as a control group. Aqueous humour was aspirated before intravitreal injection (patients’ group) or cataract surgery (control group). Inflammatory mediators in aqueous humour were measured using a multiplex bead immunoassay kit of 27 pre-mixed cytokines. Results Eotaxin, interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleukin-8 (IL-8/CXCL8) were found significantly higher in patients’ group compared to control group (p = 0.043, 0.037, 0.001, 0.015 respectively). On the contrary, interferon-gamma (IFN-gamma) and granulocyte colony-stimulating factor (G-CSF) were found significantly higher in control group than patients’ group (p = 0.003, 0.019 respectively). Basic fibroblast growth factor (Basic-FGF/FGF-2) and interleukin-1 receptor antagonist (IL-1ra) were found higher (but not statistically significant) in controls (p = 0.100 and 0.070 respectively). Additionally, a negative and significant correlation was found between Eotaxin level in aqueous humour and central macular thickness. Conclusions Some mediators might be implicated in the pathogenesis of DMO either augmenting or suppressing role. Eotaxin, IP-10, MCP-1 and IL-8 might have a role in cases not responding to standard anti-vascular endothelial growth factor (VEGF) therapy. IL-1ra might have a protective role; therefore, the effectiveness of intravitreal injection of IL-1ra homologue needs to be studied in future clinical trials.

Abstract Background The purpose of this study is to identify the proteomic differences between the aqueous humour of diabetes patients with cataracts and that of non-diabetic sufferers of cataracts in a clinical setting. Methods Patients were divided into the diabetic experimental group and the non-diabetic control group. Aqueous humour specimens were obtained via cataract surgery. Sample proteins were treated with a TMT reagent, separated using a cation chromatography column, and analysed using a C18 desalting column. Proteins were identified using HPLC-MS/MS. The differential proteins were identified using both a p value of 1.2. GO classification enrichment analysis, KEGG pathway enrichment analysis, protein interaction network analysis, and ingenuity pathway analysis were all carried out. The expression level of four differential proteins were verified by Western blot, and GC and TTR expressions were further examined using an expanded sample pool. Results The postprandial glucose levels between the experimental group (9.40 ± 1.35 mmol/L) and the control group (6.56 ± 0.81 mmol/L) were significantly different, with a p value of 1.16E-06. It is important to note, however, that the baseline levels of the parameters showed no statistical differences. In total, 397 aqueous humour proteins were identified; of these, 137 showed significant differences, with 63 upregulated ones and 74 down-regulated ones. The differential proteins play important roles in numerous biological processes and pathways, such as complement and coagulation cascades (p = 1.71E-09). Some of these differential proteins are associated with diabetic retinal degeneration and other diabetic complications. Differential proteins, such as HP, GC, and TTR, have high node degree in the protein interaction network. Western blot results further confirmed that GC were down-regulated while TTR was up-regulated in aqueous humour under diabetic condition. Conclusion A list of differential proteins in the human aqueous humour of diabetic patients was established. Proteins with high interaction scores as per protein interaction analysis, such as GC and TTR, were further verified and could potentially be used as early diagnostic markers for diabetic eye complications in clinical practice.

Purpose: to analyze the activity of α2-macroglobulin (α2-MG) in the tear, blood serum (BS), and aqueous humour (AH) in children with uveitis, and to assess the possibility of using α2-MG for inflammation activity estimation and prognosis.Material and methods. 76 children with uveitis (135 eyes) aged 3 to 17 (ave. 10.45 ± 3.34 years), were tested for the activity of α2-MG (300 tear samples, 154 BS samples, 39 AH samples). The dynamics of α2-MG was assessed in the tear of 56 patients, and in BS of 53 patients. α2-MG activity was determined by the enzymatic method using the specific substrate N-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The optical density was measured using a multifunctional photometer for Synergy MX microplates (BioTek, USA).Results. No correlation was found between the α2-MG activity and the inflammation activity and localization of uveitis. In contrast, a significant decrease in α2-MG activity was found in the tear after anti-inflammatory therapy (p = 0.013). Also, a significant preoperative α2-MG increase was noted in the tear of children who experienced fibrin effusion into the anterior chamber of the eye in the early postoperative period (p = 0.022) It was shown that α2-MG activity in BS drops significantly as the patients grow up (p = 0.025). As the degree of proliferation increases, the activity of α2-MG in AH also increases (p = 0.049).Conclusion. An increase of α2-MG activity in the tears in the preoperative period may be important for the prognosis of fibrin formation in the anterior chamber during the postoperative period. α2-MG in the tear is highly sensitive to anti-inflammatory drugs, which reduce its activity. In BS the highest activity of α2-MG was revealed at the age of 3–6 years, whereupon it is decreasing. In AH, the activity of α2-MG correlates with the stage of the proliferative process.

Background: The aim was to study aqueous humour inflammatory mediators' levels in a cohort of Egyptian patients with diabetic macular oedema (DMO). Methods: This was a case-control prospective study conducted on 2 groups: 25 eyes of 22 (11 females) patients seeking treatment for DMO as patients group, and 10 eyes of 10 (4 females) cataract patients as a control group. Aqueous humour was aspirated before intravitreal injection (patients' group) or cataract surgery (control group). Inflammatory mediators in aqueous humour were measured using a multiplex bead immunoassay kit of 27 pre-mixed cytokines. Results: Eotaxin, interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1/CCL2) and interleukin-8 (IL-8/CXCL8) were found significantly higher in patients' group compared to control group (p = 0.043, 0.037, 0.001, 0.015 respectively). On the contrary, interferon-gamma (IFN-gamma) and granulocyte colony-stimulating factor (G-CSF) were found significantly higher in control group than patients' group (p = 0.003, 0.019 respectively). Basic fibroblast growth factor (Basic-FGF/FGF-2) and interleukin-1 receptor antagonist (IL-1ra) were found higher (but not statistically significant) in controls (p = 0.100 and 0.070 respectively). Additionally, a negative and significant correlation was found between Eotaxin level in aqueous humour and central macular thickness. Conclusions: Some mediators might be implicated in the pathogenesis of DMO either augmenting or suppressing role. Eotaxin, IP-10, MCP-1 and IL-8 might have a role in cases not responding to standard anti-vascular endothelial growth factor (VEGF) therapy. IL-1ra might have a protective role; therefore, the effectiveness of intravitreal injection of IL-1ra homologue needs to be studied in future clinical trials. [ABSTRACT FROM AUTHOR]

Background: Viral nucleic acid testing of intraocular fluid using polymerase chain reaction (PCR) is a major laboratory examination in the diagnosis of acute retinal necrosis (ARN). Importantly, false negative PCR results may occur in several special situations. We reported a case of ARN with a negative PCR result in the aqueous humour in the very early stages of disease. Case presentation: A female patient presented to the ophthalmologist with complaints of blurred vision and redness in her left eye. Her medical history included ARN in her right eye 10 years prior. Although the result of the aqueous viral analysis by PCR in her left eye was negative the first time (one day after the appearance of ocular symptoms), ARN in her left eye was presumed based on the clinical signs. With timely antiviral and anti-inflammatory treatments, the retinal lesions diminished. The viral load of herpes simplex virus (HSV) turned positive (7.25 × 103 copies/mL) one week later, increased to 2.49 × 105 copies/mL after three weeks, and finally turned negative about five weeks after the onset of disease. The initial HSV-IgG level in the aqueous humour was 0.01 U/mL and increased to 222.64 U/mL in the final sampling. Conclusions: The results of PCR analysis can be negative in the very early stages of ARN. Diagnosis of ARN should be made based on the clinical features, and antiviral treatments should not be delayed. Repeated PCR analysis of the aqueous humour is necessary to confirm the diagnosis and monitor the disease process. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study is to identify the proteomic differences between the aqueous humour of diabetes patients with cataracts and that of non-diabetic sufferers of cataracts in a clinical setting. Methods: Patients were divided into the diabetic experimental group and the non-diabetic control group. Aqueous humour specimens were obtained via cataract surgery. Sample proteins were treated with a TMT reagent, separated using a cation chromatography column, and analysed using a C18 desalting column. Proteins were identified using HPLC-MS/MS. The differential proteins were identified using both a p value of < 0.05 and a fold change of > 1.2. GO classification enrichment analysis, KEGG pathway enrichment analysis, protein interaction network analysis, and ingenuity pathway analysis were all carried out. The expression level of four differential proteins were verified by Western blot, and GC and TTR expressions were further examined using an expanded sample pool. Results: The postprandial glucose levels between the experimental group (9.40 ± 1.35 mmol/L) and the control group (6.56 ± 0.81 mmol/L) were significantly different, with a p value of 1.16E-06. It is important to note, however, that the baseline levels of the parameters showed no statistical differences. In total, 397 aqueous humour proteins were identified; of these, 137 showed significant differences, with 63 upregulated ones and 74 down-regulated ones. The differential proteins play important roles in numerous biological processes and pathways, such as complement and coagulation cascades (p = 1.71E-09). Some of these differential proteins are associated with diabetic retinal degeneration and other diabetic complications. Differential proteins, such as HP, GC, and TTR, have high node degree in the protein interaction network. Western blot results further confirmed that GC were down-regulated while TTR was up-regulated in aqueous humour under diabetic condition. Conclusion: A list of differential proteins in the human aqueous humour of diabetic patients was established. Proteins with high interaction scores as per protein interaction analysis, such as GC and TTR, were further verified and could potentially be used as early diagnostic markers for diabetic eye complications in clinical practice. [ABSTRACT FROM AUTHOR]

Background: Neovascular age-related macular degeneration (nAMD) is a major cause of blindness in the elderly. The disease is due to the growth of abnormal blood vessels into the macula, leading to the loss of central vision. Intravitreal injection of vascular endothelial growth factor (VEGF) inhibitors (e.g., anti-VEGF) is the standard of care for nAMD. However, nearly 50% of patients do not respond or respond poorly to the therapy. More importantly, up to 70% of nAMD patients develop macular fibrosis after 10 years of anti-VEGF therapy. The underlying mechanism of nAMD-mediated macular fibrosis is unknown although inflammation is known to play an important role in the development of abnormal macular blood vessels and its progression to fibro-vascular membrane. In this study, we measured the intraocular levels of adhesion molecule VCAM-1, ICAM-1, CD44, CD62L, and CD62P in nAMD patients with and without macular fibrosis and investigated the link between the levels of adhesion molecule and clinical features (e.g., visual improvement, retinal thickness, etc.). We further investigated the effect of VCAM-1 in macrophage function in vitro and the development of subretinal fibrosis in vivo using a two-stage laser-induced protocol. Results: The aqueous levels of ICAM-1, VCAM-1, CD44, and CD62L were significantly higher in nAMD patients compared to cataract controls. The aqueous level of VCAM-1 (but not other adhesion molecules) was significantly higher in patients with macular fibrosis than those without and the level correlated positively with the retinal thickness. VCAM-1 was highly expressed at the lesion site in the mouse model of subretinal fibrosis. Blocking VCAM-1 or its receptor VLA-4 significantly prevented macrophage infiltration and reduced subretinal fibrosis in vivo. VCAM-1 induced macrophage migration and upregulated the expression of Arg-1, Mmp12 and Il6 but down-regulated the expression of iNOS and Il1b in macrophages. Conclusions: VCAM-1 may contribute to the development of macular fibrosis in nAMD patients by modulating macrophage functions, including migration and profibrotic polarization. [ABSTRACT FROM AUTHOR]

Abstract Background Ocular manifestations in dogs with leishmaniasis are frequent and complications in affected tissues can lead to blindness. Immune processes play a very important role in the pathogenesis of ocular inflammation. Therefore, the immunology of ocular manifestations in dogs with leishmaniasis remains complex and poorly understood. Objectives Estimation and characterisation of ocular and periocular manifestations in dogs naturally infected with Leishmania infantum and investigation of the production site of specific anti‐Leishmania infantum IgG. Methods The present investigation used 53 confirmed dogs infected with Leishmania infantum, presenting ocular and periocular lesions, and 10 control non‐infected dogs. Complete macroscopic ophthalmic examination of eyelids and globes was performed. Both total and anti‐Leishmania infantum IgG antibodies were studied in sera and aqueous humour (AH) of all dogs by ELISA technique. A Goldmann–Witmer coefficient (C value) was calculated. Results The main ophthalmological findings were keratoconjunctivitis (71.7%; 38/53), hyperplasia of conjunctival lymphoid follicles (54.7%; 29/53), blepharitis (50.9%; 27/53) and uveitis (20.7%; 11/53). Ocular production of anti‐Leishmania infantum IgG was detected in 73.6% (39/53) of infected dogs. There was no correlation between the antibody levels in AH and sera of the same dog. The mean anti‐Leishmania infantum IgG in AH was higher in uveitis, followed by lesions affecting only the adnexa (p < 0.0001). The highest mean C values were observed for uveitis, conjunctivitis and keratitis. Conclusions Our findings suggest that production of anti‐Leishmania IgG in dogs infected with Leishmania infantum with ocular manifestations begin in situ and follows by a transfer of antibodies from the bloodstream to the AH.

Purpose: To investigate the relationship between the intraocular levels of complement proteins and myopia-related retinal neuronal and vascular degeneration. Methods: Aqueous humour from 147 myopic patients, including 60 lowmyopia and 87 high-myopia were collected during Implantable Collamer Lens implantation surgery. All participants received comprehensive ophthalmic examinations, including logMAR best corrected visual acuity, axial length measurement, fundus photography and ocular B-scan ultrasonography. The myopic eyes were further classified into simple myopia (SM, n = 78), myopic posterior staphyloma (PS, n = 39) and PS with myopic chorioretinal atrophy (PS + CA, n = 30). Retinal thickness and vascular density in the macula (6 mm 6 mm) and optic nerve head (4.5 mm 4.5 mm) were measured using Optical Coherence Tomography (OCT) and OCT angiography (OCTA). The levels of complement proteins including C1q, C3, C3b/iC3b, C4, CFB, CFH, C2, C4b, C5, C5a, CFD, MBL and CFI in the aqueous humour were measured using the Luminex Multiplexing system. The real-time RT-PCR was conducted to examine the expression of complement genes (C1q, C2, C3, C4, CFI and CFD) in the guinea pig model of long-term form deprivation-induced myopic retinal degeneration. Results: OCTA showed that retinal neuronal thickness and vascular density in superficial and deep layers of the macular zone as well as vascular density in the optic nerve head were progressively decreased from SM to PS and PS + CA (p < 0.05). The aqueous humour levels of C1q, C3, C3b/iC3b, C4, CFB, CFH, C2, C4b, C5 and CFI were significantly higher in high-myopic eyes compared to those in low-myopic eyes. Further subgroup analysis revealed the highest levels of complement components/fragments in the PS + CA group. The intraocular levels of complement factors particularly C3b/iC3b and C4 were negatively correlated with macular zone deep layer retinal thickness and vascular density and optic nerve head vascular density. The expression of C2, C3 and C4 genes was significantly higher in guinea pig eyes with myopic retinal degeneration compared to control eyes. Conclusions: The intraocular classical pathway and alternative pathway of the complement system are partially activated in pathological myopia. Their activation is related to the degeneration of retinal neurons and the vasculature in the macula and the vasculature in the optic nerve head. [ABSTRACT FROM AUTHOR]

Introduction: To measure copper (Cu), lysyl oxidase (LOX) activity, and collagen levels in aqueous humour (AH) of primary glaucoma patients and correlate with clinical parameters. Methods: 120 patients with 40 each of primary open angle glaucoma (POAG), primary angle closure glaucoma (PACG), and cataract controls were recruited in this case-control study. AH samples were collected during the trabeculectomy and cataract surgeries. Cu levels were measured using an atomic absorption spectrophotometer. LOX unit activity was determined by Amplex Red assay and collagen concentration by Sirius red assay. Results: Significantly higher levels of Cu expressed as median (IQR) µmol/L were observed in POAG (p = 0.008) and PACG (p = 0.005) compared to controls. The LOX activity was increased in POAG and PACG (p = 0.04) compared to controls represented as median (IQR) µmol/min. The collagen levels given as median (IQR) mg/ml showed an insignificant increase in POAG and PACG compared to controls (p = 0.78). The LOX unit activity was correlated with visual field index (VFI), which showed a significant increase with the progression of the diseases (p < 0.05), whereas Cu levels were negatively correlated with LOX activity in AH. Cu and LOX activity showed weak correlation with YAG peripheral iridotomy (YAGPI), duration of anti-glaucoma medications, and highest preoperative intraocular pressure. Conclusion: Elevated Cu and LOX activity was observed in both POAG and PACG groups compared to controls. LOX activity showed notable increase with VFI as the severity of the disease. Although Cu levels are increased in glaucoma, it’s insufficient to significantly increase the activity of LOX. [ABSTRACT FROM AUTHOR]

Background: Ocular manifestations in dogs with leishmaniasis are frequent and complications in affected tissues can lead to blindness. Immune processes play a very important role in the pathogenesis of ocular inflammation. Therefore, the immunology of ocular manifestations in dogs with leishmaniasis remains complex and poorly understood. Objectives: Estimation and characterisation of ocular and periocular manifestations in dogs naturally infected with Leishmania infantum and investigation of the production site of specific anti‐Leishmania infantum IgG. Methods: The present investigation used 53 confirmed dogs infected with Leishmania infantum, presenting ocular and periocular lesions, and 10 control non‐infected dogs. Complete macroscopic ophthalmic examination of eyelids and globes was performed. Both total and anti‐Leishmania infantum IgG antibodies were studied in sera and aqueous humour (AH) of all dogs by ELISA technique. A Goldmann–Witmer coefficient (C value) was calculated. Results: The main ophthalmological findings were keratoconjunctivitis (71.7%; 38/53), hyperplasia of conjunctival lymphoid follicles (54.7%; 29/53), blepharitis (50.9%; 27/53) and uveitis (20.7%; 11/53). Ocular production of anti‐Leishmania infantum IgG was detected in 73.6% (39/53) of infected dogs. There was no correlation between the antibody levels in AH and sera of the same dog. The mean anti‐Leishmania infantum IgG in AH was higher in uveitis, followed by lesions affecting only the adnexa (p < 0.0001). The highest mean C values were observed for uveitis, conjunctivitis and keratitis. Conclusions: Our findings suggest that production of anti‐Leishmania IgG in dogs infected with Leishmania infantum with ocular manifestations begin in situ and follows by a transfer of antibodies from the bloodstream to the AH. [ABSTRACT FROM AUTHOR]

This paper aimed at devising an intelligence-based method to select compounds that can distinguish between open-angle glaucoma patients, type 2 diabetes patients, and healthy controls. Taking the concentration of 188 compounds measured in the aqueous humour (AH) of patients and controls, linear discriminant analysis (LDA) was used to identify the right combination of compounds that could lead to accurate diagnosis. All possibilities, using the leave-one-out approach, were considered through ad hoc programming and in silico massive data production and statistical analysis. Our proof of concept led to the selection of four molecules: acetyl-ornithine (Ac-Orn), C3 acyl-carnitine (C3), diacyl C42:6 phosphatidylcholine (PC aa C42:6), and C3-DC (C4-OH) acyl-carnitine (C3-DC (C4-OH)) that, taken in combination, would lead to a 95% discriminative success. 100% success was obtained with a non-linear combination of the concentration of three of these four compounds. By discarding younger controls to adjust by age, results were similar although one control was misclassified as a diabetes patient. Methods based on the consideration of individual clinical chemical parameters have limitations in the ability to make a reliable diagnosis, stratify patients, and assess disease progression. Leveraging human AH metabolomic data, we developed a procedure that selects a minimal number of metabolites (3–5) and designs algorithms that maximize the overall accuracy evaluating both positive predictive (PPV) and negative predictive (NPV) values. Our approach of simultaneously considering the levels of a few metabolites can be extended to any other body fluid and has potential to advance precision medicine. Artificial intelligence is expected to use algorithms that use the concentration of three to five molecules to correctly diagnose diseases, also allowing stratification of patients and evaluation of disease progression. In addition, this significant advance shifts focus from a single-molecule biomarker approach to that of an appropriate combination of metabolites.

Abstract Background Transforming growth factor (TGF) is a cytokine that acts on the proliferation, migration, differentiation, and apoptosis of cells and the accumulation of extracellular matrix components. Very few studies have precisely evaluated the concentration of TGF-β in the aqueous humour (AH) of diabetic and cataract (DMC) eyes due to the low expression of proteins in the AH or other reasons. The concentrations of TGF-β1, -β2, and -β3 in the AH of the DMC group were compared with those of the age-related cataract (ARC) group. Methods We collected AH and lens epithelium samples from 33 DMC patients and 36 ARC patients. Luminex liquid suspension chip detection was applied to detect the concentration of TGF-β1, -β2, and -β3 in the AH samples. The expression of TGFB1/2/3 in lens epithelium samples was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results The concentrations of TGF-β1 and TGF-β2 in AH samples of DMC eyes were higher than those of ARC eyes. The differences in TGF-β1 and TGF-β2 between the two groups were statistically significant (P value = 0.001 for TGF-β1, P value = 0.023 for TGF-β2). The difference of the correlation between TGF-β1 and glycosylated haemoglobin was significant (P value = 0.011, and Pearson correlation coefficient = 0.306). The difference of the correlation between TGF-β2 and glycosylated haemoglobin was significant (P value = 0.026, and Pearson correlation coefficient = 0.269). The mRNA expression levels of TGFB1 and TGFB2 were upregulated in DMC epithelium samples compared with ARC epithelium samples. The differences in TGFB1 and TGFB2 between the two groups were statistically significant (P value for TGFB1 = 0.041, P value for TGFB2 = 0.021). Conclusions The concentrations of TGF-β1 and TGF-β2 in AH samples were significantly higher in DMC eyes than in ARC eyes. The higher the glycosylated haemoglobin was, the higher the concentrations of TGF-β1 and -β2 were. The mRNA expression of TGFB1 and TGFB2 was significantly upregulated in DMC epithelial samples compared with ARC epithelial samples, suggesting the proinflammatory status of the anterior chamber of DMC eyes.

Maintaining healthy aqueous humour outflow (AHO) is important for intraocular cellular health and stable vision. Impairment of AHO can lead to increased intraocular pressure, optic nerve damage and concomitant glaucoma. An improved understanding of AHO will lead to improved glaucoma surgeries that enhance native AHO as well as facilitate the development of AHO-targeted pharmaceuticals. Recent AHO imaging has evolved to live human assessment and has focused on the structural evaluation of AHO pathways and the functional documentation of fluid flow. Structural AHO evaluation is predominantly driven by optical coherence tomography, and functional evaluation of flow is performed using various methods, including aqueous angiography. Advances in structural and functional evaluation of AHO are reviewed with discussion of strengths, weaknesses and potential future directions.

IntroductionCurrently, the detection of pathogens or mutations associated with intraocular lymphomas heavily relies on prespecified, directed PCRs. With metagenomic deep sequencing (MDS), an unbiased high-throughput sequencing approach, all pathogens as well as all mutations present in the host's genome can be detected in the same small amount of ocular fluid.MethodsIn this cross-sectional case series, aqueous fluid samples from two patients were submitted to MDS to identify pathogens as well as common and rare cancer mutations.ResultsMDS of aqueous fluid from the first patient with vitreal lymphoma revealed the presence of both Epstein-Barr virus (HHV-4/EBV) and human herpes virus 8 (HHV-8) RNA. Aqueous fluid from the second patient with intraocular B-cell lymphoma demonstrated a less common mutation in the MYD88 gene associated with B-cell lymphoma.ConclusionMDS detects pathogens that, in some instances, may drive the development of intraocular lymphomas. Moreover, MDS is able to identify both common and rare mutations associated with lymphomas.

Clearance of ocular fluid and metabolic waste is a critical function of the eye in health and disease. The eye has distinct fluid outflow pathways in both the anterior and posterior segments. Although the anterior outflow pathway is well characterized, little is known about posterior outflow routes. Recent studies suggest that lymphatic and glymphatic systems play an important role in the clearance of fluid and waste products from the posterior segment of the eye. The lymphatic system is a vascular network that runs parallel to the blood circulatory system. It plays an essential role in maintenance of fluid homeostasis and immune surveillance in the body. Recent studies have reported lymphatics in the cornea (under pathological conditions), ciliary body, choroid, and optic nerve meninges. The evidence of lymphatics in optic nerve meninges is, however, limited. An alternative lymphatic system termed the glymphatic system was recently discovered in the rodent eye and brain. This system is a glial cell-based perivascular network responsible for the clearance of interstitial fluid and metabolic waste. In this review, we will discuss our current knowledge of ocular lymphatic and glymphatic systems and their role in retinal degenerative diseases. [ABSTRACT FROM AUTHOR]

Introduction: Myopia is most common refractive error leading to visual dysfunctions worldwide. Oxygen deficit due to environmental stress leads to increase in anaerobic glycolysis which results in lactate production. Increase in Reactive Oxygen Species (ROS) and inability of defense system to eliminate them is also responsible for alteration of regulatory pathways. The patient with elevated lactate levels are at significant risk of morbidity and hence, require a prompt treatment. Aim: To estimate and study the lactate levels in tear in various degree of myopia. Materials and Methods: This case-control study was conducted in tertiary care center, Pt. B.D. Sharma PGIMS, Rohtak, Haryana, India. Twenty five patients each with low, medium, high myopia, age and sex matched healthy controls (i.e, Group 1-4). Diagnosis was established with help of detailed history, clinical and ocular examination. Lactate levels were estimated in tear samples by enzymatic method on autoanalyzer within 30 minutes of collection. Independent Student's t-test was used to determine the significance between myopia and controls. Results: Out of total 100 subjects, 68% were males and 32% were females. The mean±SD age was 24.56±6.35, 24.12±5.45, and 23.24±5.17, 21.69±3.95 in the Group 1, Group 2, Group 3 and Group 4 respectively in the control group, group with low, medium and high myopia respectively which was statistically not significant. The results of this study showed that lactate levels in tears were significantly higher (p-value=0.013) in myopia patients as compared to controls (1.34±0.46 versus 1.06±0.50). Additionally, the lactate levels increased with the progression of degree of myopia. Conclusion: Increase in the level of lactate in tear was directly proportional to the degree of myopia. Tear lactate levels can be used as a non invasive method to study oxidative stress in individuals. Further studies with more oxidative markers in tears can also be compared with blood values in larger groups of patients with long-term follow-up to validate the finding. [ABSTRACT FROM AUTHOR]

In a healthy eye, the aqueous humour (AH) flows via the ciliary body and trabecular meshwork into the collector channels, which carry it to the episcleral veins. In glaucoma, a heterogeneous group of eye disorders affecting approximately 60 million individuals worldwide, the juxtacanalicular meshwork offers greater resistance to the outflow of the AH, leading to an increase in outflow resistance that gradually results in elevated intraocular pressure (IOP). The present review comprehensively covers the morphology of Schlemm's canal (SC) and AH pathways. The path of the AH from the anterior chamber through the trabeculum into suprascleral and conjunctival veins via collector channels is described, and the role of SC in the development of glaucoma and outflow resistance is discussed. Finally, channelography is presented as a precise method of assessing the conventional drainage pathway and facilitating localization of an uncollapsed collector and aqueous veins. Attention is also given to the relationship between aqueous and episcleral veins and heartbeat. Possible directions of future research are proposed. [ABSTRACT FROM AUTHOR]

Glaucoma is an irreversible condition resulting from the increase in intraocular pressure (IOP); which leads to permanent loss of vision with the destruction of retinal ganglion cells (RGCs). The IOP elevations are controlled in normal by the physiological flow of aqueous humour. A population with age above 40 is more susceptible to glaucoma. Other factors like gender, genetics, race etc. plays major roles in the development of the disease. Current treatment methods available for the disease includes drugs come under the classes of beta receptor blockers, carbonic anhydrase inhibitors, cholinergic agonists, prostaglandins etc. N-methyl-D-aspartate (NMDA) antagonists, inducible nitric oxide synthase (iNOS) inhibition, cytoskeletal agents, Rho-kinase inhibitors etc are few novel targets sites which are in research focus for the treatment of the disease. Developments in nanomedicine are also being evaluated for their potential in treating the growing glaucomatous population. Nanosystems are suggested to avoid the difficulties in tackling the various ocular barriers to a limit, help to decrease the instillation frequency of topical medication and can provide drug delivery in a sustained or controlled manner. This review focuses on the current and emerging treatment methods for glaucoma along with some of the nanoformulations for ocular drug delivery.

Introduction: Myopia is most common refractive error leading to visual dysfunctions worldwide. Oxygen deficit due to environmental stress leads to increase in anaerobic glycolysis which results in lactate production. Increase in Reactive Oxygen Species (ROS) and inability of defense system to eliminate them is also responsible for alteration of regulatory pathways. The patient with elevated lactate levels are at significant risk of morbidity and hence, require a prompt treatment. Aim: To estimate and study the lactate levels in tear in various degree of myopia. Materials and Methods: This case-control study was conducted in tertiary care centre, Pt. B.D. Sharma PGIMS, Rohtak, Haryana, India. Twenty five patients each with low, medium, high myopia, age and sex-matched healthy controls (i.e, group 1-4). Diagnosis was established with help of detailed history, clinical and ocular examination. Lactate levels were estimated in tear samples by enzymatic method on autoanalyser within 30 minutes of collection. Independent Student’s t-test was used to determine the significance between myopia and controls. Results: Out of total 100 subjects, 68% were males and 32% were females. The mean±SD age was 24.56±6.35, 24.12±5.45, and 23.24±5.17, 21.69±3.95 in the group 1, group 2, group 3 and group 4, respectively, in the control group, group with low, medium and high myopia, respectively, which was statistically not significant. The results of the present study showed that lactate levels in tears were significantly higher (p-value=0.013) in myopia patients as compared to controls (1.34±0.46 vs 1.06±0.50). Additionally, the lactate levels increased with the progression of degree of myopia. Conclusion: Increase in the level of lactate in tear was directly proportional to the degree of myopia. Tear lactate levels can be used as a non invasive method to study oxidative stress in individuals. Further studies with more oxidative markers in tears can also be compared with blood values in larger groups of patients with long-term follow-up to validate the finding.

Purpose: To monitor the intraocular proangiogenic and profibrotic cytokine profiles within 7 days after intravitreous injection of conbercept (IVC) for patients with proliferative diabetic retinopathy (PDR). Methods: This prospective, randomized controlled, consecutive, comparative study included 157 eyes with PDR. Participant eyes underwent sham IVC or IVC and subsequent vitrectomy at days 2, 3, 4, 5, 6, 7 postinjection. The intraocular cytokines profiles were measured using beaded assay methods. Results: After IVC, the vascular endothelial growth factor (VEGF)‐A level in PDR vitreous decreased rapidly by approximately 10 times at day 2 (p = 0.00001) and kept at a low level at days 3, 4, 5, 6, 7 (p < 0.001, each compared with IVC‐sham group). Similar tendency of the change in VEGF‐A was observed in aqueous humour. The level of placenta growth factor (PIGF) in aqueous humour decreased 2 days after IVC whereas returned to baseline level after 5 days. The vitreous profibrotic cytokines, tissue growth factor (TGF)‐β1, TGF‐β2, TGF‐β3 and connective tissue growth factor did not increase after IVC in each group. Conclusion: We observed a remarkable and rapid decrease in intraocular VEGF‐A, temporal decrease in PIGF from day 2 to day 4, increase in VEGF‐C and VEGF‐D from day 2 onwards, but no profibrotic switch in PDR eyes after IVC. The findings might suggest that ideal vitrectomy timing might be around 3 days after IVC. [ABSTRACT FROM AUTHOR]

Background: Transforming growth factor (TGF) is a cytokine that acts on the proliferation, migration, differentiation, and apoptosis of cells and the accumulation of extracellular matrix components. Very few studies have precisely evaluated the concentration of TGF-β in the aqueous humour (AH) of diabetic and cataract (DMC) eyes due to the low expression of proteins in the AH or other reasons. The concentrations of TGF-β1, -β2, and -β3 in the AH of the DMC group were compared with those of the age-related cataract (ARC) group.Methods: We collected AH and lens epithelium samples from 33 DMC patients and 36 ARC patients. Luminex liquid suspension chip detection was applied to detect the concentration of TGF-β1, -β2, and -β3 in the AH samples. The expression of TGFB1/2/3 in lens epithelium samples was determined by quantitative real-time polymerase chain reaction (qRT-PCR).Results: The concentrations of TGF-β1 and TGF-β2 in AH samples of DMC eyes were higher than those of ARC eyes. The differences in TGF-β1 and TGF-β2 between the two groups were statistically significant (P value = 0.001 for TGF-β1, P value = 0.023 for TGF-β2). The difference of the correlation between TGF-β1 and glycosylated haemoglobin was significant (P value = 0.011, and Pearson correlation coefficient = 0.306). The difference of the correlation between TGF-β2 and glycosylated haemoglobin was significant (P value = 0.026, and Pearson correlation coefficient = 0.269). The mRNA expression levels of TGFB1 and TGFB2 were upregulated in DMC epithelium samples compared with ARC epithelium samples. The differences in TGFB1 and TGFB2 between the two groups were statistically significant (P value for TGFB1 = 0.041, P value for TGFB2 = 0.021).Conclusions: The concentrations of TGF-β1 and TGF-β2 in AH samples were significantly higher in DMC eyes than in ARC eyes. The higher the glycosylated haemoglobin was, the higher the concentrations of TGF-β1 and -β2 were. The mRNA expression of TGFB1 and TGFB2 was significantly upregulated in DMC epithelial samples compared with ARC epithelial samples, suggesting the proinflammatory status of the anterior chamber of DMC eyes. [ABSTRACT FROM AUTHOR]

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open‐angle glaucoma patients compared to non‐glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti‐apoptotic and anti‐TGF‐β activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF‐β2‐induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride‐induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF‐α‐induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF‐β induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma. [ABSTRACT FROM AUTHOR]

To explore the role of lncRNA m 6 A methylation modification in aqueous humour (AH) of patients with pseudoexfoliation glaucoma (PXG). Patients with open-angle PXG under surgery from June 2021 to December 2021 were selected. Age- and gender-matched patients with age-related cataract (ARC) were chosen as control. Patients underwent detailed ophthalmic examinations. 0.05-0.1 ml AH were extracted during surgery for MeRIP-Seq and RNA-Seq. Joint analysis was used to screen lncRNAs with differential m 6 A methylation modification and expression. Online software tools were used to draw lncRNA-miRNA-mRNA network (ceRNA). Expression of lncRNAs and mRNAs was confirmed using quantitative real-time PCR. A total of 4151 lncRNAs and 4386 associated m 6 A methylation modified peaks were identified in the PXG group. Similarly, 2490 lncRNAs and 2595 associated m 6 A methylation modified peaks were detected in the control. Compared to the ARC group, the PXG group had 234 hypermethylated and 402 hypomethylated m 6 A peaks, with statistically significant differences (| Fold Change (FC) |≥2, p  < 0.05). Bioinformatic analysis revealed that these differentially methylated lncRNA enriched in extracellular matrix formation, tight adhesion, TGF- β signalling pathway, AMPK signalling pathway, and MAPK signalling pathway. Joint analysis identified 10 lncRNAs with differential m 6 A methylation and expression simultaneously. Among them, the expression of ENST000000485383 and ROCK1 were confirmed downregulated in the PXG group by RT-qPCR. m 6 A methylation modification may affect the expression of lncRNA and participate in the pathogenesis of PXG through the ceRNA network. ENST000000485383-hsa miR592-ROCK1 May be a potential target pathway for further investigation in PXG m 6 A methylation.

Objective: This study aimed to perform genotyping of Toxoplasma gondii strain or variant causing atypical toxoplasmic uveitis in Indonesian patients. Methods: Ocular fluid samples originating from 46 uveitis patients with non-specific ocular manifestations were analysed for Toxoplasma infection by PCR of the B1 locus. The clonal type was determined by amplification, sequencing and phylogenetic analysis of SAG2 and GRA6 loci in B1-positive samples. Clinical data were obtained from the medical records. Results: Pan uveitis was the most frequent manifestation (65.2%) and mostly unilateral (76.1%). PCR of the B1 locus identified eight positive subjects (12.5%); six had panuveitis and two of these had diabetes mellitus. Phylogenetic analysis with maximum likelihood of the SAG2 locus in the B1-positive samples resulted in Toxoplasma gondii SAG2 type III allele. No positive result was obtained from the PCR of GRA6 locus. Conclusion: Toxoplasma gondii SAG2-type III allele was identified in an atypical presentation of toxoplasmic uveitis in Indonesia.

Glaucoma is an irreversible condition resulting from the increase in intraocular pressure (IOP); which leads to permanent loss of vision with the destruction of retinal ganglion cells (RGCs). The IOP elevations are controlled in normal by the physiological flow of aqueous humour. A population with age above 40 is more susceptible to glaucoma. Other factors like gender, genetics, race etc. plays major roles in the development of the disease. Current treatment methods available for the disease includes drugs come under the classes of beta receptor blockers, carbonic anhydrase inhibitors, cholinergic agonists, prostaglandins etc. N-methyl-D-aspartate (NMDA) antagonists, inducible nitric oxide synthase (iNOS) inhibition, cytoskeletal agents, Rho-kinase inhibitors etc are few novel targets sites which are in research focus for the treatment of the disease. Developments in nanomedicine are also being evaluated for their potential in treating the growing glaucomatous population. Nanosystems are suggested to avoid the difficulties in tackling the various ocular barriers to a limit, help to decrease the instillation frequency of topical medication and can provide drug delivery in a sustained or controlled manner. This review focuses on the current and emerging treatment methods for glaucoma along with some of the nanoformulations for ocular drug delivery. [ABSTRACT FROM AUTHOR]

Abstract Background Inflammation in anterior uveitis is characterised by breakdown of the blood-ocular barrier, which allows leakage of blood constituents of higher molecular weight into the aqueous humour. In routine clinical care, increase in aqueous protein levels can be observed at the slit lamp as ‘flare’ and the severity can be graded using various clinical grading systems, of which the Standardization of Uveitis Nomenclature (SUN) grading system is most commonly used. Alternative instrument-based technologies are available, which can detect aqueous protein levels in an objective and quantifiable way. This review will identify instruments capable of measuring anterior chamber inflammation in this way, their level of reliability, and how well the measurements correlate with clinical grading and/or actual aqueous protein concentration. Methods Standard systematic review methodology will be used to identify, select and extract data from studies that report the use of any instrument-based technology in the assessment of aqueous protein levels. Searches will be conducted through bibliographic databases (MEDLINE, EMBASE and Cochrane Library), clinical trial registries and the grey literature. No restrictions will be placed on language or year of publication. The outcomes of interest are the level of correlation between identified instrument-based test measurements, clinical grading and/or actual aqueous protein concentration, as well as the reliability of each index test identified. Study quality assessment will be based on QUADAS2. Correlation and reliability outcomes will be pooled and meta-analysed if appropriate. Discussion The assessment of inflammation in anterior chamber protein levels currently relies on crude and subjective clinical examination. The findings of this review will identify non-invasive technologies which show good correlation with actual protein concentration, which could be used in routine clinical practice for objective monitoring of AC inflammation. Systematic review registration PROSPERO CRD42017084167. Study screening stage has just been completed.

Glaucoma is a multifactorial neurodegenerative disease that causes impaired vision and, in advanced cases, blindness. The increasing prevalence of glaucoma due to an ageing population has necessitated the identification of suitable biomarkers for the early detection of the disease. Aqueous humour (AH) has been proposed as a source of biomarkers, but it can only be collected using a minor, yet invasive surgical intervention. Tears, however, are constantly available and can be collected any time via noninvasive methods. In order to examine the utility of tear as a surrogate for aqueous humour in biomarker development, we compared the levels of 27 cytokines and chemokines in paired samples of tear and aqueous humour using a Luminex multiplex immunobead‐based technique. Significantly higher levels of cytokines in tear compared to aqueous humour were detected suggesting that tear and aqueous humour are not identical in terms of inflammation response. Furthermore, the levels of IFN‐γ, GM‐CSF and IL‐5 in tear were significantly lower in patients who developed complications after one year, but no statistically significant changes in cytokine levels were observed in aqueous humour. These three molecules may have potential as predictive biomarkers for the appearance of late flap‐related complications of trabeculectomy.

Abstract Background Uveitis describes a group of inflammatory conditions affecting the eye. The ability to monitor inflammatory changes in anterior uveitis is crucial in clinical practice for making treatment decisions and in clinical trials for testing therapeutic agents. The current standard for quantifying anterior segment inflammation is clinical slit-lamp examination findings classified using the Standardisation of Uveitis Nomenclature (SUN) grading system. Such clinical grading systems rely on a subjective estimation using the slit lamp and are often non-linear and non-continuous scales, with large increases in cell count between each grade. Novel instrument-based technologies have emerged over the last few decades, which can provide objective and quantifiable measurements. This review will evaluate the reliability of such technologies and their level of agreement with anterior chamber (AC) cell count using clinical slit-lamp examination. Methods Standard systematic review methodology will be used to identify, select and extract data from studies that report the use of any instrument-based technology in the assessment of AC cells. Searches will be conducted through bibliographic databases (MEDLINE, EMBASE and Cochrane Library), clinical trial registries and the grey literature. No restrictions will be placed on language or year of publication. The outcomes of interest are the correlation of index test measurements of AC cells with clinical grading systems using slit-lamp examination and the reliability of each index test identified. Quality assessment will be undertaken using QUADAS2. Degree of correlation between the index and reference test measures will be pooled and meta-analysed if appropriate. Discussion A number of instrument-based tools are available for measuring AC cells. This review will evaluate the technologies available and measure the level of correlation of these alternative methods with clinical grading systems as well as their performance in reliability and repeatability. The findings of this review will identify those objective, instrument-based technologies which show good utility for measuring AC cells in a quantifiable way and which warrant further exploration for their sensitivity and reliability over the current standard. Systematic review registration PROSPERO CRD42017084156 (Liu X, Moore DJ, Denniston AK). Instrument-based tests for measuring anterior chamber (AC) cells in uveitis: a systematic review. 2017). Study screening stage is complete. Data extraction stage has not yet commenced.

Abstract Background The purpose of this study is to examine the clinical outcomes achieved by using initial high-dose intravitreal ganciclovir injections to treat cytomegalovirus retinitis in patients without human immunodeficiency virus (HIV) infection. Methods Twenty-four eyes (24 patients) with cytomegalovirus retinitis received multiple intravitreal injections of ganciclovir in weekly intervals. A higher dose (6 mg) of ganciclovir was applied at the first intravitreal injection, and a lower dose was used for maintenance. Anterior aqueous humour was obtained before each injection. The best-corrected visual acuity and cytomegalovirus loads in the anterior aqueous humour were measured. Results The mean cytomegalovirus DNA load in aqueous humour decreased significantly from (2.59 ± 2.28) × 105 copies/mL at baseline to (1 ± 1.76) × 104 copies/mL one month later. The aqueous cytomegalovirus DNA load was negative in 17 eyes (70.8%) one month later. No obvious improvement of best-corrected visual acuity was found during the treatment. A positive correlation was proven between initial cytomegalovirus DNA titers in aqueous humour and the total number of intravitreal injections of ganciclovir, as well as between the baseline and final best-corrected visual acuities. No severe complications developed. Conclusions An initial high dose of ganciclovir (6 mg) and continuous intravitreal injections of ganciclovir could significantly decrease the cytomegalovirus load in HIV-negative patients with cytomegalovirus retinitis. Trial registration http://clinicaltrials.gov, NCT03598452, retrospectively registered on 24 July 2018.

Primary open‐angle glaucoma (POAG) is characterized by irreversible neurodegeneration accompanied by visual field defects and high intraocular pressure. Currently, an effective treatment is not available to prevent the progression of POAG, other than treatments to decrease the high intraocular pressure. We performed proteomic analysis of aqueous humour (AH) samples from patients with POAG combined with cataract and patients with cataract to obtain a better understanding of the pathogenesis of POAG and explore potential treatment targets for this condition. Samples were collected from 10 patients with POAG combined with cataract and 10 patients with cataract. Samples from each group were pooled. A high‐resolution, label‐free, liquid chromatography‐tandem mass spectrometry‐based quantitative proteomic analysis was performed. In total, 610 proteins were identified in human AH samples from the two groups. A total of 48 up‐regulated proteins and 49 down‐regulated proteins were identified in the POAG combined with cataract group compared with the control group. Gene Ontology (GO) analysis revealed key roles for these proteins in inflammation, immune responses, growth and development, cellular movement and vesicle‐mediated transport in the biological process category. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the down‐regulated expression of glutathione S‐transferase P (GSTP1) in the glutathione metabolism signalling pathway in the POAG combined with cataract group. Additionally, certain significantly differentially expressed proteins in the proteomic profile were verified by enzyme‐linked immunosorbent assay (ELISA). GSTP1 levels were reduced in the human AH samples from the POAG combined with cataract group, based on the results of ELISA and proteomic profiling. Therefore, GSTP1, a redox‐related marker, may be involved in the pathological process of POAG and may become a treatment target in the future. [ABSTRACT FROM AUTHOR]

• Toxoplasma gondii infection caused significant visual impairment. • Toxoplasma infection should be suspected in atypical presentation of panuveitis and posterior uveitis. • Certain clonal type of Toxoplasma gondii were associated with the ocular manifestation. • This is the first report of human ocular toxoplasmosis genotyping from Indonesia. This study aimed to perform genotyping of Toxoplasma gondii strain or variant causing atypical toxoplasmic uveitis in Indonesian patients. Ocular fluid samples originating from 46 uveitis patients with non-specific ocular manifestations were analysed for Toxoplasma infection by PCR of the B1 locus. The clonal type was determined by amplification, sequencing and phylogenetic analysis of SAG2 and GRA6 loci in B1 -positive samples. Clinical data were obtained from the medical records. Pan uveitis was the most frequent manifestation (65.2%) and mostly unilateral (76.1%). PCR of the B1 locus identified eight positive subjects (12.5%); six had panuveitis and two of these had diabetes mellitus. Phylogenetic analysis with maximum likelihood of the SAG2 locus in the B1 -positive samples resulted in Toxoplasma gondii SAG2 type III allele. No positive result was obtained from the PCR of GRA6 locus. Toxoplasma gondii SAG2 -type III allele was identified in an atypical presentation of toxoplasmic uveitis in Indonesia. [ABSTRACT FROM AUTHOR]

Purpose: To characterize profile of cytokines in aqueous humour of common macular diseases during intravitreal anti‐VEGF therapy. Methods: Aqueous humour from eyes with central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic macular oedema (DME), neovascular age‐related macular degeneration (nAMD) or pathologic myopia associated choroidal neovascularization (pmCNV) was sampled prior to 1st (n = 144) and 2nd (n = 48) intravitreal anti‐VEGF therapy. Cytokines including vascular endothelium growth factor (VEGF), intercellular adhesion molecule 1 (ICAM‐1) and interleukin 6 (IL‐6) were quantitated and analysed along with retinal thickness data by optical coherence tomography (OCT) across two intravitreal injections and five macular disease types. Results: ICAM‐1, IL‐6 and VEGF are positively associated in the aqueous humour of naive eyes (r = 0.39–0.77, p = 0.018 to <0.0001). ICAM‐1, VEGF and IL‐6 were significantly higher in CRVO and DME while lowest in pmCNV (p < 0.0001). Reduction of central retinal thickness (CRT) as a favourable response to anti‐VEGF therapy was in the order of CRVO, BRVO, DME and nAMD/pmCNV (p < 0.0001). The strongest predictor for favourable CRT reduction was baseline CRT (p < 0.0001) followed by baseline ICAM‐1 (p = 0.04). After the 1st intravitreal anti‐VEGF therapy, VEGF in aqueous humour lowered significantly but ICAM‐1 and IL‐6 levels remained unchanged. ICAM‐1 was not predictive for CRT reduction following 2nd anti‐VEGF therapy. Conclusion: Rate of cytokine production is disease‐dependent and higher in CRVO and DME. Anatomical response to intravitreal anti‐VEGF therapy is disease‐specific and best in RVO patients. A combination therapy using both anti‐VEGF and anti‐inflammatory therapeutics may be superior to single anti‐VEGF therapy, at least for RVO and DME. [ABSTRACT FROM AUTHOR]

Elevated intraocular pressure (IOP) is a risk factor in glaucoma, a group of irreversible blinding diseases. Endogenous lipids may be involved in regulation of IOP homeostasis. We present comparative fold analysis of phospholipids and sphingolipids of aqueous humour and trabecular meshwork from human control vs primary open‐angle glaucoma and mouse control (normotensive) vs ocular hypertensive state. The fold analysis in control vs disease state was based on ratiometric mass spectrometric data for above classes of lipids. We standardized in vitro assays for rapid characterization of lipids undergoing significant diminishment in disease state. Evaluation of lipids using in vitro assays helped select a finite number of lipids that may potentially expand cellular interstitial space embedded in an artificial matrix or increase fluid flow across a layer of cells. These assays reduced a number of lipids for initial evaluation using a mouse model, DBA/2J with spontaneous IOP elevation. These lipids were then used in other mouse models for confirmation of IOP lowering potential of a few lipids that were found promising in previous assessments. Our results provide selected lipid molecules that can be pursued for further evaluation and studies that may provide insight into their function. [ABSTRACT FROM AUTHOR]

Abstract Background Glaucoma is classified as a neurodegenerative disease. However, the biomarkers of neurodegeneration in the aqueous humour of primary open angle glaucoma (POAG) eyes have not been quantitatively examined yet. In this study, levels of neurodegeneration-related cytokines in the aqueous humour of POAG eyes were measured and compared with those of non-glaucoma (senile cataract) control eyes. Methods This cross-sectional study included 24 patients (24 eyes) with POAG and 22 patients (22 eyes) with cataract. Aqueous humour samples were collected before the commencement of phacoemulsification surgery. The concentrations of brain-derived neurotrophic factor (BDNF), cathepsin D, myeloperoxidase (MPO), soluble intercellular adhesion molecule-1 (sICAM-1), soluble neural cell adhesion molecule (sNCAM), soluble vascular cell adhesion molecule-1 (sVCAM-1), and plasminogen activator inhibitor-1 (PAI-1) were measured using the Luminex suspension array technique. The clinical characteristics of the patients were also obtained for correlation analysis. Results Compared with the cataract group, the levels of cathepsin D (P

Abstract Background To assess the sensitivity, specificity, positive predictive value and negative predictive value of anterior chamber tap for the diagnosis of bacterial endophthalmitis on a population with high prevalence. Methods Retrospective, single centre, case series study. We reviewed all medical records with clinical diagnosis of bacterial endophthalmitis in our hospital from January 1st, 2000 to December 31st 2014. From each record, we documented general demographic data, best corrected visual acuity and vitreous and aqueous tap microbiological results. All cases were further divided according to the endophthalmitis aetiology to perform individual calculations of sensitivity, specificity, positive predictive value, negative predictive value, accuracy and prevalence. We used the results of the vitreous tap as the gold standard for diagnosis of bacterial endophthalmitis. We excluded those records in which the aqueous and vitreous samples were not taken simultaneously or had an incomplete microbiological report. Significance were assessed with chi squared statistics, with an alpha value of 0.05 for statistical significance. Results A total of 190 cases fulfilled the inclusion/exclusion criteria. Positive culture rate from vitreous samples was 64.74%. Positive culture rate from aqueous sample was 32.11%. Bacteria isolated from aqueous samples matched those isolated from vitreous samples 78.68% of the time. The overall sensitivity was 38.21%, specificity: 75.51%, positive predictive value: 79.66%, negative predictive value: 32.74% (p = 0.08). Subgroup analysis showed that anterior chamber taps in cases of post-surgical endophthalmitis had a moderate to low sensitivity (37.73%), high specificity (93%) and high positive predictive value (95%) (p

Purpose: The quality of biological fluid samples is vital for optimal preanalytical procedures and a requirement for effective translational biomarker research. This study aims to determine the effects of storage duration and freeze-thawing on the levels of various cytokines in the human aqueous humour and vitreous samples., Methods and Analysis: Human ocular aqueous humour and vitreous samples were obtained from 25 eyes and stored at -80°C for analysis. All samples were assayed for 27 cytokine biomarker concentrations (pg/mL) using a multiplex assay. Four sample storage durations following sample collection were evaluated (1 week, 3 months, 9 months and 15 months). Additionally, samples underwent up to three freeze-thaw cycles within the study period., Results: Among the 27 cytokine biomarkers, concentrations of four cytokines (Interleukin (IL)-2, IL-10, IL-12 and platelet-derived growth factor-BB) were significantly decreased by storage duration at all time points, as early as 3 months following sample collection (range of 9%-37% decline between 1 week and 15 months, p<0.001). Freeze-thawing of up to three cycles did not significantly impact the cytokine biomarker concentrations in aqueous humour or vitreous. Separability of patient-specific cytokine biomarker profiles in the principal component analysis remained relatively the same over the 15 months of storage duration., Conclusion: The findings from this study suggest that several intraocular cytokine biomarkers in human aqueous humour and vitreous samples may be susceptible to degradation with long-term storage, as early as 3 months after collection. The overall patient-specific cytokine biomarker profiles are more stable than concentrations of individual cytokines. Future studies should focus on developing guidelines for optimal and standardised sample handling methods to ensure correct research findings about intraocular biomarkers are translated into clinical practice., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)